Differential expression of cytochrome P450 enzymes from the CYP2C subfamily in the human brain

Abstract

Cytochrome P450 enzymes from the CYP2C subfamily play a prominent role in the metabolic clearance of many drugs. CYP2C enzymes have also been implicated in the metabolism of arachidonic acid to vasoactive epoxyeicosatrienoic acids. CYP2C8, CYP2C9, and CYP2C19 are expressed in the adult liver at significant levels; however, the expression of CYP2C enzymes in extrahepatic tissues such as the brain is less well characterized. Form-specific antibodies to CYP2C9 and CYP2C19 were prepared by affinity purification of antibodies raised to unique peptides. CYP2C9 and CYP2C19 were located in microsomal fractions of all five human brain regions examined, namely the frontal cortex, hippocampus, basal ganglia, amygdala, and cerebellum. Both CYP2C9 and CYP2C19 were detected predominantly within the neuronal soma but with expression extending down axons and dendrites in certain regions. Finally, a comparison of cortex samples from alcoholics and age-matched controls suggested that CYP2C9 expression was increased in alcoholics.

Fig. 1.

CYP2C protein expression in human brain microsomes. (A–C) Affinity-purified polyclonal antibodies raised against CYP2C9, CYP2C19, and recombinant CYP2C18 (“All CYP2C”) were used to assess CYP2C9 (A), CYP2C19 (B), and total CYP2C expression (C) in the cortex, hippocampus, basal ganglia, amygdala, and cerebellum. For CYP2C9 and CYP2C19 blots, 20 μg protein pooled from the three samples described in Supplemental Table 2 were loaded in each lane. For the blot incubated with the all-2C antibody, 10 μg protein was loaded from one representative brain sample. Recombinant proteins used as standards in (A) and (B) contained truncated N-terminal peptide sequences compared with the full-length, native proteins in liver and brain fractions. Recombinant CYP2C9 and CYP2C18 in (C) were expressed from full-length constructs (Cuttle et al., 2000; Shukla et al., 2005). 2C9, recombinant CYP2C9; 2C18, recombinant CYP2C18; 2C19, recombinant CYP2C19; AM, amygdala; BG, basal ganglia; CB, cerebellum; CTX, cortex; HL, human liver microsome; HP, hippocampus; Lµs, human liver pellet microsomal fraction from centrifugation at 110,000 × g; LP10, human liver pellet fraction from centrifugation at 110,000 × g.

Fig. 2.

(A and B) Immunohistochemical detection of CYP2C9 and CYP2C19 expression in the human frontal cortex (A) and hippocampus (B). Negative controls incubated with preimmune sera for CYP2C9 and CYP2C19 were performed with layers 5 and 6 (A) and CA4 (B), respectively, and were representative of other layers. (A) CYP2C9 and CYP2C19 proteins were detected in layer 2 through layer 6 of the frontal cortex, predominantly in the soma of neuronal cells. Expression of CYP2C9 was also observed in neuronal axons and dendrites, predominantly in layers 5 and 6. (B) CYP2C9 and CYP2C19 were both located throughout the hippocampus; however, expression appeared to be highest in the CA1 region in both cases. In addition, CYP2C9 was also highly expressed in the CA3 region. Bar, 50 μm.

Fig. 3.

Immunohistochemical detection of CYP2C9 and CYP2C19 expression in the human amygdala, basal ganglia, and cerebellum. Negative controls incubated with preimmune sera for CYP2C9 and CYP2C19 were performed with the molecular layer of the cerebellum but were representative of other areas. Both P450s were expressed in the amygdala, basal ganglia, and cerebellum. In the amygdala, expression was predominantly localized in the soma of neurons, whereas both somatic and axonal expression of both CYP2C9 and CYP2C19 was observed in the basal ganglia. Expression of both CYP2C9 and CYP2C19 was localized to the pyramidal cells and the granular cell layer of the cerebellar cortex. Bar, 50 μm.

Fig. 4.

(A and B) Immunoblot of microsomal fractions (pellets from centrifugation at 110,000 × g) of the frontal cortex of six individual control and six individual alcoholic case-matched human brain samples using antibodies detecting CYP2C9 (A) and CYP2C19 (B). An equivalent amount (20 μg) of total protein was loaded in each lane. (C) Individual sample densities were corrected against beta-actin and the corrected data were subjected to a t test with a 95% confidence interval. Data are expressed as mean ± S.E.M. The asterisk indicates that CYP2C9 expression in samples from alcoholics was significantly elevated over expression in controls (n = 6; P < 0.05, 95% confidence interval). No significant differences were seen in CYP2C19 expression between samples from alcoholics and controls.