Induction of CYP1A by benzo[k]fluoranthene in human hepatocytes: CYP1A1 or CYP1A2?

While fresh human hepatocyte cultures are widely used to model hepatic cytochrome P450 (CYP) regulation and activity, their CYP1A subfamily composition induced by, e.g., polycyclic aromatic hydrocarbons is ambiguous. CYP1A1, CYP1A2, or both have been reported to be expressed, and their varied roles in chemical carcinogenesis makes resolution of which CYPs are expressed essential. We have used an immunoblot system with Bis-Tris-HCl-buffered polyacrylamide gel, which clearly resolves human CYP1A1 and CYP1A2, and polyclonal goat anti-human CYP1A1/CYP1A2 and rabbit anti-human CYP1A2 antibodies to probe the expressed CYP1A1 and CYP1A2 composition of seven individual human hepatocyte cultures induced with 5 microM benzo[k]fluoranthene (BKF) for 24 h. In six of the cultures only CYP1A1 was detected, and in the seventh both CYPs were detected. In most vehicle-treated hepatocyte cultures, neither CYP1A1 nor CYP1A2 was detected. In three additional hepatocyte cultures treated individually with BKF and 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD), the resultant induced CYP1A1/1A2 profiles were essentially not influenced by the nature of the inducing agents. To develop an activity-based assay to differentiate between CYP1A1 and CYP1A2 expression in human hepatocytes, our previously published R warfarin assay (Drug Metab. Disp. (1995) 23, 1339-1345) was applied to TCDD (10 nM)-treated hepatocyte culture. The low concentration of TCDD did not produce inhibition of the warfarin metabolism-such inhibition could confound the results. Based on the ratios of 6- to 8-hydroxywarfarin formed in two cultures, the ratios of CYP1A1/CYP1A2 expressed in these cultures were determined and they agreed with the ratios determined by immunoblot analysis. Thus each individual human hepatocyte culture must be characterized for induced CYP1A1 and CYP1A2 expression in studies of CYP1A activity. The warfarin assay provides a means of characterizing the cultures.     

Production of ω-hydroxy palmitic acid using CYP153A35 and comparison of cytochrome P450 electron transfer system in vivo

Bacterial cytochrome P450 enzymes in cytochrome P450 (CYP)153 family were recently reported as fatty acid ω-hydroxylase. Among them, CYP153As from Marinobacter aquaeolei VT8 (CYP153A33), Alcanivorax borkumensis SK2 (CYP153A13), and Gordonia alkanivorans (CYP153A35) were selected, and their specific activities and product yields of ω-hydroxy palmitic acid based on whole cell reactions toward palmitic acid were compared. Using CamAB as redox partner, CYP153A35 and CYP153A13 showed the highest product yields of ω-hydroxy palmitic acid in whole cell and in vitro reactions, respectively. Artificial self-sufficient CYP153A35-BMR was constructed by fusing it to the reductase domain of CYP102A1 (i.e., BM3) from Bacillus megaterium, and its catalytic activity was compared with CYP153A35 and CamAB systems. Unexpectedly, the system with CamAB resulted in a 1.5-fold higher yield of ω-hydroxy palmitic acid than that using A35-BMR in whole cell reactions, whereas the electron coupling efficiency of CYP153A35-BM3 reductase was 4-fold higher than that of CYP153A35 and CamAB system. Furthermore, various CamAB expression systems according to gene arrangements of the three proteins and promoter strength in their gene expression were compared in terms of product yields and productivities. Tricistronic expression of the three proteins in the order of putidaredoxin (CamB), CYP153A35, and putidaredoxin reductase (CamA), i.e., A35-AB2, showed the highest product yield from 5 mM palmitic acid for 9 h in batch reaction owing to the concentration of CamB, which is the rate-limiting factor for the activity of CYP153A35. However, in fed-batch reaction, A35-AB1, which expressed the three proteins individually using three T7 promoters, resulted with the highest product yield of 17.0 mM (4.6 g/L) ω-hydroxy palmitic acid from 20 mM (5.1 g/L) palmitic acid for 30 h.     

Regio- and stereo-selective 1β-hydroxylation of lithocholic acid by cytochrome P450 BM3 mutants

Regio- and stereo-selective hydroxylation of bile acids is a valuable reaction but often lacks suitable catalysts. In the research, semi-rational design in protein engineering techniques had been applied on cytochrome P450 monooxygenase CYP102A1 (P450 BM3) from Bacillus megaterium, and a mutation library had been set up for the 1β-hydroxylation of lithocholic acid (LCA) to produce 1β-OH-LCA. After four rounds of mutagenesis, a key residue at W72 was identified to regulate the regio- and stereo-selectivity at C1 of LCA. A quadruple variant (G87A/W72T/A74L/L181M) was identified to reach 99.4% selectivity of 1β-hydroxylation and substrate conversion of 68.1% resulting in a 21.5-fold higher level of 1β-OH-LCA production than the template LG-23. Molecular docking indicated that introducing hydrogen bonds at W72 was responsible for enhancing selectivity and catalytic activity, which gave some insights into the structure-based understanding of Csp³-H activation by the developed P450 BM3 mutants.     

Substrate analog studies of the ω-regiospecificity of Mycobacterium tuberculosis cholesterol metabolizing cytochrome P450 enzymes CYP124A1, CYP125A1 and CYP142A1

We report the synthesis and evaluation of a series of cholesterol side-chain analogs as mechanistic probes of three important Mycobacterium tuberculosis cytochrome P450 enzymes that selectively oxidize the ω-position of the methyl-branched cholesterol side-chain. To probe the structural requirements for the thermodynamically disfavored ω-regiospecificity we compared the binding of these substrate analogs to each P450, determined the turnover rates, and characterized the enzymatic products. The results are discussed in the context of the structure-activity relationships of the enzymes and how their active sites enforce ω-oxidation.     

Inhibition and inactivation of cytochrome P450 2A6 and cytochrome P450 2A13 by menthofuran, β-nicotyrine and menthol

Nicotine is the primary addictive agent in tobacco products and is metabolized in humans by CYP2A6. Decreased CYP2A6 activity has been associated with decreased smoking. The extrahepatic enzyme, CYP2A13 (94% identical to CYP2A6) also catalyzes the metabolism of nicotine, but is most noted for its role in the metabolic activation of the tobacco specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, the inhibition and potential inactivation of CYP2A6 and CYP2A13 by two tobacco constituents, 1-methyl-4-(3-pyridinyl) pyrrole (β-nicotyrine) and (-)-menthol were characterized and compared to the potent mechanism based inactivator of CYP2A6, menthofuran. The effect of these compounds on CYP2A6 and CYP2A13 activity was significantly different. (-)-Menthol was a more efficient inhibitor of CYP2A13 than of CYP2A6 (KI, 8.2 μM and 110 μM, respectively). β-Nicotyrine was a potent inhibitor of CYP2A13 (KI, 0.17 μM). Neither menthol nor β-nicotyrine was an inactivator of CYP2A13. Whereas, β-nicotyrine was a mechanism based inactivator of CYP2A6 (KI(inact), 106 μM, kinact was 0.61 min(-1)). Similarly, menthofuran, a potent mechanism based inactivator of CYP2A6 did not inactivate CYP2A13. Menthofuran was an inhibitor of CYPA13 (KI, 1.24 μM). The inactivation of CYP2A6 by either β-nicotyrine or menthofuran was not due to modification of the heme and was likely due to modification of the apo-protein. These studies suggest that β-nicotyrine, but not menthol may influence nicotine and NNK metabolism in smokers.     

Characterization of the physiological turnover of native and inactivated cytochromes P450 3A in cultured rat hepatocytes: a role for the cytosolic AAA ATPase p97?

Mammalian hepatic cytochromes P450 (P450s) are endoplasmic reticulum (ER)-anchored hemoproteins engaged in the metabolism of numerous xeno- and endobiotics. P450s exhibit widely ranging half-lives, utilizing both autophagic-lysosomal (ALD) and ubiquitin-dependent 26S proteasomal (UPD) degradation pathways. Although suicidally inactivated hepatic CYPs 3A and "native" CYP3A4 in Saccharomyces cerevisiae are degraded via UPD, the turnover of native hepatic CYPs 3A in their physiological milieu has not been elucidated. Herein, we characterize the degradation of native, dexamethasone-inducible CYPs 3A in cultured primary rat hepatocytes, using proteasomal (MG-132 and MG-262) and ALD [NH4Cl and 3-methyladenine (3-MA)] inhibitors to examine their specific degradation route. Pulse-chase with immunoprecipitation analyses revealed a basal 52% 35S-CYP3A loss over 6 h, which was stabilized by both proteasomal inhibitors. By contrast, no corresponding CYP3A stabilization was detected with either ALD inhibitor NH4Cl or 3-MA. Furthermore, MG-262-induced CYP3A stabilization was associated with its polyubiquitylation, thereby verifying that native CYPs 3A were also degraded via UPD. To identify the specific participants in this process, cellular proteins were cross-linked in situ with paraformaldehyde (PFA) in cultured hepatocytes. Immunoblotting analyses of CYP3A immunoprecipitates after PFA-cross-linking revealed the presence of p97, a cytosolic AAA ATPase instrumental in the extraction and delivery of ubiquitylated ER proteins for proteasomal degradation. Such native CYP3A-p97 interactions were greatly magnified after CYP3A suicidal inactivation (which accelerates UPD), and/or proteasomal inhibition, and were confirmed by proteomic and confocal immunofluorescence microscopic analyses. These findings clearly reveal that native CYPs 3A undergo UPD and implicate a role for p97 in this process.     

Allosteric activation of cytochrome P450 3A4 by α-naphthoflavone: branch point regulation revealed by isotope dilution analysis

Cytochrome P450 3A4 (CYP3A4) is the dominant xenobiotic metabolizing CYP. Despite great interest in CYP enzymology, two in vitro aspects of CYP3A4 catalysis are still not well understood, namely, sequential metabolism and allosteric activation. We have therefore investigated such a system in which both phenomena are present. Here we report that the sequential metabolism of Nile Red (NR) is accelerated by the heterotropic allosteric effector α-naphthoflavone (ANF). ANF increases the rates of formation for NR metabolites M1 and M2 and also perturbs the metabolite ratio in favor of M2. Thus, ANF has as an allosteric effect on a kinetic branch point. Co-incubating deuterium-labeled NR and unlabeled M1, we show that ANF increases k(cat)/k(off) ~1.8-fold in favor of the k(cat) of M2 production. Steady-state metabolic experiments are analyzed using a kinetic model in which the enzyme and substrates are not in rapid equilibrium, and this distinction allows for the estimation of rates of catalysis for the formation of both the primary (M1) and secondary (M2) products, as well as the partitioning of enzyme between these states. These results are compared with those of earlier spectroscopic investigations of NR and ANF cooperativity, and a mechanism of ANF heteroactivation is presented that involves effects on substrate off rate and coupling efficiency.     

A new regulation of IL-6 production in adult cardiomyocytes by β-adrenergic and IL-1β receptors and induction of cellular hypertrophy by IL-6 trans-signalling

The sympathetic nervous system and pro-inflammatory cytokines play key roles in numerous cardiovascular disorders. Chronic β-adrenergic receptor (β-AR) stimulation in myocardium induces expression of pro-inflammatory cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6), which contribute to cardiac hypertrophy and failure. To evaluate the relationship between β-AR stimulation and pro-inflammatory cytokines, we studied the effects of the β-AR agonist isoprenaline (ISO) on IL-1-induced IL-6 production in adult rat ventricular myocytes (ARVMs). We report that ISO and IL-1 synergistically enhanced IL-6 gene expression and secretion. The synergistic effect of ISO was mimicked by cAMP elevating agents and involved the G_s protein/cAMP/PKA signalling pathway, but not the exchange factor EPAC. To evaluate the contribution of IL-6 to cellular hypertrophy, we examined the signalling pathways stimulated by the membrane-bound IL-6 receptor (IL-6R), and the IL-6 soluble receptor (sIL-6R) involved in the mechanism named IL-6 trans-signalling. The IL-6/sIL-6R complex promoted a rapid and persistent phosphorylation of STAT3^Tyr705 in ARVMs. Moreover, IL-6 trans-signalling increased protein synthesis, c-fos gene expression and B-type natriuretic peptide secretion, three markers of cardiac hypertrophy. IL-6 trans-signalling also increased cell size. In contrast, IL-6 alone had no significant effect on either cell size or STAT3 phosphorylation although it induced phosphorylation of ERK1/2, AKT and S6K, demonstrating the presence of a functional IL-6R in ARVMs. Taken together, these results demonstrate that β-AR stimulation synergises with IL-1 for IL-6 secretion in adult ventricular myocytes and indicate that IL-6 induces cardiac hypertrophy only via IL-6 trans-signalling. The IL-6 soluble receptor may thus serve as a switch for IL-6 to activate STAT3 phosphorylation and hypertrophy.     

A new insight into the role of rat cytochrome P450 24A1 in metabolism of selective analogs of 1α,25-dihydroxyvitamin D₃

We examined the metabolism of two synthetic analogs of 1α,25-dihydroxyvitamin D? (1), namely 1α,25-dihydroxy-16-ene-23-yne-vitamin D? (2) and 1α,25-dihydroxy-16-ene-23-yne-26,27-dimethyl-vitamin D? (4) using rat cytochrome P450 24A1 (CYP24A1) in a reconstituted system. We noted that 2 is metabolized into a single metabolite identified as C26-hydroxy-2 while 4 is metabolized into two metabolites, identified as C26-hydroxy-4 and C26a-hydroxy-4. The structural modification of adding methyl groups to the side chain of 1 as in 4 is also featured in another analog, 1α,25-dihydroxy-22,24-diene-24,26,27-trihomo-vitamin D? (6). In a previous study, 6 was shown to be metabolized exactly like 4, however, the enzyme responsible for its metabolism was found to be not CYP24A1. To gain a better insight into the structural determinants for substrate recognition of different analogs, we performed an in silico docking analysis using the crystal structure of rat CYP24A1 that had been solved for the substrate-free open form. Whereas analogs 2 and 4 docked similar to 1, 6 showed altered interactions for both the A-ring and side chain, despite prototypical recognition of the CD-ring. These findings hint that CYP24A1 metabolizes selectively different analogs of 1, based on their ability to generate discrete recognition cues required to close the enzyme and trigger the catalytic mechanism.     

Inhibition by triptoquinone-A of LPS- and IL-1β-primed induction of no synthase in rat thoracic aorta

We investigated the effect of triptoquinone-A (TQA), an active principal of Triptergium wilfordii, on the induction of nitric oxide synthase (NOS) promoted by endotoxin (LPS) and interleukin-1β (IL-1β). Prophylactic application of TQA selectively prevented LPS-primed initiation of L-arginine (Arg)-induced relaxation, and cGMP formation of rat thoracic aorta, and LPS-stimulated nitrite production by cultured aortic smooth muscle cells, which appear to be mediated by NOS expressed by LPS in vascular smooth muscle. TQA also prevented IL-1β-triggered initiation of Arg-induced relaxation and nitrite accumulation. These results suggest that TQA prevents LPS- or IL-1β-promoted induction of NOS in vascular smooth muscle, thus inhibiting development of Arg-induced vasorelaxation.     

6α-Hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The V_max for 6α-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent K_m was 90 μM. Cytochrome b₅ was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6α-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r²=0.97) and testosterone 6β-hydroxylation (r²=0.9). There was also a strong correlation between 6α-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6β-hydroxylation (r²=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6α-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 μM concentration. Other inhibitors, such as α-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent K_m for 6α-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 μM). This might give an explanation for the limited formation of 6α-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver.     

Action of E. coli endotoxin,IL-1β and TNF-α on antioxidant status of cultured hepatocytes

We have previously reported that endotoxin induces in vivo oxidative stress in liver and a significant increase in hepatic and plasma glutathione concentrations during the acute phase of reversible endotoxic shock in rats. In the present study we examined the in vitro effects of E. coli 0111:B4 endotoxin (lipopolysaccharide, LPS), IL-1 and TNF- on antioxidant status of cultured hepatocytes in order to differentiate between the direct and mediated endotoxin action. LPS increased total glutathione (tGSH) levels after 2 h treatment but decreased oxidized glutathione (GSSG) content which lead to a marked decrease in GSSG/tGSH index. At shorter treatment times a biphasic and dose-dependent behaviour was observed. Cytokines (IL-1 and TNF-) produced significant decreases in tGSH and GSSG after 30 min treatment. Despite its prooxidant effect, TNF- significantly reduced GSSG/tGSH index. Although no significant effects were observed on glutathione reductase activity, both LPS and cytokines induced an important inhibition of glutathione peroxidase which can justify the lipid peroxidation previously observed both in liver during reversible endotoxic shock and in cultured hepatocytes after treatment with endotoxin. The inhibition of hepatic glutathione peroxidase, besides the stimulation of GSH synthesis by LPS and GSH efflux by cytokines, guarantees the export of hepatic glutathione in its reduced form for other organs, contributing to the interorgan homeostasis. On the other hand, the results presented here support a new role for GSSG/tGSH index different from a mere indicator of oxidative stress.     

Hypertension, cardiovascular risk and polymorphisms in genes controlling the cytochrome P450 pathway of arachidonic acid: A sex-specific relation?

Hypertension is a multifactorial disease in which the interplay of genetic and environmental factors that maintain blood pressure stable throughout life is altered. Cytochrome P450 (CYP)-derived metabolites of arachidonic acid such as epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE), active on vascular tone, endothelial function and renal sodium reapportion, have been identified as candidate mediators in the development of hypertension in several animal models, with remarkable sex-specific effect. Several SNPs, some recognized as functional, in human genes implicated in EETs/20-HETE biosynthesis and metabolism, such as CYP2J2 and CYP4A11, have been tested for association with blood pressure, hypertension and its long-term cardiovascular consequences in different populations, with conflicting results. A sex-specific effect, related to CYP4F2 polymorphisms and expression, has been observed in association studies. This finding indicates that altered 20-HETE bioactivity underlay the excess of hypertension and associated vascular events observed in men with respect to women and is consistent with the results from experimental models. Further epidemiological and mechanistic studies are required to confirm the effect of lipid mediators on blood pressure in humans and define the mechanisms of a putative sex-specific effect.     

The relationship between δ-aminolaevulinate synthetase induction and the concentrations of cytochrome P-450 and catalase in rat liver

The porphyrinogenic drug 2-allyl-2-isopropylacetamide causes the degradation of microsomal cytochrome P-450 and inhibits the synthesis of catalase in rat liver. The inhibition of catalase synthesis follows the induction of delta-aminolaevulinate synthetase and the consequent overproduction of haem. The allylisopropylacetamide-mediated breakdown of cytochrome P-450 is a rapid event and has a reciprocal relationship to the pattern of delta-aminolaevulinate synthetase induction. Breakdown of cytochrome P-450 appears to be one of the conditions leading to the ;derepression' of delta-aminolaevulinate synthetase.     

A new insight into the role of rat cytochrome P450 24A1 in metabolism of selective analogs of 1α,25-dihydroxyvitamin D3

We examined the metabolism of two synthetic analogs of 1α,25-dihydroxyvitamin D₃ (1), namely 1α,25-dihydroxy-16-ene-23-yne-vitamin D₃ (2) and 1α,25-dihydroxy-16-ene-23-yne-26,27-dimethyl-vitamin D₃ (4) using rat cytochrome P450 24A1 (CYP24A1) in a reconstituted system. We noted that 2 is metabolized into a single metabolite identified as C26-hydroxy-2 while 4 is metabolized into two metabolites, identified as C26-hydroxy-4 and C26a-hydroxy-4. The structural modification of adding methyl groups to the side chain of 1 as in 4 is also featured in another analog, 1α,25-dihydroxy-22,24-diene-24,26,27-trihomo-vitamin D₃ (6). In a previous study, 6 was shown to be metabolized exactly like 4, however, the enzyme responsible for its metabolism was found to be not CYP24A1. To gain a better insight into the structural determinants for substrate recognition of different analogs, we performed an in silico docking analysis using the crystal structure of rat CYP24A1 that had been solved for the substrate-free open form. Whereas analogs 2 and 4 docked similar to 1, 6 showed altered interactions for both the A-ring and side chain, despite prototypical recognition of the CD-ring. These findings hint that CYP24A1 metabolizes selectively different analogs of 1, based on their ability to generate discrete recognition cues required to close the enzyme and trigger the catalytic mechanism.     

FTIR studies of the redox partner interaction in cytochrome P450: The Pdx–P450cam couple

Recently we have developed a new approach to study protein–protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam–Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP–FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in β-sheets and α-helix content, a decrease in the population of random coil/3₁₀-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam–Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx–P450cam complex.     

Rapid loss of δ-aminolevulinic acid dehydratase activity in primary cultures of adult rat hepatocytes: A new model of zinc deficiency

Nonproliferating cultures of adult rat hepatocytes were found to lose 60–70% of cell-associated zinc during their first 24 h of incubation in standard, serum-free medium. The loss of zinc was accompanied by a profound loss (95%) in the activity of the zinc metalloenzyme, δ-aminolevulinic acid dehydratase, as well as a loss (>85%) in the cellular content of immunoreactive δ-aminolevulinic acid dehydratase protein. Restoration of cellular zinc content by the addition of zinc to the culture medium partially prevented the losses of both δ-aminolevulinic acid dehydratase activity and immunoreactive protein. Since the spontaneous, selective loss of cellular zinc appears to have specific effects on a relevant hepatic function, this culture system constitutes a novel $\text{in}$$\text{vitro}$ model of zinc deficiency in mature liver.