Effects of ionic strength on the functional interactions between CYP2B4 and CYP1A2

The presence of one P450 can influence the catalytic characteristics of a second enzyme through the formation of heteromeric P450 complexes. Such a complex has been reported for mixed reconstituted systems containing NADPH-cytochrome P450 reductase, CYP2B4, and CYP1A2, where a dramatic inhibition of 7-pentoxyresorufin-O-dealkylation (PROD) was observed when compared to simple reconstituted systems containing reductase and a single P450 enzyme. The goal of the present study was to characterize this interaction by examining the potential of the CYP1A2-CYP2B4 complex to be formed by charge-pair interactions. With ionic interactions being sensitive to the surrounding ionic environment, monooxygenase activities were measured in both simple systems and mixed reconstituted systems as a function of ionic strength. PROD was found to be decreased at high ionic strength in both simple and mixed reconstituted systems, due to disruption of reductase-P450 complexes. Additionally, the inhibition of PROD in mixed reconstituted systems was relieved at high ionic strength, consistent with disruption of the CYP2B4-CYP1A2 complex. When ionic strength was measured as a function of CYP1A2 concentration, a shift to the right in the inflection point of the biphasic curve occurred at high ionic strength, consistent with a loss in CYP1A2 affinity for CYP2B4. When this analysis was applied to the same systems using a different substrate, 7-EFC, evidence for a high-affinity complex was not observed, demonstrating that the characteristics of the CYP1A2-CYP2B4 complex are influenced by the substrates present. These results support the role for a substrate specific electrostatic interaction between these P450 enzymes.     

Age-related changes in the mRNA levels of CYP1A1, CYP2B1/2 and CYP3A1 isoforms in rat small intestine

It has been established beyond doubt that, as well as the liver, the small intestine is an important site of first-pass metabolism of numerous drugs, food components and toxic xenobiotics. However, there is not much information available about age-dependent changes of intestinal biotransformation pathways. In the present paper, we evaluated the relationships between intestinal cytochrome P450 complex activity and the age of animals. The study was carried out on male Sprague–Dawley rats (n = 5) from 5 age series: 0.5-, 2-, 4-, 20-, and 28 months old. Animals at every age series were divided into 4 groups: control and three groups of rats treated with the CYP450 specific inducers: phenobarbital, β-naphtoflavone and dexamethasone, respectively. RNA was isolated from intestinal mucosa, and then standard RT-PCR was used for the analysis of CYP1A1, CYP2B1/2 and CYP3A1 mRNA expression. Additionally, the activities of NADPH-cytochrome P450 and NADH-cytochrome b₅ reductases in the microsomal fraction were biochemically estimated. The constitutive intestinal CYP1A1 mRNA expression changes during maturation and aging. Inducibility of CYP1A1 gene was evident in intestinal mucosa at 2-, 4- and 20-month-old rats. A similar pattern of changes was observed for CYP2B1/2 isoforms. CYP3A1 mRNA expression was not detected in small intestine of 2-week-old rats. In matured rats, constitutive intestinal CYP3A1 expression was low, although after induction, significant increases in CYP3A1 mRNA amount were noted in aged individuals. Intestinal activity of both analyzed reductases was lowest in immature rats and highest in 28-month-old animals. In conclusion, the activity of cytochrome P450 complex in rat small intestine was not decreased by the aging processes, so the high rate of oxidative metabolic reactions in intestinal mucosa can be maintained till the advanced life stage.     

Hormone controlled phosphorylation and degradation of CYP2B1 and CYP2E1 in isolated rat hepatocytes

Addition of adrenalin to primary rat hepatocytes caused a 3- and 2-fold increase in [32P]-incorporation into CYP2E1 and CYP2B1, respectively. Adrenalin also increased the rate of CYP2E1 degradation at similar concentrations as needed for phosphorylation of the protein (r = 0.93), but did not influence the degradation rate of CYP2B1. Ethanol (75 mM) completely protected from adrenalin dependent phosphorylation and degradation of CYP2E1, but did not influence CYP2B1 on these parameters. Examination of para-nitrophenol hydroxylase revealed that ethanol stabilized the catalytically active form of CYP2E1. Insulin treatment caused a stabilization of CYP2E1, but did not affect CYP2B1 degradation. It is concluded that degradation of CYP2E1 is the subject of hormonal control, whereas CYP2B1 decomposition is accomplished in a different and a less regulated manner.     

Heteromeric complex formation between CYP2E1 and CYP1A2: evidence for the involvement of electrostatic interactions

Mixed reconstituted systems containing CYP2B4, CYP1A2, and NADPH-cytochrome P450 reductase were previously shown to exhibit a dramatic inhibition of 7-pentoxyresorufin O-dealkylation (PROD) when compared to simple reconstituted systems containing reductase and a single P450 enzyme, results consistent with the formation of CYP1A2-CYP2B4 complexes where the reductase binds with high affinity to the CYP1A2 moiety of the complex. In this report, we provide evidence for an interaction between CYP1A2 and CYP2E1. Synergism of 7-ethoxyresorufin O-deethylation (EROD) and PROD was observed when these P450s were combined in mixed reconstituted systems at subsaturating reductase concentrations. Higher ionic strength attenuated the synergistic stimulation of both PROD and EROD in mixed reconstituted systems, consistent with disruption of heteromeric CYP2E1-CYP1A2 complexes. The effect of ionic strength was further examined as a function of reductase concentration. At lower ionic strength, there was a significant synergistic stimulation of EROD. This synergistic stimulation diminished with increasing reductase concentration, resulting in an additive response as reductase became saturating. Interestingly, at high ionic strength, the synergism of EROD in the mixed reconstituted system was not observed. In contrast, mixed reconstituted systems containing CYP2E1 and CYP2B4 did not provide evidence for the formation of these heteromeric P450-P450 complexes. The synergistic stimulation observed with the reductase-CYP1A2-CYP2E1 mixed reconstituted system is consistent with the formation of a CYP1A2-CYP2E1 complex. Taken together with the lack of a kinetically detectable interaction between CYP2B4 and CYP2E1, and the previously reported CYP1A2-CYP2B4 interaction, these results suggest that CYP1A2 may facilitate the formation of complexes with other P450 enzymes.     

NF-kappaB site interacts with Sp factors and up-regulates the NR1 promoter during neuronal differentiation

The NR1 gene undergoes induction in neurogenesis mainly via promoter de-repression, and up-regulation during neuronal differentiation by undefined mechanism(s). Here, we show that in the distal region the NR1 promoter has an active NF-kappaB site sharing the consensus with the immunoglobulin (Ig)/human immunodeficiency virus NF-kappaB site. Mutation of this site significantly reduced NR1 promoter up-regulation during neuronal differentiation of P19 cells. Electrophoretic mobility shift assays revealed that P19 nuclei constitutively contained p50 and that neuronal differentiation not only increased nuclear p50 but also induced p65 nuclear translocation. Responding to this change was an up-regulation of NF-kappaB-dependent promoter activity. However, inhibition of NF-kappaB nuclear translocation by an IkappaBalpha super-repressor or decoy DNA only moderately inhibited NR1 promoter up-regulation. Interestingly, the NR1 NF-kappaB site strongly interacted with Sp3/Sp1, instead of NF-kappaB factors, in P19 nuclear extracts. This interaction was reduced for Sp3 following neuronal differentiation, accompanied by dynamic expression of Sp factors. Cotransfection of Sp factors (Sp1, 3, or 4) upregulated the NR1 NF-kappaB site dramatically in differentiated neurons, but only moderately in undifferentiated P19 cells. This up-regulation was strong for Sp1 in differentiated cells and for Sp3 in undifferentiated cells. Chromatin-immunoprecipitation assays further demonstrated that Sp1 and Sp3 interacted with the NR1 NF-kappaB site in situ, and Sp3 lost its interaction after neuronal differentiation. We conclude that the NF-kappaB site positively regulates the NR1 promoter during neuronal differentiation via interacting mainly with Sp factors and neuronal differentiation reduces the effect of Sp3 factor on this site.