Induction of Mrp3 and Mrp4 transporters during acetaminophen hepatotoxicity is dependent on Nrf2

Abstract

The transcription factor NFE2-related factor 2 (Nrf2) mediates detoxification and antioxidant gene transcription following electrophile exposure and oxidative stress. Mice deficient in Nrf2 (Nrf2-null) are highly susceptible to acetaminophen (APAP) hepatotoxicity and exhibit lower basal and inducible expression of cytoprotective genes, including NADPH quinone oxidoreductase 1 (Nqo1) and glutamate cysteine ligase (catalytic subunit, or Gclc). Administration of toxic APAP doses to C57BL/6J mice generates electrophilic stress and subsequently increases levels of hepatic Nqo1, Gclc and the efflux multidrug resistance-associated protein transporters 1-4 (Mrp1-4). It was hypothesized that induction of hepatic Mrp1-4 expression following APAP is Nrf2 dependent. Plasma and livers from wild-type (WT) and Nrf2-null mice were collected 4, 24 and 48 h after APAP. As expected, hepatotoxicity was greater in Nrf2-null compared to WT mice. Gene and protein expression of Mrp1-4 and the Nrf2 targets, Nqo1 and Gclc, was measured. Induction of Nqo1 and Gclc mRNA and protein after APAP was dependent on Nrf2 expression. Similarly, APAP treatment increased hepatic Mrp3 and Mrp4 mRNA and protein in WT, but not Nrf2-null mice. Mrp1 was induced in both genotypes after APAP, suggesting that elevated expression of this transporter was independent of Nrf2. Mrp2 was not induced in either genotype at the mRNA or protein levels. These results show that Nrf2 mediates induction of Mrp3 and Mrp4 after APAP but does not affect Mrp1 or Mrp2. Thus coordinated regulation of detoxification enzymes and transporters by Nrf2 during APAP hepatotoxicity is a mechanism by which hepatocytes may limit intracellular accumulation of potentially toxic chemicals.

Figure 1. Plasma ALT activity in wild-type and Nrf2-null mice after APAP

Plasma was isolated from WT and Nrf2-null mice 24 and 48 hrs following APAP (200, 400 mg/kg) or vehicle administration. The data are presented as mean plasma ALT activity (U/L) ± SE (n = 3 – 16 animals). Asterisks (*) represent a statistical difference (p < 0.05) between pooled 0 hr control and APAP-treated groups and daggers (†) represent a statistical difference (p < 0.05) between WT and Nrf2-null mice.

Figure 2. Western blot analysis of Nrf2 in nuclear liver preparations from wild-type and Nrf2-null mice after APAP

An immunoblot for Nrf2 protein was performed using nuclear extracts (60 μg protein/lane) from WT and Nrf2-null mice 4 hrs following treatment with APAP (400 mg/kg) or vehicle. The data are presented as an individual blot (A) and as mean relative protein expression (normalized to control WT mice) ± SE (B). Asterisks (*) represent a statistical difference (p < 0.05) between control and APAP-treated groups (n = 2 animals). ND, not detected.

Figure 3. Immunofluorescent localization of Nrf2 in livers from wild-type and Nrf2-null mice after APAP

Indirect immunofluorescence to detect Nrf2 (green) and actin (red) was conducted on liver cryosections (5 μm) from WT and Nrf2-null mice 4 hrs following treatment with APAP (400 mg/kg) or vehicle. Sections were mounted in Prolong Gold containing DAPI for nuclear staining (blue). Representative images are shown. Panel A: control WT; B: APAP-treated WT; C: control Nrf2-null; D: APAP-treated Nrf2-null. Bar 31.74 μm.

Figure 4. Nqo1 and Gclc mRNA expression in livers from wild-type and Nrf2-null mice after APAP

Total RNA was isolated from livers of WT and Nrf2-null mice 24 and 48 hrs following treatment with APAP (200, 400 mg/kg) or vehicle. RNA was analyzed by the bDNA assay for expression of Nqo1 and Gclc. The data are presented as mean RLU ± SE (n = 3 – 16 animals). Asterisks (*) represent a statistical difference (p < 0.05) between pooled 0 hr control and APAP-treated groups and daggers (†) represent a statistical difference (p < 0.05) between WT and Nrf2-null mice.

Figure 5. Nqo1 and Gclc protein expression in livers from wild-type and Nrf2-null mice after APAP

Western blots for Nqo1 and Gclc were performed using liver cytosol (50 μg protein/lane) from WT and Nrf2-null mice 48 hrs following treatment with APAP (200 mg/kg) or vehicle. The data are presented as individual blots (A) and as mean relative protein expression (normalized to control WT mice) ± SE (n = 3 animals) (B). Asterisks (*) represent a statistical difference (p < 0.05) between control and APAP-treated groups and daggers (†) represent a statistical difference (p < 0.05) between WT and Nrf2-null mice.

Figure 6. Mrp1-4 mRNA expression in livers from wild-type and Nrf2-null mice after APAP

Total RNA was isolated from livers of WT and Nrf2-null mice 24 and 48 hrs following treatment with APAP (200, 400 mg/kg) or vehicle. RNA was analyzed by the bDNA assay for expression of Mrp1-4. The data are presented as mean RLU ± SE (n = 3 – 16 animals). Asterisks (*) represent a statistical difference (p < 0.05) between pooled 0 hr control and APAP-treated groups and daggers (†) represent a statistical difference (p < 0.05) between WT and Nrf2-null mice.

Figure 7. Mrp1-4 protein expression in livers from wild-type and Nrf2-null mice after APAP

Western immunoblots for Mrp1-4 were performed using liver membrane fractions (50 μg protein/lane) from WT and Nrf2-null mice 48 hrs following treatment with APAP (200 mg/kg) or vehicle. The data are presented as individual blots (A) and as mean relative protein expression (normalized to control WT mice) ± SE (n = 3 animals) (B). Equal protein loading was confirmed by detection of β-actin. Asterisks (*) represent a statistical difference (p < 0.05) between control and APAP-treated groups and daggers (†) represent a statistical difference (p < 0.05) between WT and Nrf2-null mice.

Figure 8. Nrf2-mediated gene transcription during APAP hepatotoxicity

APAP is bioactivated to N-acetyl-p-benzoquinone imine (NAPQI) by various cytochrome P450 isoforms in the hepatocyte. Generation of NAPQI alters cellular homeostasis by increasing oxidative stress and depleting GSH stores. The net result is release of Nrf2 from Keap1 and translocation of Nrf2 to the nucleus. Once in the nucleus, Nrf2 heterodimerizes with small Maf or Jun proteins, binds ARE sequences in the upstream region of target genes and activates gene transcription. The Nrf2-mediated antioxidant defense in hepatocytes is composed of numerous genes involved in cell stress response, drug metabolism, detoxification, and efflux transport.