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Alcohol use disorder (AUD) is a clinical problem of great significance that cost the United States $249 billion in 2010, with of the cost related to binge drinking or a pattern of drinking that brings blood alcohol concentration ≥ 80 mg/dL (or 0.8 mg/mL; NIAAA, 2004). Excessive alcohol use is the fourth leading preventable cause of death in the United States, but globally, it accounts for 5.9% of all deaths (∼3.3 million in 2012) and is the first leading risk factor for premature death and disability among people between the ages of 15 and 49 (NIAAA, 2017). Epidemiological evidence indicates that women develop alcohol-related heart disease, liver damage, and peripheral neuropathy after fewer years of heavy drinking, and that women may be more vulnerable to AUD-induced brain damage (Wiren, 2013 and references therein).

Behavioral, biochemical, and molecular pharmacological evidence indicates that N-methyl-D-aspartate (NMDA) receptors are one of the primary targets of ethanol. Other primary targets include γ-aminobutyric acid_A (GABA_A), glycine, serotonin-3, and neuronal nicotinic acetylcholine receptors, as well as L-type calcium channels and G protein-activated inwardly rectifying potassium channels (reviewed in Spanagel, 2009; Cui and Koob, 2017). Concentrations as low as 1 mM produce alterations in the function of these receptors and ion channels, which initiate a cascade of intracellular events and lead to the acute behavioral effects of ethanol that range from disinhibition to sedation and hypnosis (depending on the dose). Given that practically all neurons in the brain are estimated to possess glutamatergic inputs, glutamatergic neurotransmission is in a position to regulate or influence a diverse array of neuronal processes (see Chandler, 2003; Lau and Zukin, 2007; Bell et al., 2016). A large body of evidence also implicates activity-dependent changes in the efficacy of glutamatergic neurotransmission as a major underlying event in the addicted brain (e.g., reviewed in Chandler, 2003; Tzschentke and Schmidt, 2003; Kauer and Malenka, 2007; Szumlinski et al., 2008a; Kalivas, 2009; Bell et al., 2016). Importantly, we recently found that repeated binge drinking recruited sexually divergent signaling cascades downstream of phosphoinositide 3-kinase (PI3K) in the nucleus accumbens (NAc) in C57BL/6J mice, with significant changes in males and females relatively resistant to these changes (Cozzoli et al., 2016). The functional implication of the changes was confirmed by the demonstration that intra-NAc rapamycin, which inhibits mammalian target of rapamycin (mTOR) in the PI3K signaling cascade, significantly decreased binge ethanol drinking in male but not in female mice (Cozzoli et al., 2016). Taken in conjunction with evidence that rapamycin (see Neasta et al., 2014 and references therein) and newly developed mTOR complex 1 inhibitors (Morisot et al., 2018) significantly reduce high ethanol drinking in male rodents, the results by Cozzoli et al. (2016) highlight sex differences in the influence of binge drinking on signaling cascades downstream of PI3K and presumably, metabotropic Group 1 glutamate receptors (mGluR1).

Neuroadaptive responses to binge ethanol consumption are not limited to effects on neurotransmitter systems. Microarray expression analysis in whole brain or in brain regions such as the medial prefrontal cortex (mPFC), NAc, ventral tegmental area (VTA), and amygdala have found that various models of binge drinking produced changes in expression of genes in male rodents that were involved in some of the following networks: glutamate signaling, BDNF (brain derived neurotrophic factor), synaptic vesicle fusion, synaptic transmission, apoptosis, glucocorticoid receptor (GR) signaling, anti-apoptosis, regulation of G-protein receptor signaling, transcription factors, neurogenesis, and neuroimmune-related pathways (e.g., Rodd et al., 2008; Bell et al., 2009; McBride et al., 2010; Mulligan et al., 2011; Wolstenholme et al., 2011; Agrawal et al., 2014). Binge drinking in female rodents produced changes in VTA gene expression that were associated with neuroimmune and epigenetic functions, a pro-inflammatory response, and an enhanced response to glucocorticoids and steroid hormones (McBride et al., 2013; Marballi et al., 2016), and the two top networks identified were neurological/psychological disorders and lipid/nucleic acid metabolism (Marballi et al., 2016). Changes in NAc and amygdala protein expression in female rats following binge drinking were associated with functional categories such as the cytoskeleton, cellular stress response, membrane transport, and neurotransmission (Bell et al., 2006). Although male and female rodents were never directly compared in the same study, binge-like ethanol drinking changed the expression of genes and proteins that likely alter neuronal function in several ways and that can be either adaptive or deleterious.

Chronic ethanol intoxication that results in physical dependence via continuous or intermittent ethanol vapor exposure also produces gene expression changes, with a different transcriptional response in the cortex during acute withdrawal (8 h) than after a period of abstinence (3 weeks) in male and female rodents. Studies conducted in male mice during acute withdrawal found that transcriptionally responsive genes in the PFC were involved in the Ras/MAPK (mitogen-activated protein kinase) pathway, notch signaling, and ubiquitination (Melendez et al., 2012) and that dysregulation in the expression of several chromatin remodeling genes in PFC was primarily evident during acute withdrawal rather than during a period of abstinence (Hashimoto et al., 2017). Pathways identified in cingulate cortex of male rats after a period of abstinence were involved in neurotransmission (e.g., glutamatergic, endocannabinoid, monoaminergic), signal transduction (e.g., MAPK, ERK2 or extracellular signal-related kinase 2), and synaptic plasticity (Rimondini et al., 2002). Additionally, a study designed to discover master regulator genes (i.e., key genes that drive the expression of the specific transcriptional response associated with physical dependence) during abstinence in male rats identified Nr3c1, the gene encoding the GR, as one of the highest master regulators in the mPFC, NAc, VTA, and central nucleus of the amygdala (Repunte-Canonigo et al., 2015). Importantly, several studies were conducted in male and female mPFC with the goal of examining sex and ethanol withdrawal severity genotype differences in gene expression profiles in mice selectively bred for high and low withdrawal. During acute withdrawal, sex rather than withdrawal genotype, correlated best with the transcriptional response in dependent mice. Females showed regulation of genes associated with cell death/neurodegeneration, DNA/RNA binding, and inflammation/immune function whereas males showed regulation of genes associated with protein degradation, calcium ion binding pathways, inflammation/immune function, and nervous system disorders/development (Hashimoto and Wiren, 2008; Wilhelm et al., 2014). However, while NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling was identified as a significant signaling node in both males and females, the interacting proteins were completely distinct between the sexes, which was indicative of a sexually dimorphic immune response during acute withdrawal. Subsequent studies during acute withdrawal focused on glucocorticoid signaling, and bioinformatics of genes regulated in dependent mice identified activation of inflammatory signaling and cell death pathways in females, while males exhibited disease and disorder pathways that were associated with endocrine and neurological diseases (Wilhelm et al., 2015). In contrast, abstinence produced a transcriptional response that varied by withdrawal genotype rather than sex. In the low withdrawal genotype, genes associated with the biological processes thyroid hormone metabolism, glutathione metabolism, axonal guidance, and DNA damage response were identified. Classes of genes associated with acetylation and histone deacetylase were highly dimorphic between mice with a high versus (vs.) low withdrawal genotype. The top pathway identified was Death Receptor Signaling, with apoptosis as a central node, but both sexes of the withdrawal resistant genotype had increased apoptotic signaling and more up-regulated transcripts whereas the high withdrawal genotype mice had less apoptotic signaling (Hashimoto et al., 2011; Wilhelm et al., 2014). Collectively, the available data indicate that acute withdrawal following chronic intoxication or binge drinking produces sexually divergent transcriptional responses and activation of distinct networks.

Based on the above evidence for a strong dichotomy between male and female rodents in the response to ethanol during acute withdrawal, the purpose of the present study was to characterize RNA expression differences from male and female C57BL/6J mice following 7 binge ethanol sessions. Tissue was harvested from the NAc, as this brain region is a central mediator of addiction (e.g., Tzschentke and Schmidt, 2003; Kauer and Malenka, 2007; Kalivas, 2009; Koob and Volkow, 2010). Focused quantitative PCR (qPCR) array analysis was employed to quantify expression levels of 384 genes identified as important in “Mood Disorders.” The results indicated that there was a largely divergent regulation of genes by binge drinking in males and females, reflecting different neuroadaptive responses in the NAc that would result in dysregulation of distinct biological pathways between the sexes.

The present results add to a body of evidence indicating that binge drinking and chronic ethanol intoxication leading to the development of physical dependence both produce neuroadaptive changes in neurotransmitter systems as well as numerous other cellular pathways that can alter neuronal function in a manner that can be either adaptive or deleterious (see section “Introduction”). Importantly, because we directly tested males and females following repeated binge drinking, the present results show for the first time that repeated binge drinking experience produces sexually divergent transcriptional responses and activation of distinct networks, similar to what has been reported for males and females tested during acute withdrawal following chronic intoxication (Hashimoto and Wiren, 2008; Wilhelm et al., 2014, 2015). Of the 106 genes significantly affected by binge drinking in the present study, only 4 were regulated similarly in males and females, demonstrating a profound sex difference in neuroadaptive responses in the NAc that would result in dysregulation of distinct biological pathways between the sexes. For instance, IPA identified Psychological Disorders and Neurological Disease as one of the top two networks, based on the expression differences following repeated binge drinking in male and female mice. However, the relationships between genes identified distinct molecules as significant signaling nodes, suggestive of a sexually dimorphic response that also may be related to mood disorders.

It was not surprising that neurotransmission was significantly affected by binge drinking. The current results are consistent with prior microarray studies that identified networks or biological processes related to glutamate signaling, BDNF and synaptic transmission in the NAc and central nucleus of the amygdala (Rodd et al., 2008; McBride et al., 2010) or in the PFC (Wolstenholme et al., 2011) from male rodents following binge drinking and networks related to neurotransmission in the NAc and amygdala from female rats following binge drinking (Bell et al., 2006). Neurotransmission also was one pathway identified in the cingulate cortex of dependent male rats after a period of abstinence, which included the glutamatergic and monoaminergic systems (Rimondini et al., 2002). Based on the results in dependent male rats during abstinence, in conjunction with the binge drinking-related changes in expression of glutamatergic and dopaminergic genes in male mice in the present study, it is possible that repeated binge drinking experience produces neuroadaptive changes in glutamatergic and dopaminergic signaling that continue through the development of dependence and a period of abstinence, at least in male rodents. Consistent with this idea, 3 months of chronic ethanol intake produced a significant increase in NAc Homer2 protein levels that persisted at 2 months of abstinence in male C57BL/6J mice, and Homer2 overexpression in the NAc facilitated the effect of single or repeated ethanol injections on extracellular glutamate and dopamine levels in the NAc (Szumlinski et al., 2008b). Likewise, NAc Homer2 and mGluR5 protein levels were significantly elevated at 1 month of abstinence after 6 months of chronic ethanol drinking (Obara et al., 2009). Collectively, a large body of evidence indicates that changes in glutamate receptors, transporters, enzymes, and scaffolding proteins are critical for the development of dependence and addiction (see reviews by Szumlinski et al., 2008a; Kalivas, 2009; Bell et al., 2016).

Earlier work with the Scheduled High Alcohol Consumption model of binge drinking found that repeated bouts of binge drinking increased NAc protein levels of Homer2, NMDA receptor 2A and 2B subunits, and PI3K activation in male C57BL/6J mice, without altering protein levels of mGluR1 and mGluR5 at 24 h after the final binge session (Cozzoli et al., 2009). Recently, we replicated the lack of effect of binge drinking on NAc protein levels of mGluR1 and mGluR5, but we also observed an ethanol-induced decrease in RNA expression of Grm1 and Grm5 in male C57BL/6J mice, and comparable changes were not found in female C57BL/6J mice (Cozzoli et al., 2016). In the present study, binge drinking significantly increased Homer2 expression and tended to decrease Grm5 expression only in male mice (Grm1 was not on the arrays), and there was a non-significant increase in Pik3r1 expression in female (↑ 1.48-fold, p = 0.12) and male (↑ 1.17-fold, p < 0.20) mice. As we discuss in Cozzoli et al. (2016), it is possible that some of the differences between studies were due to whether the experiments were conducted in the circadian dark vs. light phase. The majority of studies that observed a binge drinking-induced activation of PI3K at 24 h of abstinence were conducted during the circadian dark phase (Cozzoli et al., 2009, 2012; Neasta et al., 2010 – but see Neasta et al., 2011), whereas binge ethanol drinking occurred during the circadian light phase in the current and our recent studies (Cozzoli et al., 2016). In the studies by Cozzoli et al. (2016), 24 h of abstinence following repeated binge drinking significantly decreased activation of PI3K and mammalian target of rapamycin (mTOR) protein levels in NAc tissue from male but not female mice. It is interesting that intra-NAc administration of rapamycin to inhibit mTOR signaling significantly decreased binge drinking in male but not female mice (Cozzoli et al., 2016). This result suggested that rapamycin blocked a binge ethanol-induced activation of mTOR in males, because it was administered prior to the binge ethanol session, and that the ethanol-induced activation of mTOR (and presumably PI3K) in males was more transient in our studies that were conducted during the circadian light phase than what was reported in other studies that were conducted during the circadian dark phase. Regulation of circadian clock genes has been shown to influence ethanol and drug sensitivity, and ethanol also can disrupt circadian gene expression (reviewed in Parekh et al., 2015). In the present study, we did observe changes in expression of some circadian genes following binge drinking, with a significant decrease in Per2 in females (p < 0.05, Supplementary Table S1) and trends for an opposite effect on Clock in females and males (females: ↑ 1.5-fold, p < 0.10; males: ↓ 1.5-fold, p < 0.09). Regardless, the results add to evidence for sex differences in the effects of binge drinking on glutamatergic signaling.

It is interesting that Akt (many times associated with PI3K) was identified as a central node in female mice following binge drinking. However, as mentioned above, we recently found that females were insensitive to the ability of intra-NAc rapamycin (inhibits mTOR, in signaling cascade downstream of PI3K and Akt) to decrease binge drinking, whereas intra-NAc rapamycin significantly decreased binge drinking in males (Cozzoli et al., 2016). The reduction in binge drinking in males is consistent with prior work (Neasta et al., 2010, 2011, 2014), so the insensitivity of females suggests that an alternate signaling pathway that is independent of PI3K and that links Group 1 mGluRs to transcriptional changes in the nucleus (i.e., protein kinase A, calcium calmodulin dependent protein kinase, or MAPK; see Wang and Zhuo, 2012) is influenced by binge drinking in females. One possible mechanism would be via the ability of membrane estrogen receptors (mER) to stimulate mGluRs, as coupling of mERα to mGluRs can initiate independent signal transduction pathways (see review by Meitzen and Mermelstein, 2011). Related to this point, the present study found that binge drinking produced a fivefold upregulation in Esr1 (which encodes ERα) only in female mice (p < 0.05, Table 3), and evidence indicates that ERα also can localize to the plasma membrane and initiate signal transduction through PI3K (reviewed in Levin, 2009). So, it is not known whether the identification of Akt (and presumably the Akt-PI3K pathway) as a central node in females following binge drinking is related to signaling downstream of mERα or downstream from the coupling of mERα to mGluRs.

Characterization of the expression differences following binge drinking also identified hormone signaling in female mice, with a trend for regulation of CRH signaling in male mice. In fact, “CRH signaling” was identified as a canonical pathway of interest from the analysis of genes that were regulated by binge drinking in both males and females (Figure 3). Importantly, the effects of binge drinking on CRH signaling were divergent for all the genes listed in Figure 3D. An examination of the genes responsible for the initiation of CRH signaling indicate that binge drinking reduces activity of the pathway in females (↓ Crhr1 and Crhr2), while it increased activity of the pathway in males (↑ Crh and Crhr2). The decreased activity of the CRH pathway in females is interesting, given evidence for sex differences in the coupling of CRHR1 with the Gs and β-arrestin 2 proteins that render females more responsive to acute stress and less able to adapt to chronic stress as a result of compromised CRHR1 internalization (Valentino et al., 2013a,b). Consistent with this, we found that 1 month of continuous ethanol drinking with intermittent traumatic stress exposure upregulated protein levels of CRHR1 in the hippocampus and protein levels of GR in the hippocampus and PFC of female but not male C57BL/6J mice (Finn et al., 2018). Taken in conjunction with the present results, it is possible that binge drinking alone produces an opposite effect on CRH signaling than the combination of stress and ethanol consumption.

The transcriptional response to repeated binge drinking identified “MIF-mediated glucocorticoid regulation” in the NAc of females in the present study, despite the divergent and non-significant binge drinking-related changes in expression of the gene encoding GR (Nr3c1; females: ↓ 1.1-fold, p = 0.19; males: ↑ 1.2-fold, p < 0.19) and the gene encoding the chaperone heat shock protein 90 (Hsp90b1; females: ↑ 2-fold, p < 0.08; males: ↓ 1.3-fold, p = 0.18). Similarly, the identification of GR signaling in the NAc and central nucleus of the amygdala of male rats (McBride et al., 2010) and an enhanced response to glucocorticoids in the VTA of female rats (McBride et al., 2013) following chronic binge drinking experience (8 weeks for male rats; 10 weeks for female rats) was based on significant binge ethanol-induced regulation of the expression of genes that did not include GR. It is well documented that glucocorticoids can act via a nuclear GR to regulate many transcriptional pathways, including homeostasis, metabolism, and inflammation (reviewed in Biddie et al., 2012). But, while glucocorticoids can have anti-inflammatory and immunosuppressive properties, long term and/or high dose glucocorticoid administration can lead to symptoms of depression and decreased immunological function. Relevant to the pathway identified in females in the present study, MIF is able to directly regulate the immunosuppressive action of glucocorticoids (reviewed in Flaster et al., 2007). MIF can be produced at all levels of the hypothalamic-pituitary-adrenal axis, and plasma MIF levels fluctuate in a circadian rhythm relative to cortisol. Early studies found that MIF counteracted the glucocorticoid-induced suppression of inflammatory cytokine secretion in activated macrophages (e.g., TNF, IL-1, IL-6, IL-8) and completely blocked the protective effect of the synthetic glucocorticoid dexamethasone in a model of lethal, endotoxic shock induced by lipopolysaccharide (Calandra et al., 1995), providing evidence that the regulatory effect of MIF on glucocorticoid immunosuppression occurs in vivo. Additionally, cross-talk between GR and NF-κB occurs via a physical interaction that produces a dose-dependent and mutual antagonism effect mediated by the p65 (RelA, encoded by Rela) subunit of NF-κB (McKay and Cidlowski, 1998). Glucocorticoids also inhibit NF-κB activation, in part by increasing the expression of the IκB complex that maintains NF-κB in an inactive state until IκB dissociates from NF-κB following its phosphorylation (Flaster et al., 2007; Lang et al., 2015; simplified NF-κB and IκB interaction depicted in Figure 5A). And, one effect of MIF is to prevent glucocorticoids from increasing the expression of IκB, which would offset the glucocorticoid-mediated inhibition of NF-κB (Flaster et al., 2007; Lang et al., 2015). Since the immunosuppressive and anti-inflammatory effects of glucocorticoids are thought to depend on the inhibition of NF-κB, which is a transcription factor that plays a role in immune signaling and cell survival (Li and Verma, 2002), counteracting this effect with MIF could result in sustained inflammatory signaling in females with binge drinking experience.

Acute withdrawal following chronic intoxication affected pathways related to inflammatory activation and apoptotic/cell death signaling in PFC from females vs. males (Hashimoto and Wiren, 2008; Wilhelm et al., 2014, 2015). Acute withdrawal from repeated binge drinking also affected several pathways related to immune function only in female NAc in the present study (“MIF regulation of innate immunity,” “TNFR1 signaling,” and “TNFR2 signaling”). And, “TNFR2 signaling” was identified as a canonical pathway of interest from the analysis of genes that were regulated by binge drinking in the NAc from both males and females (Figure 5). Notably, binge drinking produced a significant and opposite change in the expression of Lta (encodes TNF-β or lymphotoxin-α), with a decrease in females and increase in males (Table 2 and Figure 5G), and this protein also is involved in cell survival, proliferation, differentiation, apoptosis, and immune regulation. So, the sex difference in significant expression change in Lta by binge drinking (↓ in females, ↑ in males, Table 2 and Figure 5G) would be predicted to have opposite effects on cell survival and immune responses. In addition, binge drinking significantly decreased expression of Il6 only in females; an ethanol-induced reduction in levels of these two cytokines in females would be consistent with a decrease in the initiation of signaling at TNFR1 (see Figure 2 in Mayfield et al., 2013) and TNFR2 (Figure 5A). Overall, the results in females demonstrate that binge drinking produced a fairly consistent decrease in expression of genes in the signaling cascade through the TNF superfamily, with the exception of a trend for an increase in expression of Tnf (encodes TNF-α). These binge ethanol-induced changes in females, including the downregulation of the RelA subunit of NF-κB, likely produce a decrease in the activation of NF-κB. A more complex pattern of changes was identified in males following binge drinking, so the influence on NF-κB activation in males is unclear. Regardless, the results in females in the present study would be consistent with a decrease in cell survival, and an increase in apoptosis and inflammation via a decrease in the activation of the NF-κB. It is interesting that acute withdrawal from chronic intoxication also identified NF-κB as a central node in both male and female networks, but the interacting gene sets were completely distinct between the sexes (Wilhelm et al., 2014). An examination of the genes regulated by chronic intoxication in these pathways revealed that several of the genes in females were indicative of a proinflammatory response, while the genes in males were suggestive of overall immunosuppression (Wilhelm et al., 2014). Collectively, binge drinking experience and chronic intoxication leading to the development of physical dependence both produced sexually divergent changes in inflammatory signaling in the NAc and PFC from mice.

The repeated binge drinking sessions produced sexually divergent transcriptional responses and activation of distinct networks. These results add to a body of evidence indicating that binge drinking and chronic ethanol intoxication both produce neuroadaptive changes in neurotransmitter systems and in many cellular pathways that likely alter neuronal function in a manner that can be either adaptive or deleterious. The opposite effects of binge drinking on immune function in the present study, with changes in females consistent with a decrease in cell survival and an increase in inflammation and apoptosis, have important implications, given the evidence for a role of neuroimmune signaling in the acute and chronic effects of ethanol, including neurodegeneration (reviewed in Mayfield et al., 2013; Crews et al., 2015). Related to this point, chronic intoxication activated inflammatory signaling and cell death pathways in female but not male mice, and confirmation studies showed that ethanol dependent females exhibited significant neuronal degeneration in cortical regions, whereas cell death in males was significantly reduced (Hashimoto and Wiren, 2008; Wilhelm et al., 2015). Finally, a broader implication of the current findings is pertinent to sex differences in the immune system and the relationship to mood disorders (reviewed in Rainville and Hodes, 2018). Taken in conjunction with sex differences in mood and anxiety disorders (e.g., Altemus et al., 2014), future studies examining potential immune or stress-related mechanisms that may contribute to stress and ethanol susceptibility and associated mood disorders will be important.

One limitation of the present investigation is that we did not conduct confirmation studies to identify protein changes, neuronal degeneration, or behavioral changes that could possibly account for the sex-specific gene expression profiles that we observed. However, we did examine the expression of additional select genes that were not present on the arrays but that were implicated in the downstream signaling cascades of the IPA-identified pathways to strengthen conclusions about the select pathways that were altered by binge drinking. Future studies will determine whether the current gene expression changes correspond to behavioral and/or physiological differences.

Importantly, an increased understanding of sexually dimorphic molecular pathways influenced by binge drinking and chronic intoxication leading to dependence may identify novel treatment options for males and females. The current study is contributing data sets that can be used to generate sex-specific bioinformatics tools, which have the potential to enormously accelerate the discovery of sex-specific changes associated with AUD. Finally, we recently reported that binge drinking produced sex differences in the regulation of PI3K signaling in the NAc and in the ability of intra-NAc rapamycin to decrease binge drinking, with females resistant to these molecular changes (Cozzoli et al., 2016). The functional implication of the report by Cozzoli et al. (2016) emphasizes that targeting a pathway that is unaffected by binge drinking in females will not be an effective pharmacotherapeutic strategy. Collectively, the fundamental sex differences identified in the present and prior work provide evidence for distinct pathways that could be targeted therapeutically for the treatment of AUD in males and females.

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

DF and KW contributed conception and study design. DF, DC, MN, and MK conducted the drinking study. DC dissected accumbens tissue. JH isolated RNA and prepared samples for qPCR array analysis. DF conducted preliminary analysis of drinking data and identified animals for the qPCR analysis. DF, MN, and MK participated in final analysis of drinking data. MH conducted follow-up qRT-PCR analysis, with assistance from JH. JH and KW performed Pathway Analysis. JH, KW, and MG assisted in the interpretation of the array and pathway analyses. DF wrote the first draft of the manuscript, but JH and KW wrote sections of the “Materials and Methods and Results.” All authors contributed to the final version of the manuscript, read, and approved the final version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.