Effect of beta-naphthoflavone on AhR-regulated genes (CYP1A1, 1A2, 1B1, 2S1, Nrf2, and GST) and antioxidant enzymes in various brain regions of pig.
The constitutive and inducible expression of aryl hydrocarbon receptor (AhR) and of the AhR-regulated genes coding for CYP1A1, CYP1A2, CYP1B1, CYP2S1, and Nrf2 was investigated by real-time or traditional PCR in cerebral areas (cortex, cerebellum, midbrain, and hippocampus), blood-brain interfaces (meninges and brain microvessels) and liver obtained from control pigs and from pigs treated with beta-naphthoflavone (betaNF), a potent AhR agonist. The enzymatic activities of ethoxyresorufin-O-deethylase (EROD), and methoxyresorufin-O-deethylase (MEROD), marker for CYP1A1 and CYP1A2, the GST and various antioxidant enzymes (catalase, superoxide dismutase, GSSG-reductase, and GSH-peroxidase) were also determined in the same CNS regions. The AhR, CYP1A1, CYP1A2, CYP1B1, Nrf2 mRNAs were detected, although at different extent, in all the CNS regions, while CYP2S1 mRNA was detected only in midbrain. In the blood-brain interfaces, the constitutive basal expression of AhR and CYP1A1 was comparable to the hepatic one and even higher for CYP1B1 and Nrf2. The treatment with betaNF determined the induction of CYP1A1 and 1B1 (but not of AhR, CYP1A2, and Nrf2) mRNA levels in various CNS areas; notably, CYP1A1 mRNA was increased to about 300-fold in the microvessels. The analysis of enzymatic activities revealed that EROD, but not MEROD, was induced in microsomes but not in mitochondria of all the CNS areas. However, the mitochondrial EROD activities were comparable (in midbrain, meninges) or higher (in cortex, cerebellum, hippocampus) than the microsomal ones, suggesting an important metabolic function of CYP1A1 in this subcellular localization. The activities of GST and antioxidant enzymes were detected in all CNS tissues, with levels lower than the hepatic ones, but found quite evenly distributed and marginally affected by betaNF treatment. The high expression of metabolic enzymes found in blood-brain interfaces could represent a very important defence toward toxins of CNS.
Istituto di Fisiologia Clinica, CNR, via Moruzzi 1, 56100 Pisa, Italy.
Annalisa
A
Nannelli
Francesco
F
Rossignolo
Roberto
R
Tolando
Paolo
P
Rossato
Vincenzo
V
Longo
P Giovanni
PG
Gervasi
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000201:enzymology*; D002374:Catalase / Q000032:analysis; D003433:Crosses, Genetic; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D018076:DNA, Complementary / Q000096:biosynthesis; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005979:Glutathione Peroxidase / Q000032:analysis; D005980:Glutathione Reductase / Q000032:analysis; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics*; D007527:Isoenzymes; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D051267:NF-E2-Related Factor 2 / Q000096:biosynthesis / Q000235:genetics*; D009919:Orchiectomy; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon / Q000187:drug effects / Q000235:genetics*; D013482:Superoxide Dismutase / Q000032:analysis; D034421:Sus scrofa; D013552:Swine; D019324:beta-Naphthoflavone / Q000494:pharmacology*
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D051267:NF-E2-Related Factor 2; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D019324:beta-Naphthoflavone; D012313:RNA; D003577:Cytochrome P-450 Enzyme System; D002374:Catalase; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; D005980:Glutathione Reductase; D005982:Glutathione Transferase