Mitochondrial Metabolism in Astrocytes Regulates Brain Bioenergetics, Neurotransmission and Redox Balance

FAs are stored within cells as energy-rich triacylglycerides localized in lipid droplets, removing excess free FAs from the cytoplasm, which are toxic and disrupt mitochondrial membrane integrity. In addition, LDs deliver FAs into mitochondria for their consumption as an alternative energy source during periods of nutrient depletion. Neurons do not typically make LDs, while astrocytes do (Schonfeld and Reiser, 2013), and it has been shown that toxic FAs produced in hyperactive neurons are transferred to astrocytic LDs where they are consumed by via mitochondrial FAO (Ioannou et al., 2019). Importantly, under nutrient deprivation conditions, the survival of astrocytes depends on LD-fueled FAO (Cabodevilla et al., 2013).

Peroxisomes are located throughout the brain but are primarily found in astrocytes and oligodendrocytes (Troffer-Charlier et al., 1998). VLCFAs cannot be transported into the mitochondria via the carnitine shuttle and thus, are broken down first by peroxisomes. As in mitochondria, FAs must be esterified to CoA before undergoing FAO in peroxisomes (Figures 1.4, 4). Because peroxisomes lack an electron transport chain (ETC), electrons from FADH2 are transferred directly to O2 creating hydrogen peroxide (H2O2) that is scavenged by catalase (CAT). Within the peroxisome, FAs are shortened to Ac-CoA, Prop-CoA, and a wide range of LCFAs, MCFAs, and short chain FAs (SCFAs, 6 carbons or less). A variety of export systems exist within the peroxisomes, each with different affinities for different lengths of FAs, including carnitine-transferases [carnitine acetyltransferases (CRATs), carnitine octanoyltransferases (CROTs)], and thiolases [acyl-CoA thioesterases (ACOTs)] (Figure 1.4; Antonenkov and Hiltunen, 2012). Additionally, peroxisomes have been shown to possess ketogenic enzymes, implicating peroxisomes as another potential source of ketone bodies and cholesterol (Hovik et al., 1991; Antonenkov et al., 2000; Olivier et al., 2000).

Dysfunction of peroxisomal processes has been linked to demyelination, oxidative stress, inflammation, cell death, and abnormalities in neuronal migration and differentiation (Trompier et al., 2014). Defects in the ATP binding cassette subfamily D member 1 (ABCD1) transporter that translocates very LCFAs into peroxisomes (Figure 1.4), leads to the accumulation of these fats in cells and tissues. VLCFAs and branched chain FAs can interfere directly with the ETC by opening the permeability transition pore, disrupting Ca2+ balance and depolarizing the mitochondria (Hein et al., 2008; Kruska et al., 2015; Schonfeld and Reiser, 2016). LCFAs activate PPAR transcription factors that stimulate both FAO and ketogenesis as well as mitochondrial and peroxisome biogenesis. Recent reports have also demonstrated that peroxisome biogenesis requires the generation of pre-peroxisomes from mitochondrial derived vesicles (Sugiura et al., 2017).

Previous studies have demonstrated that astrocytes have the machinery to metabolize MCFAs, LCFAs and VLCFAs as evidenced not only by the higher expression of FAO-related enzymes but also by the higher content of peroxisomes (Fecher et al., 2019; Fitzner et al., 2020). For example, the MCFA octanoate (C8) that composes 13% of the normal free fatty acid pool and can cross the BBB is avidly metabolized by astrocytes (Ebert et al., 2003). In brain slices, astrocytes have been shown to contain palmitate (C16) and oleate (C18) LCFAs (Hofmann et al., 2017).

Alterations in FA metabolism in astrocytes have been reported in different disorders. A recent report demonstrated that in a Huntington’s disease mouse model, astrocytes within the striatum consume FAs as an alternative fuel, which results in increased oxidative damage (Polyzos et al., 2019). Astrocytes carrying the ApoE4 allele, the strongest genetic risk factor for the development of Alzheimer’s disease, observe profound alterations in FA metabolism and LD formation (Farmer et al., 2019). Stimulation of FAO protects against ischemic injury as well (Sayre et al., 2017). Pharmacological modulation of FAO has been suggested as a therapeutic approach against glioblastoma cells (Lin et al., 2017). Many chemical compounds are considered to be inhibitors or activators of FAO enzymes. However, the current knowledge on their specificities, potencies and metabolic impacts is limited and controversial. For example, at high concentrations, etomoxir, the inhibitor of CPT1a, has off-target effects linked to the inhibition of the ETC (Yao et al., 2018). Other FAO modulators include the CPT1a inhibitor oxfenicine and PPARs agonists/antagonists. Accordingly, the impairment in FAO observed in astrocytes derived from induced pluripotent stem cells isolated from Alzheimer’s disease patients is corrected by PPARβ/δ agonists (Konttinen et al., 2019). The specificity of the metabolic modifiers ranolazine (Ranexa) and trimetazidine, initially described as partial FAO inhibitors that target the HAD (or 3-KAT) activity of the trifunctional protein HADHA, has been recently questioned (Ma et al., 2020).

Astrocyte processes extending to synaptic clefts play an important role in regulating synaptic transmission, particularly glutamatergic signaling. After its vesicle-mediated release, the excitatory neurotransmitter glutamate is taken from the synaptic cleft by astrocytes in a Na+ dependent manner via EAAT1 (glutamate aspartate transporter, GLAST) or EAAT2 (glutamate transporter 1, GLT-1) (Figure 3.4). In the cytosol, glutamate is then converted into non-excitatory glutamine via GS, and transferred back to neurons via the Na+-coupled neutral amino acid transporters (SNATs) to be converted back into glutamate in neurons via phosphate-activated glutaminase (PAG) (Figure 3.4; Norenberg and Martinez-Hernandez, 1979; Anderson and Swanson, 2000; Schousboe et al., 2013; Leke and Schousboe, 2016). In astrocytes, despite the presence of a weak mitochondrial targeting sequence, GS is localized in the cytosol because the mitochondrial membrane potential is not negative enough to drive GS into the mitochondria (Figures 1.6, 3.2; Matthews et al., 2010). Neurons lack PC activity (Yu et al., 1983), restricting their ability to replenish TCA intermediates lost during neurotransmitter release (anaplerotic reaction). Thus, the glutamate-glutamine cycle between neurons and astrocytes, not only terminates/modulates glutamatergic signaling, preventing excitotoxicity triggered by excessive or prolonged exposure to glutamate, but it also replenishes the neurotransmitter pool in neurons (Figure 3.4; Belanger et al., 2011).

Not all the glutamate taken from the synaptic cleft by astrocytes becomes glutamine. There is evidence that a significant portion is incorporated into the TCA cycle via oxidative metabolism (Figure 2.4). Glutamate transport into the mitochondria is mediated via the aspartate/glutamate carrier antiporter (AGC or Aralar 1) or via the glutamate carrier (GC1 or GC2), a symporter for glutamate and H+. Controversy still exists about the contribution of AGC to the uptake of glutamate in astrocytic mitochondria, as it has been shown to be primarily expressed in neurons. However, while the AGC activity in astrocytes corresponds only to 7% of the total brain AGC activity, it is calculated to be twice the minimum required for glutamate production and degradation (Figure 2.6; Hertz, 2011). AGC is also central to the malate-aspartate shuttle (Figure 1.6), a system that allows the transfer of NADH electrons generated in the cytosol, from processes such as glycolysis, to the mitochondria (Amoedo et al., 2016). On the other hand, knockdown of GC1 suggests that this carrier is the main gate for net glutamate entry into mitochondria for oxidative metabolism in astrocytes (Figure 2.6; Goubert et al., 2017).

In the mitochondria, glutamate is metabolized to αKG by transamination via aspartate aminotransferase (GOT2), and to a lesser extent by alanine (ALAT) and branched chain aminotransferases (BCAT) (Figure 2.4). In addition, αKG can also be synthesized from glutamate via glutamate dehydrogenase (GDH), which is also a source of NADPH. While glutamate transamination via GOT2 generates a truncated TCA cycle due to the requirement of OAA as a co-substrate, GDH-mediated dehydrogenation can serve as an anaplerotic reaction to replenish TCA intermediates (Figures 2.1, 2.4; Sonnewald et al., 1993; Olsen and Sonnewald, 2015; McKenna et al., 2016). Astrocytes deficient in GDH increase their reliance on GOT2 and their glycolytic input to PC, which is paralleled by an accumulation of intracellular glutamate, underlying the importance of GDH in anaplerosis (Nissen et al., 2015; Pajecka et al., 2015). It should be noted that humans, unlike most other animals including mice and rats, have an additional GDH2 isoform that is expressed solely in astrocytes. Expression of GDH2 in rat cortical astrocytes increased glutamate uptake and metabolism, reducing their dependence on glucose and increasing their capacity to utilize branched chain amino acids (BCAA) (Nissen et al., 2017). Importantly, glutamate to glutamine metabolism is not determined by the extracellular glutamate concentration. However, the entry of glutamate into the TCA cycle seems to be triggered by high extracellular glutamate concentrations (high μM) (McKenna et al., 1996).

The homeostasis of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) is similarly linked to the glutamate-glutamine cycle. GABA is synthesized in neurons from the decarboxylation of glutamate by glutamate decarboxylase (GAD). After its vesicular release, GABA is uptaken by astrocytes through the GABA transporter (GAT3) (Figure 3.4). GABA is then metabolized by GABA transaminase (GABA-T) with αKG as cofactor to produce succinate semialdehyde (SSA) and glutamate (Figure 2.4). SSA is metabolized to succinate via SSA-dehydrogenase (SSADH), which can then be incorporated back into the TCA cycle. This anaplerotic addition of succinate compensates the cataplerotic loss of carbons from the generation of glutamate by the GABA-T (Figure 2.1; Schousboe et al., 2013).

In addition to metabolism, evidence indicates that astrocyte mitochondrial bioenergetics are important for the regulation of neurotransmitter balance. Both glutamate and GABA uptake systems in astrocytes are driven by Na+ gradients that are established by the electrogenic action of the Na+/K+ ATPase (Figure 3.4). PC and GS activities also require ATP as well, and are critical enzymes in replenishing neuronal glutamine/glutamate (FIgures 2.1, 3.4). Importantly, it has been demonstrated that mitochondria co-localize with EAAT1/2 and glycolytic enzymes on the plasma membrane of astrocytes (Genda et al., 2011; Bauer et al., 2012), and that glutamate transporters physically interact with the Na+/K+ ATPase (Figure 3.6; Rose et al., 2009), suggesting that while mitochondrial-derived ATP is not required for astrocyte survival (Supplie et al., 2017), functional mitochondrial metabolism is important for neurotransmitter uptake and homeostasis (Figures 3.2, 3.4, 3.6). In fact, astrocyte processes enveloping synaptic terminals contain abundant mitochondria (Jackson et al., 2014; Stephen et al., 2015; Jackson and Robinson, 2018), and inhibition of mitochondrial function by fluorocitrate in astrocytes increases glutamate excitotoxicity in co-cultures with neurons (Voloboueva et al., 2007).

Astrocytes contain higher levels of endogenous antioxidants and antioxidant systems being more resistant to oxidative stress than neurons, which is explained by the activation of the antioxidant response via the nuclear factor erythroid-2-related factor 2 (Nrf2) transcription factor (Shih et al., 2003; Jimenez-Blasco et al., 2015). Astrocytes also have higher levels of NADPH and G6PD (glucose 6-phosphate dehydrogenase) glucose 6-phosphate dehydrogenase (G6PD) (Garcia-Nogales et al., 2003). In the mitochondria, superoxide anion (O2•–) is generally considered to be generated in the matrix by electron leakage from complex I (Grivennikova and Vinogradov, 2006), and in both the matrix and the intermembrane space (IMS) by electron leakage from complex III (Chen et al., 2003). O2•– generated in the matrix and the IMS has been proposed to be released to the cytosol via the mitochondrial permeability transition pore (mPTP) (Wang et al., 2008) and the VDAC (Han et al., 2003), respectively. However, at least 10 different additional sites for O2•–/H2O2 production in the mitochondria have been described. The relative and absolute contribution of specific sites in the mitochondria to the production of ROS seems to depend on the availability of the substrate being oxidized (Cardoso et al., 2012; Quinlan et al., 2013). Of importance, one ROS can lead to the generation of distinct more reactive species. As such, O2•– can be dismutated in an enzymatic (superoxide dismutases [SODs]) or non-enzymatic manner to H2O2. While H2O2 is thought to diffuse across membranes, it has also been reported that it can be transported by aquaporins (Bienert et al., 2006, 2007).

Surprisingly, astrocytes have less efficient mitochondrial respiration and increased ROS formation when compared to neurons, in part due to a lower incorporation of complex I into supercomplexes in astrocytes caused by lower expression of the NADH-ubiquinone oxidoreductase core subunit S1 (NDUFS1) (Lopez-Fabuel et al., 2016).

The physiological role of increased mitochondrial ROS production in astrocytes had not been explored before, but an elegant recent investigation using mitochondria-targeted antioxidants demonstrated that high ROS production in astrocytes regulates GSH metabolism via Nrf2, and conversely, represses glucose metabolism via the PPP to decrease NADPH-dependent ROS production by NADPH-dependent oxidases (Nox1 and 2), which together modulate the redox status and survival of neurons (Vicente-Gutierrez et al., 2019). Interestingly, glutamate (N-methyl-d-aspartate) receptors (NMDA-R) couple neurotransmission to Nrf2 activation, where NMDA-R signaling in astrocytes promotes Nrf2 phosphorylation by cyclin-dependent kinase 5 (Cdk5) and its translocation to the nucleus, a novel mechanisms of intercellular feedback communication from neurons to astrocytes (Jimenez-Blasco et al., 2015).

While both neurons and astrocytes can synthesize GSH, astrocytes have been reported to protect neurons via regulation of GSH metabolism (Chen et al., 2001; Shih et al., 2003). This protective effect is thought to be dependent on the supply of cysteine from astrocytes as a precursor for de novo GSH synthesis (Dringen et al., 1999; Wang and Cynader, 2000), and in part due to neurons being unable to uptake significant amounts of extracellular cystine (Sagara et al., 1993; Kranich et al., 1996). GSH has been proposed to be released from astrocytes via the ATP-binding cassette transporters subfamily C member 1 transporter (ABCC1, or multidrug-resistance-associated protein 1 [MRP1]) (Figures 1.6, 3.2; Hirrlinger et al., 2002; Hirrlinger and Dringen, 2005). Indeed, inhibition of MRP1 has been reported to reduce the extracellular accumulation of GSH in cultured astrocytes (Hirrlinger and Dringen, 2005). We have failed to observe a reciprocal accumulation of GSH in astrocytess when MRP1 activity is reduced (unpublished data), but this effect might be ascribed to feedback inhibition of GSH synthesis by GSH.

Once released, GSH is degraded by the γ-glutamyl transpeptidase (γGT) to produce l-cysteine-l-glycine (CysGly) (Dringen et al., 1997). CysGly is cleaved further by the neuronal aminopeptidase N (ApN) into the amino acids glycine and cysteine that are taken up by neurons for de novo GSH synthesis via the Na+-Cl–-dependent glycine transporter 1 (GlyT1) and EAAT3, respectively (Figures 3.2, 3.3; Dringen et al., 1999, 2001; Aoyama et al., 2008; Belanger et al., 2011). The export of GSH should imply a loss of carbons that must be replenished in astrocytes to maintain cellular redox homeostasis. Cysteine acquisition in astrocytes is mediated by the Na+-dependent XAG-/EAAT3 and -independent Xc-/xCT uptake systems, but their exact contribution is still under debate (Shanker et al., 2001; Seib et al., 2011). De novo synthesis via the TSP pathway is also an important source for cysteine in astrocytes, and was shown to be upregulated and utilized during oxidative stress (Figures 3.2, 4.5; Vitvitsky et al., 2006). In most cells, cysteine is considered the limiting substrate for GSH synthesis. However, glutamate might also be considered a limiting in astrocytes as it is metabolized to glutamine (Figure 1.6). Because glutamate synthesis depends on mitochondrial anaplerotic metabolism, mitochondrial function might as well be essential for GSH homeostasis (Figure 2.1, 2.4). Interestingly, GSH depletion upregulates mitochondrial activity and expression of complex I in astrocytes (Vasquez et al., 2001), but the exact mechanisms that regulate this phenomenon are still unclear.

Central carbon metabolism in the mitochondria is also important for the maintenance of redox balance as it fuels the regeneration of NADPH. In addition to the anaplerotic activity of GDH mentioned above (Figure 2.4), within the mitochondria, the malic enzyme (ME) and isocitrate dehydrogenase isoform 2 (IDH2) regenerate NADPH coupled to the formation of OAA and αKG, respectively (Figures 2.1, 2.6). While higher levels of mitochondrial IDH activity have been reported in astrocytes (Minich et al., 2003), conflicting results exists in regards to the activity levels of mitochondrial ME (McKenna et al., 1995; Vogel et al., 1998). Metabolic flux analysis has also demonstrated that transfer of H from NADH via nicotinamide nucleotide transhydrogenase (NNT) contributes to the regeneration of the mitochondrial NADPH pool (Figure 2.5; Lewis et al., 2014), which is highly expressed in the brain (Arkblad et al., 2002). A comparative analysis demonstrated that these three metabolic pathways actively contribute to NADPH replenishment in brain mitochondria (Vogel et al., 1999), but other studies have reported a predominant role for the NNT pathway, particularly under metabolic stress (Lopert and Patel, 2014).

Folate metabolism supports one-carbon metabolism that serves to activate and transfer one-carbon units for biosynthetic processes. Mammals lack the capacity for folate synthesis whose requirements are largely met through dietary sources. Folate is not biologically active, but it is readily reduced in the intestinal mucosa or liver and converted into tetrahydrofolate (THF) molecules, such as 5-methyl-THF and 5-formy-THF. 5-methylTHF is the predominant form of folate in the blood and its transport in the brain across the BBB is mediated by the folate receptor alpha (FRα/FOLR1), the H+-coupled folate transporter (PCFT/SLC46A1), and the reduced folate carrier (RFC/SLC19A1), but whether the same mechanisms mediate folate transport in neural cells including astrocytes is unclear (Figure 3.1; Alam et al., 2020). Furthermore, because there is no dihydrofolate reductase in the brain to catalyze the formation of 5-methyl-THF (Wu and Pardridge, 1999), 5-methylTHF rather than folate is present in the CSF. Carrier-mediated energy-dependent transport of 5-methyl- and 5-formyl-THF has been described in astrocytes (Cai and Horne, 2003).

While serine can be acquired from the diet, central carbon metabolism makes substantial contribution to serine availability and as a consequence to folate metabolism in both the cytosol and the mitochondria (Figure 4.4). THF and serine are transported into the mitochondria via SLC25A32 (Lawrence et al., 2011) and sideroflexin-1 (SFXN1) (Kory et al., 2018), respectively. Mitochondrial serine hydroxymethyltransferase 2 (SHMT2) transfers the β-carbon of serine to THF, generating 5,10-methylene-THF that can also be produced from glycine by the activity of glycine decarboxylase (GLDC), an abundant enzyme in astrocytes (Figure 4.4; Li et al., 2018). GLDC deficiency induces neural tube defects and hydrocephaly (Pai et al., 2015; Leung et al., 2017; Santos et al., 2020). The bifunctional enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) or MTHFD2-like (MTHFD2L) then catalyzes the sequential production of 5,10-methenyl-THF and 10-formyl-THF. Finally, 10-formyl-THF is cleaved by MTHFD1L to generate formate, which can be exported to the cytosol (Figure 4.4). 10-formyl-THF can also be metabolized by 10-formyl-THF dehydrogenase (ALDH1L2). During this process both MTHFD2(L) and ALDH1L2 generate NADPH (Yang and Vousden, 2016; Figure 4.4). As such, knockdown of MTHFD2 decreases cellular NADPH/NADP+ and GSH/GSH disulfide (GSSG) ratios, and sensitizes cells to oxidative stress (Fan et al., 2014). The cytosolic counterpart of ALDH1L2, ALDH1L1 is expressed abundantly in astrocytes (Neymeyer et al., 1997; Foo and Dougherty, 2013). ALDH1L2 mutations seem to induce astrocytic stress (Sarret et al., 2019). Interestingly, complex I deficiency has been shown to decrease NADPH synthesis via ALDH1L2 due to the impaired generation of NAD+ (Balsa et al., 2020).

Despite the bidirectionality of this pathway, most of the cytosolic one-carbon units derived from serine are catabolized in the mitochondria, as this seems to be favored by the more oxidative mitochondrial redox potential, when compared to the cytosol. However, when the mitochondrial folate pathway is lost the directionality of the cytosolic one-carbon flux is reversed to compensate the synthesis of NADPH (Ducker et al., 2016).

In the cytosol, the trifunctional enzyme MTHFD1 regenerates THF and the intermediate 5,10-methylene-THF links the folate pathway and thus, central carbon metabolism, with the remethylation of homocysteine within the TSP. As mentioned before the TSP is an important source for cysteine in astrocytes (Figures 3.2, 4.5; Vitvitsky et al., 2006). While remethylation of homocysteine to methionine can be accomplished by the activity of methionine synthase, in the liver and kidney mitochondria, betaine-homocysteine S-methyltransferase (BHMT) also produces methionine from homocysteine, linking the mitochondrial oxidation of choline to betaine with the TSP (Figure 4.5). Brain genetic variants of BHMT have been linked to schizophrenia (Ohnishi et al., 2019). However, the presence of BHMT and its significance to the remethylation of homocysteine hasn’t been confirmed in astrocytes (Kempson et al., 2014).

The scientific process has been aptly described as blind people attempting to describe an elephant while only focusing on particular parts of the animal. Each person is contextually correct, but only when the data is combined can we achieve an accurate understanding of the whole. While there are far more parts of the proverbial elephant to explore to complete the picture, the recent evidence indicates that we must adjust our current understanding of brain metabolism, particularly as it pertains to astrocytes and neurons.

Current research indicates that metabolism in the brain extends far beyond glucose. FAs, and their derived ketone bodies, are important sources of bioenergetic potential for the brain, particularly during development, as shown by the experiments in vivo and in vitro that follow its oxidation and integration into biomolecules. Glutamate is more than just a neurotransmitter kept in strict stoichiometry by the glutamate-glutamine cycle; it is a resource the brain uses for amino acid synthesis by transamination, for redox balance by generation of NADPH, and for anaplerosis to maintain the TCA cycle.

The brain is far from a homogeneous body of cells, and the literature paints a clear picture of metabolic specialization and intracellular cooperation/dependence. In this review, we have centered our attention to astrocytic mitochondrial metabolism and its role in the regulation of brain bioenergetics, neurotransmission and redox balance via neuronal-astrocyte metabolic interactions. Astrocytes have been demonstrated to be functional without OXPHOS in vivo, to provide a GSH-dependent protective effect to neurons, to function as neutralizing and cycling agents of glutamatergic/GABAergic signaling, and to be synthesizers/exporters of OXPHOS bioenergetic molecules (lactate/ketone bodies). In contrast, neurons have a metabolic specialization toward OXPHOS, spurning FAO, restricting glucose oxidation while maintaining the PPP for redox homeostasis, and limiting their ability to conduct mitochondrial biosynthesis due to a lack of pyruvate carboxylase for anaplerosis. Together, neurons shoulder the bioenergetic burden of neurotransmission by controlled alterations in cell potential, while astrocytes conduct biosynthesis and high ROS oxidative processes to supplement neuronal functions.

The key organelle in this differential specialization is the mitochondria. While the broad mitochondrial role in OXPHOS is well known in mammalian cells, specialization is evident within neurons and astrocytes. Astrocytes restrict the conversion of pyruvate to Ac-CoA by their phosphorylation of PDH, limiting their utilization of glucose for OXPHOS but instead directing these carbons to biosynthesis through the TCA cycle by PC. Additionally, the efficiency of the astrocyte ETC is far less than neurons, resulting in higher ROS generation in astrocyte compared to neurons. Astrocytic mitochondria have been demonstrated to utilize FAO for the production of ketone bodies, a process that has not been shown to be significant in neurons. Further, evidence shows that astrocytes localize mitochondria to endfeet processes in conjunction with glutamate transporters and enzymes related to glycolysis. Coupled with the evidence that OXPHOS is not required in astrocytes in vivo, this paints a unique picture for astrocyte mitochondria compared to the prototypical mammalian cell. It is a mitochondria specializing in synthesis at the expense of OXPHOS, while the neuronal mitochondria specializes in OXPHOS at the expense of synthesis.

JR and CB contributed to the first draft and figures. AP, MP, and RF contributed to the edition of the final version of the manuscript and figures. All authors contributed to the article and approved the submitted 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.

adenosine-triphosphate-binding cassette transporter subfamily C member 1 or multidrug-resistance-associated protein 1