Epitranscriptomic marks in the form of RNA modifications are catalyzed by specific enzymes or RNA-protein complexes on the base or ribose sugar of the canonical adenosine, cytosine, guanosine and uridine nucleosides. RNA modifications can include simple changes such as methylation or acetylation, or hyper modifications involving multiple enzymes and cofactors. Transfer RNA (tRNA) is the most heavily modified RNA species with an average of 13 RNA modifications comprising ~17% of the 80-90 ribonucleosides. Epitranscriptomic marks can be located throughout tRNA molecules and can affect tRNA stability, folding, localization, transport, processing, and function (Jackman & Alfonzo, 2013) . Modifications in the anticodon loop of tRNA can function as regulators of translation by promoting or preventing codon-anticodon interaction, by altering charge or base-pairing potential. Global studies using cell systems have demonstrated that anticodon specific epitranscriptomic marks, as well as others, are dynamically regulated during cellular stress. Further it has been shown that the collection of epitranscriptomic marks change depending on the mechanism of action of the toxicants (C. T. Y. Chan et al., 2015 Chan et al., , 2010 Dewe, Fuller, Lentini, Kellner, & Fu, 2017) .
Toxicant specific reprogramming of the epitranscriptome housed on tRNA has been demonstrated in bacterial, yeast and human cell studies (C. T. Y. Chan et al., 2015 Chan et al., , 2010 Dewe et al., 2017) . For example, in response to oxidizing agents such as γradiation, hydrogen peroxide (H 2 O 2 ), tert-Butyl hydroperoxide (TBHP) or peroxynitrile (ONOO -), there is a significant increase in m 5 C, ncm 5 U, and i 6 A levels in yeast tRNA while the same modifications are unaffected in response to alkylating agents. In contrast, Um, mcm 5 U, mcm 5 s 2 U, m 2 G, and m 1 A modification levels increase in response to alkylating agents ethylmethanesulfonate (EMS), methylmethanesulfonate (MMS), isopropyl methanesulfonate (IMS) and N-methyl-N'-nitro-N-mitrosoguanidine (MNNG) , and are conversely unaffected by treatment with oxidizing agents. These data support the idea that there is a highly predictive and toxicant-dependent system of translational control used to respond to cellular stress. Specifically the reprogrammed tRNA promotes the selective translation of codon-biased mRNAs in order to translate critical survival proteins. Codon biased transcripts being more efficiently translated by increased epitranscriptomic marks has been observed in bacteria (Chionh et al., 2016) , yeast , mouse embryonic fibroblasts (Endres et al., 2015) and human melanoma cells (Rapino et al., 2018) . A specific example of epitranscriptomic marks driving codon biased translation is found with the RNA modifications 5-methoxycarbonylmethyluridine (mcm 5 U) and 5-methoxycarbonylmethyluridine-2'-O-methyluridine (mcm 5 Um) housed on wobble uridine of selenocysteine tRNA (tRNA Sec ) in mice and humans. The epitranscriptomic marks mcm 5 U and mcm 5 Um are an integral part of the specialized addition of the selenocysteine (Sec) amino acid into proteins. The resulting selenoproteins are made by stop codon recoding (Copeland, 2003; Copeland, Fletcher, Carlson, Hatfield, & Driscoll, 2000; Endres et al., 2015; Low & Berry, 1996; Songe-Moller et al., 2010) , where mcm 5 U and mcm 5 Um promote binding of the tRNA Sec anticodon to the UGA stop codon on mRNA. Corresponding selenoprotein transcripts are inherently codon-biased because of this unusual in-frame UGA stop codon as well as the actual stop codon (Doyle et al., 2016) . Importantly though, other core elements are required for stop codon recoding and they include a stem-loop structure in the 3' untranslated region (3'UTR) of the transcript termed the Sec Insertion Sequence (SECIS) element, a SECIS Binding Protein (SBP2), and SEC-specific elongation factors (EFSec) (Driscoll & Copeland, 2003) .
for the formation of modified wobble uridines on tRNA Sec to promote UGA stop codon recoding (Dragony Fu et al., 2010; Songe-Moller et al., 2010) . ALKBH8 has three functional domains, an RNA recognition motif (RRM), a 2-oxogluterate Fe 2+ -dependent oxygenase (2-OG-Fe(II)) domain, sometimes referred to as the AlkB domain, and a methyltransferase domain (Fig. 1) . The Elongator complex which includes six proteins (Elp1-6) is required for the carboxymethylation of uridine (U) to 5-carbonylmethyluridine (cm 5 U) Karlsborn et al., 2014) . ALKBH8 methyltransferase activity on the wobble uridine of tRNA Sec uses 5-carbonylmethyluridine (cm 5 U) as a substrate to produce mcm 5 U. ALKBH8 also catalyzes wobble mcm 5 U in tRNA Lys(UUU) , tRNA Gln(UUG) and tRNA Glu(UUC) and tRNA Arg(UCU) (D. Hatfield & Gladyshev, 2002; Songe-Moller et al., 2010; Sprinzl & Vassilenko, 2005) . Furthermore, mcm 5 U can undergo additional modifications to mcm 5 Um (tRNA Sec ) (Endres et al., 2015;  Songe-Moller et al., 2010), mcm 5 s 2 U (tRNA Glu(UUC, tRNA Arg(UCU) and tRNA Lys(UUU) ) (Sprinzl & Vassilenko, 2005) , and mchm 5 U (tRNA Gly(UCC) and tRNA Arg(UCG) )(Van Den Born et al., 2011) . Among these further modifications to mcm 5 U, only mchm 5 U is known to be catalyzed directly by ALKBH8 via its 2OG-Fe(II) oxygenase domain (Van Den Born et al., 2011) . The mcm 5 s 2 U modification depends on the CTU1/CTU2 enzyme complex, while formation of mcm 5 Um is dependent on ALKBH8 (Dewez et al., 2008; Endres et al., 2015; Songe-Moller et al., 2010) .
The mcm 5 U and mcm 5 Um modifications on tRNA Sec help translationally regulate the production of selenoproteins, which comprise an important group of activities involved in development, reproduction and protection against oxidative stress. Selenoproteins such as glutathione peroxidase (GPX) family members catalyze the reduction of hydrogen peroxide and lipid peroxides by glutathione and play an essential role in protecting cells against oxidative damage. Thioredoxin reductases (TRXRs) are involved in maintaining the reduced state of thioredoxins (TRXs), which function to reduce oxidative stress and play critical roles in development.
We have previously shown that deficiency in ALKBH8 Similar to other important stress and selenoprotein deficiency (Haan et al., 1998) , the ALKBH8 deficient mice may need to experience an external stress to display observable phenotypes.
Naphthalene (NA) is a polyaromatic hydrocarbon (PAH) and exposure to this environmental toxicant can promote ROS, DNA damage and disrupt cellular glutathione levels (ATSDR, 2005; D. Bagchi, Bagchi, Balmoori, Vuchetich, & Stohs, 1998; M. Bagchi, Bagchi, Balmoori, Ye, & Stohs, 1998; Carratt et al., 2019; Lin et al., 2005; Plopper et al., 2013a) . NA is found in cigarette smoke, gasoline, and mothballs (Jia & Batterman, 2010; Kakareka & Kukharchyk, 2003; Sudakin, Stone, & Power, 2011) Li et al., 2008) . NA is currently classified as a possible human carcinogen (IARC, 2002) and has been shown to cause lesions and tumors in rodents, although the mechanism of toxicity and carcinogenicity is still under debate (Abdo et al., 1992; Abdo, Grumbein, Chou, & Herbert, 2001) . Notably NA has been shown to cause oxidative DNA damage (Lin et al., 2005) , and DNA adduct formation Carratt et al., 2019) . All tumor formation in both mice and rats was accompanied by cytotoxicity, with recurrent damage and repair/proliferation thought to be one of the driving forces behind NA-induced tumor formation in the lung (DeStefano-Shields, Morin, & Buckpitt, 2010; Van Winkle, Buckpitt, Nishio, Isaac, & Plopper, 1995) . Importantly, glutathione (GSH) depletion is a major determinant in NA-induced lung toxicity, and precedes observable injury. Mice treated with the GSH depleter dimethylmaleate before exposure suffered significantly more injury to the nasal and intrapulmonary airway epithelium (Phimister, Lee, Morin, Buckpitt, & Plopper, 2004) and mice treated with the GSH synthesis inhibitor buthionine sulfoximine exhibited similar effects (West et al., 2003 (West et al., , 2002 . Treatment of mice with glutathione prodrugs prevent lung cell toxicity and necrosis (Phimister, Nagasawa, Buckpitt, & Plopper, 2005) . Selenoprotein H plays a role in glutathione synthesis by acting as a transcription factor to promote transcription of genes critical for glutathione synthesis as well as exhibiting oxidoreductase activity (Panee, Stoytcheva, Liu, & Berry, 2007) . As such, disrupted ALKBH8 epitranscriptomic systems leading to dysregulated translation of selenoproteins required for GSH synthesis, use and redox balance are likely to sensitize mice to NA.
In the following study we have tested the hypothesis that the ALKBH8 epitranscriptomic writer plays a protective role against NA-induced lung toxicity in vivo. We have characterized the lungs of Alkbh8 deficient (Alkbh8 def ) mice under normal conditions to elucidate how these mice adapt to an epitranscriptomic defect. We have also performed NA exposure studies on WT, Alkbh8 def , and Cyp2abfgs-null mice. The Cyp2abfgs-null mice are a total-body knockout of a 12-gene cluster of lung preferential CYPs needed for NA bioactivation in the lung (Lei Li, Megaraj, Wei, & Ding, 2014) . As NA bioactivation is a well-documented requirement of NA toxicity as previously described, the Cyp2abfgsnull mice served as our negative controls (L. Li et al., 2011; Lei Li et al., 2017) . acceptor sequence upstream of a β-geo cassette (β-galactosidase/neomycin phosphotransferase fusion), which was inserted into intron 7 at chromosome position 9:3349468-3359589, creating a fusion transcript of Alkbh8-β-geo (Stryke et al., 2003) , as previously described (Endres et al., 2015) . Multiplex qRT-PCR and relative cycle threshold analysis (ΔΔ CT) on genomic DNA derived from tail biopsies was used to determine animal zygosity for Alkbh8 def with TaqMan oligonucleosides specific for neomycin (Neo, target allele) and T-cell receptor delta (Tcrd, endogenous control). Neo Fig. 1 ).
Lung tissue was solubilized in extraction buffer (0.1 g per 1 mL phosphate buffered saline + 0.1% v/v Triton-X-100) using a rotor/stator tissue homogenizer. 30 mL of lysate was then applied to a filter-based sensor inserted into the RedoxSYS® galvanostat (RedoxSYS, Aytu BioScience, Inc., Englewood, CO). An electrochemical circuit was made when the sample (drawn by liquid flow) entered the sensor cell, after which an initial ORP measurement was made followed by the application of a current sweep through the sample, which exhausts the antioxidants present in the sample; the amount of current required to do this was determined over a 3-minute period. The primary output was a measurement of static ORP (mV), also known as the redox potential, representing the potential for electrons to move from one chemical species to another.
WT, Alkbh8 def , Cyp2abfgs-null and Alkbh8 def /Cyp2abfgs-null double mutant mice were exposed to NA as described above and sacrificed 24h after last injection by CO 2 asphyxation. Lungs and trachea were exposed, a small incision was placed in the trachea, and the lungs were slowly inflated with 0.8 mL Z-Fix formalin based fixative Quantitative analysis and imaging of γ-H2AX foci (FITC: 570-620nm) and nuclei (DRAQ5: >655 nm) was performed on 10,000 cells per mouse, with bright field and fluorescence images quantified in separate channels and then merged. A compensation matrix was established with γ-H2AX and DRAQ5 stains in separate samples to eliminate overlap in emission spectra between channels. The IDEAS Spot Count analysis program was used to quantify γ-H2AX foci and γ-H2AX channel fluorescence. DRAQ5
fluorescence was used to ensure that all spots counted were in the nucleus.
8-isoprostane was measured as an indicator of ROS in mouse lungs using an 8- Tissue homogenates were processed using protein precipitation and centrifugation as follows: 40µL internal standard, GSH-13 C2, 15 N (3 µg/mL in 10% acetonitrile), was added to 40µL of tissue homogenate, followed by the addition of 200µL methanol and 200µL
water. The samples were mixed and centrifuged at 4°C and 14,000 rpm for 10 minutes.
The supernatant was collected. 300µL 0.1N HCL was added to 100µL supernatant and vortexed for 30 s and centrifuged at 4°C and 14,000 rpm for 10 minutes. The supernatant was collected and 3 µL injected into the LC-MS system.
The LC-MS system consisted of an Agilent HPLC-1290 (Agilent, USA), and a Qtrap 6500 plus mass spectrometer (Sciex, Ontario, Canada) equipped with a Turbo IonSpray source. For data acquisition and processing, Analyst Software 1.6.3 was used.
Chromatographic separation was performed on a reversed-phase ZORBAX SB-C8
column ( 
Calibration standards for GSH and GSSG were prepared by diluting the stock solutions with water:acetonitrile (9:1,v/v) to the final concentrations of 1, 2, 10, 20, 50, 100 µg/mL;.
Calibration standards were processed using protein precipitation and centrifugation as follows: 40µL internal standard, GSH-13 C2, 15 N (3 µg/mL in 10% acetonitrile), was added to 40µL of calibration standards and followed by the addition of 200µL methanol and 200µL water. The samples were mixed and centrifuged at 4°C and 14,000 rpm for 10 minutes. The supernatant was collected. 300µL 0.1N HCL was added to 100µL supernatant and vortexed for 30 s and centrifuged at 4°C and 14,000 rpm for 10 minutes.
The supernatant was collected and 3 µL injected into the LC-MS system. 
WT, Alkbh8 def and Cyp2abfgs-null/Alkbh8 def were euthanized using CO 2 asphyxiation, the lungs and trachea were exposed, a small incision was placed in the trachea, intubated using a sterile, disposable blunt end needle, 22 gauge, 0.5 in (Part #B22-50, SAI Infusion Technologies, Lake Villa, IL) secured with a piece of thread. Next, the lungs were slowly inflated with 0.8 ml PBS, using a 1-ml Coviden™ Monoject™ tuberculin syringe (Fisher Scientific, Franklin, MA) and fluid removed and re-injected 2 additional times over the course of ~1 minute to draw out the maximum number of inflammatory cells. The PBS containing cells was then transferred to a 2-ml microcentrifuge tube. The PBS lavage step was repeated 2 more times with 0.5ml PBS each. The pooled Bronchoalveolar Lavage Fluid (BALF) was then mixed by gentle pipetting, dissolved 1:1 with trypan blue (Thermo Fisher), and live cells immediately counted on a hemocytometer. Cells/ml were plotted (N = 3 mice per genotype per condition), with error bars denoting standard error of the mean.
Under standard care conditions, with no external applied stress, 12-week-old WT and We also investigated molecular changes in the lungs of Alkbh8 def mice by measuring selenoprotein levels. We used a Simple Western (Wes) automated western blot tool for protein detection. Under normal growth conditions, we observed little difference in GPX1, GPX3, and Selenoprotein S (SELS) protein levels. In contrast we observed a significant decrease in TRXR1, TRXR2 and TRX2 protein levels in the lungs of Alkbh8 def mice. TRXRs are Sec-containing proteins critical for all signaling pathways in which thioredoxin (TRX) is involved as a reducing agent as TRXRs maintain TRX in its reduced and active state. As such, they support other TRX-dependent antioxidants such as methionine sulfoxide reductases (MSRS) and peroxiredoxins (PRXs). Dysfunctions in the TRXR system have been linked to oxidative stress, mitochondrial dysfunction, apoptosis and sensitivity to added stress (Lopert, Day, & Patel, 2012) , and molecular reprogramming in the lung to adapt to the stress (Locy et al., 2012) . TRX2 is localized to the mitochondria and is an essential member of the thioredoxin system, with deletions associated with massive dysregulation of redox balance, apoptosis and early embryonic lethality in mice, corresponding to the time when mitochondria develop to begin oxidative phosphorylation (Nonn, Williams, Erickson, & Powis, 2003; Tanaka et al., 2002) . TRX2 is a non-selenoprotein enzyme containing cysteine in the active site and functions to maintain protein thiols in a reduced state, thereby becoming oxidized. As such, we predicted that molecular reprogramming of ROS and protein-damage response genes may play a factor in Alkbh8 def mouse lungs in response to TRX2 and TRXR1 and TRXR2
deficiency.
Next we used mRNA-seq (Fig. 2F) Oxidation-reduction potential (ORP) is a direct measure of the balance between ROS and antioxidants in a system. The more positive the ORP, the higher the oxidant activity or alternately, the lower the antioxidant activity. ORP is considered a new marker of ROS and is used clinically as a measure of oxidative stress in blood of patients with traumatic brain injury, stroke, sepsis, metabolic disorder and liver toxicity (Bar-Or, 2009; Bjugstad & Fanale, 2016; Bobe et al., 2017; Rael et al., 2007; Spanidis et al., 2015) . Isoprostanes are a family of eicosanoids produced by the random oxidation of tissue phospholipids by ROS and provide one of the most reliable measures of oxidative stress in vivo (Gross et al., 2005; Morrow, 2005) . We observed approximately 10% increase in oxidation reduction potential (ORP) (Fig. 3A) and a near 40% increase in the average 8-isoprostane levels (Fig. 3B ) in the lungs of Alkbh8 def mice.
We did not observe differences in 2',7'-dichlorofluorescin diacetate (DCFDA) in Alkbh8 def mice (Supplemental Fig. 2A-B) , but this is a less sensitive assay compared to 8isoprostane levels. Lastly, we measured GSH and GSSG in basal mouse lungs and plotted the ratio as an indicator of oxidative stress. The Alkbh8 def mice have less than half of the GSH/GSSG ratio under basal conditions (Fig. 3C) . Separate graphs for GSH, GSSG and total glutathione can be found in Supplemental Figure 3 . Together, our ORP, 8-isoprostane and GSH/GSSG findings support the idea that there is increased ROS in the lungs of Alkbh8 def mice and possibly a GSH synthesis defect.
Increased ROS can promote increased DNA strand breaks (Møller & Wallin, 1998; Sharma et al., 2016) . γ-H2AX is recruited to sites of double stranded DNA breaks by ATM and is a classic marker for the DNA damage response (DDR) (Rogakou, Pilch, Orr, Ivanova, & Bonner, 1998) . We assayed for γ-H2AX using dissociated primary lung cells processed through an Amnis Imagestream imaging flow cytometer. The Amnis
Imagestream takes single-cell brightfield and fluorescent images, which can be spatially quantitated and set to count the number of foci in a cell. We processed 10,000 dissociated primary lung cells assayed with a γ-H2AX antibody. Example brightfield and fluorescent images are shown in Supplemental Fig. 2C for low γ-H2AX spot count and 2D for high spot count. We observed an increase in γ-H2AX foci in the lungs of Alkbh8 def mice, relative to WT. The number of Alkbh8 def lung cells show a shift toward increased spot counts (Fig. 3D) , as well as increased γ-H2AX channel fluorescence in the ≥2 spot count group (Fig. 3E) , supporting our earlier finding of increased DNA damage response transcripts in Alkbh8 def mice. Ultimately our transcript, ROS and DDR data support the idea that under normal growth conditions the lungs of Alkbh8 def are experiencing some stress but have undergone molecular reprograming to adapt to the epitranscriptomic and selenoprotein deficiency. Importantly, the Alkbh8 def mice are adapting to the stress by upregulating a variety of signaling pathways.
We next asked whether the Alkbh8 def mice would be sensitive to a challenge by the environmental toxicant NA. We treated WT, Alkbh8 def , Cyp2abfgs-null, and DM (Cyp2abfgs-null/Alkbh8 def ) mice, the last two of which lack the CYPs required for NA bioactivation, to an acute 200 mg/kg dose of NA (Fig. 4) . The 200 mg/kg dose via I.P.
injection is well established in literature as the amount of NA delivered to the lung is relevant to OSHA standard for NA exposure in the workplace (10 ppm, or 15 ppm shortterm). We observed overall appearance, lung morphology and quantified cells in the bronchoalveolar lavage fluid (BALF). Alkbh8 def mice have an observable difference in appearance after NA exposure compared to WT or DM mice. They display more fluffed fur, which is a classic marker of stress in mice. Note that DM mice, despite Alkbh8 deficiency, have a smooth coat of fur, supporting previous studies that NA bioactivation is required for toxicity (Buckpitt & Warren, 1983; Warren, Brown, & Buckpitt, 1982) . mice. However, the Alkbh8 def mice exhibit necrosis of epithelial cells in many more bronchioles. We next used bronchoalveolar lavage to measure the number of cells in the recovered fluid, so as to gauge inflammation in WT, Alkbh8 def and DM mice (Fig. 4D) .
Alkbh8 def mice have a significantly increased number of cells in the bronchoalveolar lavage fluid (BALF) compared to WT mice, under both corn oil and NA treated conditions (P < 0.01, P < 0.05, respectively).
Next we investigated the redox and transcriptional states of WT, Alkbh8 def and DM mice 24 hours after NA. ORP measures highlight a NA-induced increase in oxidants, or a deficiency in antioxidants, in Alkbh8 def mice (P = 0.058), while a reduction is observed in WT mice (P ≤ 0.05) (Fig. 5A) . We note that the increase in ORP observed in Fig. 3A in untreated Alkbh8 def mice is no longer seen in the corn oil condition. Although relative values are presented, we attribute this to a slight increase in WT ORP after corn oil injection while Alkbh8 def ORP remained the same (raw data not shown). This may suggest some metabolic differences between WT and Alkbh8 def mice. While ORP is a general indicator of oxidative stress, a more specific in vivo measure is 8-isoprostane levels. We observed an increase in 8-isoprostane levels in both WT and Alkbh8 def mice treated with NA, relative to the untreated sample (Fig. 5B) . WT NA-treated mice show a statistically significant increase (P ≤ 0.05) in 8-isoprostane levels relative to the corn-oil treated WT group, while NA-treated Alkbh8 def mice show a more significant increase (P ≤ 0.001) relative to corn oil-treated mice. Although Alkbh8 def mice show an increase in 8isoprostanes in the lung, the difference relative to the WT NA-challenged mice is not statistically significant (P = 0.23). DM mice do not have notable differences in 8isoprostane levels in the lung between corn oil and NA treatment conditions. We again note a small but statistically significant difference in the corn oil condition between WT and Alkbh8 def , supporting the idea of metabolic differences between the mice. We further measured the GSH and GSSG in CO and 200mg/kg NA-treated mouse lungs after 24 hour exposure. We observe a decreased GSH/GSSG ratio in Alkbh8 def mice under both conditions (Fig. 5C) , again supporting the idea of increased oxidative stress in the writer-deficient mice.
We also measured protein levels to determine if specific selenoproteins are upregulated in response to NA and whether this was corrupted in the Alkbh8 def lungs. WT mice upregulate the selenoproteins GPX1, TRXR1 and TRXR2, and non-selenoprotein TRX2 in response to NA stress (Fig. 5D-H) . In contrast we observed a consistent failure to upregulate selenoprotein levels in Alkbh8 def mice (Fig. 5D -H, with full blots available in Supplemental Figure 5 ). While we note decreased TRXR1 and 2 in Alkbh8 def lungs under basal conditions (Fig. 2E) , after corn oil treatment the levels are similar to WT corn oil treated lungs again suggesting that there may be some metabolic effect or some level of oxidants present in the oil causing the Alkbh8 def to upregulate TRXR proteins from baseline. Together the data support a model in which there are increased ROS levels and decreased ROS detoxifiers in the Alkbh8 def lungs.
We also used targeted mRNA sequencing of mouse lungs to determine if the transcriptional response to NA was altered in the Alkbh8 def mice, relative to WT. We observed distinct groups of transcripts that were up-regulated in WT but down-regulated in Alkbh8 def , and vice versa (Fig. 5I & Table 1 ). Inflammatory markers were greatly increased in the lungs of Alkbh8 def mice, relative to WT, but other signaling pathways were also affected. DNA damage response marker Exonuclease 1 (Exo1) was increased in Alkbh8 def mice relative to WT. Heat shock transcripts including Hspa1α, Hspa1b, and Hsph1 were upregulated in the Alkbh8 def mice, relative to WT, which suggests that there may be some protein damage occurring. Induction of pulmonary heat shock proteins after NA exposure has previously been demonstrated and is predicted to be a response to NA-protein adduction (Williams, Cruikshank, & Plopper, 2003) .
Surprisingly, little difference in transcript levels for oxidative stress signaling pathway was observed when comparing the WT and the Alkbh8 def mice at the acute dose. We predict that this is due to several reasons. First, Alkbh8 def mice function under increased oxidative stress at basal conditions (Fig. 3A-C) , which likely pre-conditions them for oxidative stress at acute doses due to molecular reprograming of oxidative stress response systems (Endres et al., 2015) . Second, acute exposure to NA results in damage to the pulmonary club cells in the airway epithelium comprising only a small fraction of all lung cells. Due to the large cellular heterogeneity of the lung material used for RNAseq, this may lead to limited sensitivity of the assay to detect changes which are only occurring in club cells. As such, we note a much greater increase in inflammatory markers in Alkbh8 def mice relative to WT mice in response to NA. As inflammatory cells, such as cytokine and chemokine-secreting macrophages, are recruited and distributed throughout the lung including airways and alveolar regions, RNAseq was able to detect the increases in inflammatory transcripts, which comprise by far the largest group of upregulated transcripts in response to NA treatment. For example, Il6 had a 23.8 fold upregulation relative to WT mouse lungs. IL6 is produced at sites of tissue inflammation and is important in the response to acute lung injury (Gadient & Patterson, 1999; Voiriot et al., 2017 Fig 4) . These data support the idea that there is clear evidence of increased stress, inflammation and lung injury in the lungs of Alkbh8 def mice 24 hours after NA.
We next used sub-chronic NA exposure conditions to determine whether there were differences in survival between WT and Alkbh8 def mice. Mice were exposed to NA daily for two weeks to test their ability to survive and the resulting Kaplan-Meier plot (Fig. 6A) provides evidence that Alkbh8 def mice have increased sensitivity to NA, relative to WT.
We observed that over 50% of the WT mice survive and start gaining weight around day 3-4 with no deaths observed after day 5 (Fig. 6A-B) . In contrast, all Alkbh8 def mice die by day 7 and do not show marked recovery in weight. As the turnaround time for WT mice was around day 3, we sent lung tissues after 3-days of exposure to a boardcertified pathologist for analysis. While WT mice had the early classic response of flattened and elongated club cells and some necrosis, they begin to recover the cuboidal structure of their airway epithelial cells around day 3, a sign of tolerance. Meanwhile, Alkbh8 def lungs remain severely damaged with complete absence of epithelial cells in many parts of the bronchioles and injury and necrosis extending to ciliated cells to a moderate degree (Supplemental Fig. 6 ). The Alkbh8 def mice also present blood clot formation in the alveoli suggesting either a greater incidence of hemolytic anemia, which NA is known to cause and which can contribute to intravascular coagulation, or injury to lung cells outside of the airways, such as endothelial cells in alveolar capillaries (ATSDR, 2005; Cappellini, 2007) .
Based on these data WT mice appear to be developing tolerance to NA by day 3, and we performed dedicated tolerance study to investigate. As a control (non-tolerant) experiment we treated mice with 5 daily doses of corn oil, followed by a NA dose of 200 mg/kg. Both WT and Alkbh8 def mice lost significant amounts of weight and show lung airway damage 24 hours after NA treatment (Fig. 6C&E) , with WT and Alkbh8 def lung airways showing a similar response to a single 24-hour NA treatment, as expected (Fig.   4C ). Cyp2abfgs-null mice show no loss in weight after the NA dose, and show a small dip in weight early on after corn oil which we attribute to handling or injection stress.
Next, we tolerated the mice with 5 daily doses of 50 mg/kg NA, followed by a high dose of 200 mg/kg on day 6 (Fig. 6D) . As opposed to non-tolerated mice (5 corn oil treatments followed by a high dose of NA), NA tolerated WT mice did not lose significant weight. In contrast Alkbh8 def mice lost a similar amount of weight as in the non-tolerant study. H&E stained lung sections from the tolerated mice provide evidence that the Alkbh8 def mice have a marked increase in lung airway damage compared to WT and Cyp2abfgs-null mice, with continued necrosis and exfoliation of epithelial cells to the airway lumen in most bronchioles compared to WT mice (Fig. 6F ).
We have previously shown that under normal growth conditions mouse embryonic fibroblasts deficient in the epitranscriptomic writer ALKBH8 have increased ROS and DNA damage but adapt to these stresses by molecular reprogramming stress-response transcript levels (Endres et al., 2015) . Our current study supports the idea that this reprogramming can be extended to the lungs of Alkbh8 def mice, as they have adapted to the epitranscriptomic deficiency and corresponding decrease in selenoprotein levels by upregulating the expression of inflammation, DNA repair and protein stress-response transcripts and markers. Molecular reprogramming of stress response systems in response to an epitranscriptomic writer deficiency has also been reported in lower organisms. For example, deficiencies in wobble uridine modification systems in budding yeast have been linked to upregulation of heat shock and unfolded protein response pathways (Patil et al., 2012) . Molecular reprogramming due to cellular stress, including oxidative stress, protein stress and DNA damage, is also a well-documented response and has implications in development, aging and disease susceptibility. For example, the nuclear factor erythroid 2-related factor 2 (NRF2) is a master regulator of cellular resistance to oxidative stress(Qiang Ma, 2013; Vomund, Schäfer, Parnham, Brüne, & Von Knethen, 2017) . In response to oxidative stress, NRF2 dissociates from its negative regulator KEAP1, translocates to the nucleus, and binds to specific antioxidant response elements (AREs) in the promoter region of antioxidant and cytoprotective protein genes, including enzymes that catalyze glutathione synthesis (Qiang Ma, 2013; Nguyen, Nioi, & Pickett, 2009 Ma & He, 2012; Motohashi & Yamamoto, 2004; Rangasamy et al., 2004) , while boosting NRF2 activity has a protective effect against oxidative damage (Talalay, Dinkova-Kostova, & Holtzclaw, 2003) . Nevertheless, mice lacking NRF2 have been shown to compensate for the deficiency by upregulating endothelial nitric oxide synthase (eNOS), which mediates cardioprotection against myocardia ischemia in conditions of decreased antioxidant capacity (Erkens et al., 2018) . eNOS synthesizes nitric oxide, which has potent antioxidant effects (Erkens et al., 2018; Wink et al., 2001) . Despite severely decreased glutathione levels and general antioxidant capacity, NRF2 -/mice were able to preserve their redox status, vascular function, and resist myocardial ischemia/perfusion damage (Erkens et al., 2018) . Nrf2 deficiency has also been shown to play a protective role against cardiomyopathy with molecular reprogramming of antioxidants at the transcript and protein levels playing a contributing role, with a noted increase in GPX1 transcript in NRF2 deficient mice with cardiomyopathy (Kannan et al., 2013) . NRF2 and selenoproteins have been shown to work in conjunction to protect against oxidative damage (Kawatani, Suzuki, Shimizu, Kelly, & Yamamoto, 2011) .
There are 25 Sec-containing proteins in humans, and 24 in rodents, with many serving as antioxidants in various thiol-dependent reactions (Hawkes & Alkan, 2010; Reeves & Hoffmann, 2009) . Mice deficient in selenoprotein TRXR1, Selenoprotein R (SELR), and Selenoprotein P (SELP) have growth delays and display markers of oxidative stress and metabolic deficiencies (Bondareva et al., 2007; Fomenko et al., 2009; Schomburg et al., 2003) . There are strong links between selenoprotein defects and inflammation.
For example, selenoprotein S is an important regulator of the inflammatory response (Curran et al., 2005; Gao et al., 2006) , with polymorphisms in the promoter region of SelS linked to decreased SELS expression and increased IL-6, IL-1β, and TNF-α levels (Curran et al., 2005) . A deficiency or mutation in SELS is also associated with inflammation-related disorders and has been linked to gastric cancer (Mao, Cui, & Wang, 2015) and inflammatory bowel disease (Seiderer et al., 2007) . In our study, we found a significant difference in SELS protein levels between WT and Alkbh8 def NA treated mice, which may also explain the dramatic increase in IL-6 and other inflammatory markers at the transcript level in the Alkbh8 def lungs. The dramatic increase in inflammation markers in writer deficient lungs can be also linked to increased ROS and decreased antioxidant enzymes in the form of specific seleno-and other proteins. In support of this idea, we observed deficiencies in selenoproteins TRXR1 and TRXR2 and non-selenoprotein TRX2 in Alkbh8 def mice and associated increase in oxidative stress markers ORP and 8-isoprostanes. TRXs primarily reduce oxidized protein cysteine residues to form reduced disulfide bonds, with TRX2 localized to the mitochondria. TRXs are maintained in their reduced and active state by TRXRs using NADPH as a reducing equivalent, with TRXR1 located in the cytoplasm and TRXR2 in the mitochondria. This system is critical for signaling pathways involved in the protection from oxidants (Saccoccia et al., 2014) . TRXRs are also required for development as complete knockout of TRXR1 and TRXR2 in mice result in severe growth abnormality and embryonic death at day 8.5 and 13, respectively (Bondareva et al., 2007; Conrad et al., 2004; Jakupoglu et al., 2005) .
GPX family members catalyze the reduction of harmful peroxides by glutathione and play an essential role in protecting cells against oxidative damage. GPX1 is the most abundant selenoprotein in mice, and its importance in NA stress response is demonstrated by WT mice significantly upregulating GPX1 protein levels after NA exposure, whereas Alkbh8 def mice fail to do so. Alkbh8 def mice have decreased GPX1
and GPX3 levels compared to their carrier-treated counterparts. Previous studies have
shown that glutathione (GSH) protects cells against NA metabolites (i.e. 1,2naphthoquinone). GSH is a key detoxification pathway and its depletion is a major determinant of NA respiratory toxicity (Phimister et al., 2004; Plopper et al., 2013b) .
Many of the selenoproteins linked to ALKBH8 use GSH, including GPXs 1-6 in humans, and 1-5 in rodents, to transform H 2 O 2 to water and lipid peroxides to alcohols. While the writer deficient lungs reprogram and upregulate other stress response systems under normal growth conditions, they are ill equipped to handle environmental challenge. These observations support the idea that dysregulated epitranscriptomic systems sensitizes lungs to NA-induced injury.
In general, cells and tissues respond to environmental stress by regulating stress Thus, inhaled environmental toxicants often have an effect that exceeds that of the lung.
We have observed this phenomime in our Alkbh8 def mice, as weigh loss is increased and animal survival decreased after NA-exposure, which is likely due to systemic issues.
Our study demonstrates that the epitranscriptomic writer ALKBH8 is required to mitigate the effects of NA. It represents one of the first examples of how a mammalian RNA modification system is vital after environmental challenge. As there are over twenty epitranscriptomic writers in mammals (Kadumuri & Janga, 2018) , there are most likely other RNA modifications, writers and perhaps erasers essential to the response to common environmental toxicants.
The highest occupational exposure to naphthalene occurs in industries dealing with wood treatment, coal tar production, mothball production and jet fuel industries. NA bioactivation by CYP enzymes is a well-documented requirement of NA toxicity (ATSDR, 2005; L. Li et al., 2011) . Bioactivation and now regulators of selenoproteins, in this case ALKBH8-dependent epitranscriptomic control, should also be considered modifiers of NA exposure effects. Age-associated decline in GSH synthesis and activity has been reported in mice and rats (Chen, Richie, & Lang, 1989; Kim et al., 2003; Liu, 2002) .
Deceased ALKBH8 has been identified in aged fibroblasts, relative to young, with deficiencies also linked to early senescence (Lee et al., 2019) . Due to age induced changes in transcription and epitranscriptomic activity, NA sensitivity may be amplified in older people, but that is speculation that needs to be further studied. In support though, age related decreases have been established for drug and xenobiotic metabolism capacity in aging mice (Kwak, Kim, Oh, & Kim, 2015) and humans (Sotaniemi, Arranto, Pelkonen, & Pasanen, 1997) .
Tolerance is defined as resistance to a high challenging dose after repeated exposures.
Upregulation of glutathione synthesis has been reported as a tolerance mechanism in lung airway epithelial club cells, (Plopper et al., 2013b; Van Winkle et al., 1995; West et al., 2003) . Glutathione depletion is a hallmark of NA toxicity (Phimister et al., 2004) . γ-GCS is the rate-limiting enzyme in GSH synthesis, and treatment of mice with buthionine sulfoximine, a γ-GCS inhibitor, eliminates the tolerant phenotype in a majority of mice post-NA exposure (West et al., 2003 (West et al., , 2002 . Selenoprotein H (SELH) has been shown to be an important transcription factor promoting expression of genes involved in GSH synthesis, including γ-GCS (Panee et al., 2007) . Decreased SELH may explain our finding that ALKBH8-deficient mice fail to develop tolerance to NA, but we were unable to identify suitable antibodies to measure it. Our study highlights the importance of ALKBH8 in promoting tolerance, most likely via translational regulation of selenoproteins and GSH levels. Dysregulated transcriptional response due to the molecular reprogramming that occurs in response to the epitranscriptomic deficiency may also prevent tolerance in the Alkbh8 def mice. It is notable though that tolerance to a high dose chemical challenge has been demonstrated for a homolog of ALKBH8, namely E.
coli AlkB (Samson & Cairns, 1977) . The adaptive response to alkylating agents was characterized under conditions that mimic tolerance, as E.coli cells pretreated with a low dose of alkylating agent were able to survive a high dose challenge, relative to naive cells. The adaptive response includes up-regulation of members of the Ada operon, the DNA glycosylase AlkA and the demethylase AlkB. A major finding of our study is the determination that the epitranscriptomic writer ALKBH8 is a key driver of tolerance, which highlights a novel role for tRNA modifications and translational regulatory mechanisms in environmental responses.
Our study supports a model (Fig. 7) A) The ALKBH8 protein contains RNA recognition motif (RRM), 2-oxogluterate and Fe 2+dependent oxygenase (2OG-Fe(II)), and methyltransferase (MT) domains. Included is the insertion area of the β-galactosidase/neomycin resistance cassette (Stryke et al., 2003) . B) Formation of mcm 5 U and mcm 5 Um. Uridine (U) is first carboxymethylated to 5-carbonyl-methyluridine (cm 5 U) by elongator (ELP) proteins. Next, ALKBH8 catalyzes the formation mcm 5 U using S-adenosyl methionine (SAM) as a methyl donor. ALKBH8 is also required for the formation of mcm 5 Um. C) Selenoprotein synthesis requires a specialized mechanism of translation. In addition to a modified tRNA Sec , elongation factors, SECIS Binding Protein 2 (SPB2), and 3' UTR Selenocysteine Insertion Sequence (SECIS) elements are required for translation of the UGA stop codon found in transcripts corresponding to selenocysteine-containing proteins. transcript levels from basal WT and Alkbh8 def lungs were quantified using qPCR analysis (N = 3 per genotype, P ≤ 0.0001). E) GPX1, GPX3, SELS, TRXR1 and TRXR2 selenoprotein expression levels and non-selenoprotein TRX2 (blot only) from basal WT and Alkbh8 def lungs were assed using Wes® Simple Western. F) Targeted RNA sequencing was performed on 350 stress response genes and fold differences in number of sequence reads after normalization to endogenous control (Ribosomal protein L13a, Rpl13a) was used to generate a heat map. Expression values are Alkbh8 def lungs relative to WT under basal conditions. Notable transcripts with fold-change values of 1.5 or greater in each signaling pathway are listed. N = 2 mice per genotype. 
Alkbh8 def lungs and we observed a significant difference (P ≤ 0.05, N = 3). B) 8isoprostane levels (pg/gram lung tissue) were increased in Alkbh8 def lungs (P = 0.0557, N = 3). C) GSH and GSSG were measured using LC-MS/MS and plotted as a ratio, with Alkbh8 def mice showing a decrease (N = 3, P ≤ 0.01) D) Basal WT and Alkbh8 def lungs were excised and enzymatically and mechanically separated into a single-cell suspension, fixed and stained with a FITC conjugated antibody specific for γ-H2AX and the DRAQ5 nuclear stain, followed by quantitative imaging with an Amnis Imagestream ISX100 flow cytometer. γ-H2AX foci were scored for intensity and number using the IDEAS Spot Count analysis program, presented as the frequency of cells with ≤1 spots or ≥2 spots (N = 3 mice per genotype, 10,000 cells per lung). E) γ-H2AX-FITC channel fluorescence intensity was quantified and normalized to WT to demonstrate there are increased levels in Alkbh8 def lungs (P = 0.098). Mice were tested for their ability to tolerate a prolonged exposure to NA. A) Kaplan-Meier plot showing survival over 14 days after daily injection with 200 mg/kg NA. The log-rank (Mentel-Cox) test was used to generate a P-value. (P < 0.01). B) Weight was recorded daily over the 14-day study and plotted relative to starting weight. Any mice with a 20% or greater weight loss relative to starting weight were euthanized. C) Mice were tested for their ability to build tolerance to NA. The non adapted mice were treated with 5 doses of the carrier control, corn oil, followed by a 200 mg/kg high dose of NA.
Average weight was plotted relative to starting weight. D) Mice were adapted to NA to build tolerance by subjecting them to 5 days of low-dose NA (50 mg/kg) followed by a 6 th day of high dose (200 mg/kg). Average weight was plotted relative to starting weight. 