Discussion

Adverse drug reactions (ADRs) have gained increasingly greater attention over the last several years. Defined by the World Health Organization as an unintentional, harmful reaction to an administered medication, ADRs remain a significant contributor to causes of death in several countries (Finney, 2008; Wooten, 2010). Type B ADRs are those that are unpredictable and are thus considered idiosyncratic (Wooten, 2010). Because of their unpredictability and often unknown etiology, type B ADRs pose a formidable threat to patient medication management. In contrast, type A ADRs are typically predictable on the basis of the known pharmacokinetic and pharmacodynamic profiles of the drug (Wooten, 2010). Predictions of drug disposition can be made based on the identification of a drug as a substrate for specific transporters. However, this predictability may prove useless for a patient population or disease demographic in which basic drug metabolism and disposition data are unavailable. Thus, for diseases that alter drug metabolizing enzyme and transporter expression and function, the predictability of type A ADRs fails. This leads to an inability to identify a particular demographic as an at-risk population for ADRs. Given that NAFLD is becoming an increasingly prevalent disease, understanding the capacity for hepatic drug metabolism and disposition in each stage of the disease is necessary to reduce the risk of ADRs in this patient population.

Our laboratory has previously investigated the effect of human NAFLD on cytochrome P450 and GSH transferase enzyme expression and functionality (Fisher et al., 2009; Hardwick et al., 2010). However, little information is available on the expression or functionality of the ABC transporters in human NAFLD. These patients represent a population that is very likely to be prescribed various pharmaceuticals to treat symptoms of the metabolic syndrome, yet little is understood about the ability of these patients to manage the metabolism and elimination of xenobiotics. To date, animal models have been used to gain an understanding of the effects of NAFLD on transporter expression and subsequent drug disposition. The current data provide new insight to the effect of NAFLD on the regulation of efflux transporter expression in human patients that could, in turn, enable better prediction of pharmacokinetics in these patients, thereby leading to a more positive therapeutic outcome.

Previous studies within our laboratory using diet-induced rodent models of NAFLD have shown that disease progression leads to significant induction of Abcc3, Abcc4, and Abcg2 mRNA in NASH, but not steatosis (Lickteig et al., 2007). Likewise, protein levels of Abcc2–4 and Abcg2 were induced in NASH rodents (Lickteig et al., 2007). These expression changes in hepatic ABC transporters were found to result in functional alterations of drug metabolite disposition. Upon administration of acetaminophen, NASH rodents exhibited a significant reduction in the biliary excretion of glucuronide-, sulfate-, and GSH-acetaminophen conjugates with a concomitant shift to sinusoidal excretion (Lickteig et al., 2007). The link between altered efflux transporter expression and changes in drug disposition is clearly demonstrated in this rodent model of NASH; however, until now, similar studies in human patients have been severely lacking.

The data presented in the current study provide a generous foundation for further investigation into drug disposition outcomes during NAFLD progression. Similar to rodent NAFLD models, ABCC4 and ABCG2 gene induction was observed in human NASH patients (Fig. 2). Additional transporters, including ABCC1, 5, and ABCB1, were also found to be induced with disease progression (Fig. 2). In general, most transporters of the ABC family followed an increasing trend in mRNA expression as the disease progressed from steatosis to end-stage NASH (not fatty). Constitutive androstane receptor (CAR) and pregnane X receptor are known to induce ABCC2, 3, ABCB1, and ABCG2 in humans (Klaassen and Aleksunes, 2010). We have previously identified induction of the CAR target gene CYP2B6 and the pregnane X receptor target genes CYP2A6 and GSTA1 in this same set of human samples (Fisher et al., 2009; Hardwick et al., 2010). Nuclear translocation of CAR and induction of CAR target genes has been shown to worsen histological features of NASH in a rodent model of the disease and possibly play a role in the transition from steatosis to NASH (Yamazaki et al., 2007). The current study has demonstrated induction of the CAR target genes ABCB1 and ABCG2, as well as a modest increase in ABCC3 mRNA. In addition, NF-E2-related factor 2 (Nrf2), in response to oxidative stress conditions, is believed to induce human ABCC2, 3, ABCB1, and ABCG2 (Klaassen and Aleksunes, 2010). We have previously shown activation of Nrf2 and several of its downstream target genes in this same set of NAFLD samples (Hardwick et al., 2010). Here, we have identified induction of several transporters that have been identified as Nrf2 target genes, including ABCB1 and ABCG2. In addition, although not significant (p = 0.066), ABCC3 exhibited a noticeable increasing trend in mRNA expression similar to ABCB1 and ABCG2. The observed induction of ABC transporter genes may be due to initiators of the second hit mediating the transition from steatosis to NASH through activation of various nuclear receptors and transcription factors.

In this study, we have identified an overarching increase in ABC transporter protein levels with progression of NAFLD. ABCC1–6, ABCB1, and ABCG2 protein all exhibited significant increasing trends (Figs. 3 and 4) with disease progression. A noticeable elevation in protein levels of ABCC1, ABCC3–6, and ABCG2 was observed between steatosis and NASH samples (Fig. 3). The increase in protein levels of numerous efflux transporters suggests a heightened ability for NASH patients to excrete drugs and drug conjugates out of hepatocytes. However, due to the overlapping substrate specificity, yet differing affinity for substrates of several of these transporters, these effects could have a variety of implications upon the hepatic elimination of xenobiotics.

Of particular note in this study is the potential altered regulation of ABCC2 in NASH (not fatty) patients. Internalization of Abcc2 to intracellular vesicles has been extensively studied in rodents, and linked to oxidative stress and cholestatic conditions. In particular, vesicular retrieval of Abcc2 has been demonstrated during estradiol-17β-glucuronide (E₂17-G)-induced cholestasis (Mottino et al., 2002), lipopolysaccharide-induced oxidative stress (Saeki et al., 2011), bile duct ligation (Kojima et al., 2008b), and following ethacrynic acid treatment (Ji et al., 2004). ABCC2 internalization has also been identified in several forms of human liver injury including drug-induced liver injury, obstructive jaundice, autoimmune hepatitis, primary sclerosing cholangitis, and primary biliary cirrhosis (Kojima et al., 2003, 2008a). Here, we provide the first evidence that ABCC2 may be internalized in the later stages of NAFLD. Previous studies have shown that internalization of hepatic Abcc2 is correlated to GSH depletion in vivo (Sekine et al., 2008). We have previously shown in these same NAFLD samples that disease progression leads to significant depletion of hepatic GSH stores (Hardwick et al., 2010). It is likely that GSH depletion in these samples is the mechanism responsible for the proposed internalization of ABCC2 observed in the current study; however, further analyses are needed to identify the specific mechanism underlying this alternative method of ABCC2 regulation.

Zhang et al. (2005) identified a role for glycosylation in the regulation of Abcc2 cellular localization. Rat hepatocytes were isolated and placed in a sandwich-culture system. At day 0 of culture, hepatocytes expressed a 180- to 190-kDa form of Abcc2, but by day 4 the molecular mass of Abcc2 had increased to 200 kDa (Zhang et al., 2005). It was discovered that the glycosylation status determined the molecular mass shift of Abcc2, and that the fully glycosylated, 200-kDa form was properly localized to the canalicular membrane (Zhang et al., 2005). In contrast, the low-molecular-mass, 180- to 190-kDa, lesser-glycosylated form was not localized in the canalicular membrane (Zhang et al., 2005). To further test our hypothesis of ABCC2 internalization in NASH, we investigated the protein expression of these two glycosylation states of ABCC2. Normal and steatotic human samples solely exhibited the high-molecular-mass, 200-kDa form of ABCC2, indicative of membrane localization. We identified the appearance of a 180-kDa band of ABCC2 protein that is preferentially expressed in NASH samples (Fig. 7), which correlates with the observation of ABCC2 internalization identified by immunohistochemical staining. This suggests that there is a portion of ABCC2 protein in NASH samples that is not fully glycosylated and not properly localized in the canalicular membrane.

The functional consequence of this method of ABCC2 regulation is extremely important in drug disposition. In conjunction with their studies identifying the association between glycosylation status and cellular localization, Zhang et al. (2005) determined that the loss of glycosylation of the ABCC2 protein results in diminished transport function due to improper localization. The biliary excretion of 5-(6)-carboxy-2′,7′-dichlorofluorescein was reduced when Abcc2 was internalized (Zhang et al., 2005). However, when the fully glycosylated, high-molecular-mass form was expressed in sandwich-cultured rat hepatocytes, Abcc2 was properly localized to the canalicular membrane and able to excrete substrates into the canalicular space (Zhang et al., 2005). The disruption of Abcc2 cellular localization and its effect on transport function has been demonstrated in vivo in estradiol-17-β-d-glucuronide-induced cholestasis. Upon administration of estradiol-17-β-d-glucuronide, Abcc2 was retrieved to pericanalicular vesicles and the biliary excretion of dinitrophenyl-S-GSH was diminished (Mottino et al., 2002). These studies indicate that the internalization of ABCC2 that was observed in our NASH samples may limit these patients' ability to excrete ABCC2 substrates into bile.

In conclusion, we have observed a general increase in the mRNA and protein expression of numerous ABC transporters after the transition from steatosis to NASH in human NAFLD samples. However, internalization of ABCC2 in end-stage NASH could lead to a reduction in functionality and ultimately elicit alterations in the hepatic elimination of xenobiotics. Considering NAFLD patients are frequently medicated for symptoms of the metabolic syndrome, such as hypertension, dyslipidemia, and insulin resistance, these patients could be at increased risk for ADRs. Despite greater recognition of this disease, the field of NAFLD research is in great need of further studies on the ability of these patients to metabolize and eliminate various pharmaceutical agents to ensure safety and improve therapeutic outcome.