Role of glutamic acid 216 in cytochrome P450 2D6 substrate binding and catalysis

Human cytochrome P450 (P450) 2D6 is an important enzyme involved in the metabolism of drugs, many of which are amines or contain other basic nitrogen atoms. Asp301 has generally been considered to be involved in electrostatic docking with the basic substrates, on the basis of previous modeling studies and site-directed mutagenesis. Substitution of Glu216 with a residue other than Asp strongly attenuated the binding of quinidine, bufuralol, and several other P450 2D6 ligands. Catalytic activity with the substrates bufuralol and 4-methoxyphenethylamine was strongly inhibited by neutral or basic mutations at Glu216 (>95%), to the same extent as the substitution of Asn at Asp301. Unlike the Asp301 mutants, the Gln216 mutant (E216Q) retained 40% enzyme efficiency with the substrate spirosulfonamide, devoid of basic nitrogen, suggesting that the substitutions at Glu216 affect binding of amine substrates more than other catalytic steps. Attempts to induce catalytic specificity toward new substrates by substitutions at Asp301 and Glu216 were unsuccessful. Collectively, the results provide evidence for electrostatic interaction of amine substrates with Glu216, and we propose that both of these acidic residues plus at least another residue(s) is (are) involved in binding the repertoire of P450 2D6 ligands.

Acute cadmium chloride administration induces hepatic and renal CYP2A5 mRNA, protein and activity in the mouse: involvement of transcription factor NRF2

Modulation of the cytochrome P450 (CYP) monooxygenase system by cadmium was investigated in male, adult DBA/2J mice treated with a single dose (16 mumol/kg body weight, i.p.) of cadmium chloride (CdCl2). Total CYP content of liver and kidney microsomes decreased maximally (56% and 85%, respectively) 24 and 18 h, respectively, after CdCl2 treatment. Progressive increases of hepatic coumarin 7-hydroxylase (COH) activity; indicative of CYP2A5 activity, relative to the total CYP content were seen at 8 h (2-fold), 12 h (3-fold), 18 h (12-fold), and 24 h (15-fold). Similar changes were seen in the kidney. Liver and kidney CYP2A5 mRNA levels increased maximally 12 and 4 h after treatment and decreased to almost half 6 h later. In contrast, kidney and liver CYP2A5 protein levels increased maximally at 18 and 24 h. The CYP2A5 mRNA levels in the kidney and liver increased after Cd treatment in Nrf2 +/+ but not in Nrf2 -/- mouse. This study demonstrates that hepatic and kidney CYP2A5 is upregulated by cadmium with a somewhat faster response in the kidney than the liver. The strong upregulation of the CYP2A5 both at mRNA and enzyme activity levels, with a simultaneous decrease in the total CYP concentration suggest an unusual mode of regulation of CYP2A5 in response to cadmium exposure, amongst the CYP enzymes. The observed decrease in the mRNA but not in protein levels after maximal induction may suggest involvement of post-trancriptional mechanisms in the regulation. Upregulation of CYP2A5 by cadmium in the Nrf2 +/+ mice but not in the Nrf2 -/- mice indicates a role for this transcription factor in the regulation. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

A commentary on the impacts of metals and metalloids in the environment upon the metabolism of drugs and chemicals

The salient feature of metals is that unlike organic compounds they do not degrade in the environment and barely move from one environmental matrix to another. Human interventions take these compounds from their stable and non-bioavailable geological matrix into situations of biological accessibility. Studies in the 1970s and the 1980s of metal bioavailability and impacts of metals and metalloids were driven by the process of abatement of lead in the environment. Humans have clear and identifiable sources of exposure from fuels, food and leaded water pipes to lead. Interventions started at that time have dramatically lowered human lead exposure. Attention has now shifted to other metals, in particular, cadmium, which has seen increasing use. It is generally accepted that food crops grown on cadmium containing soils or soils naturally rich in this metal are the major source of exposure to humans other than exposure from smoking of cigarettes. This mini-review gives a summary and commentary on early studies on effects of lead on haem metabolism that provide us the clue to why investigations of the impacts of other toxic heavy metals and metalloids such as cadmium and arsenic on different human cytochrome P450 forms have become of great interest at the current time. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Reduced hepatic extraction of palmitate in steatosis correlated to lower level of liver fatty acid binding protein

Nonalcoholic fatty liver disease is the most common of all liver diseases. The hepatic disposition [H-3]palmitate and its low-molecular-weight metabolites in perfused normal and steatotic rat liver were studied using the multiple indicator dilution technique and a physiologically based slow diffusion/bound pharmacokinetic model. The steatotic rat model was established by administration of 17alpha-ethynylestradiol to female Wistar rats. Serum biochemistry markers and histology of treated and normal animals were assessed and indicated the presence of steatosis in the treatment group. The steatotic group showed a significantly higher alanine aminotransferase-to-aspartate aminotransferase ratio, lower levels of liver fatty acid binding protein and cytochrome P-450, as well as microvesicular steatosis with an enlargement of sinusoidal space. Hepatic extraction for unchanged [H-3]palmitate and production of low-molecular-weight metabolites were found to be significantly decreased in steatotic animals. Pharmacokinetic analysis suggested that the reduced extraction and sequestration for palmitate and its metabolites was mainly attributed to a reduction in liver fatty acid binding protein in steatosis.

Promoter-, cell-, and ligand-specific transactivation responses of the VDRB1 isoform

The vitamin D receptor (VDR) mediates the effects of 1,25(OH)(2)D-3, the active form of vitamin D. The human VDRB1 isoform differs from the originally described VDR by an N-terminal extension of 50 amino acids. Here we investigate cell-, promoter-, and ligand-specific transactivation by the VDRB1 isoform. Transactivation by these isoforms of the cytochrome P450 CYP24 promoter was compared in kidney (HEK293 and COS1), tumor-derived colon (Caco-2, LS174T, and HCT15), and mammary (HS578T and MCF7) cell lines. VDRB1 transactivation in response to 1,25(OH)(2)D-3 was greater in Cost and HCT15 cells (145%), lower in HEK293 and Caco-2 cells (70-85%) and similar in other cell lines tested. By contrast, on the cytochrome P450 CYP3A4 promoter, 1,25(OH)(2)D-3-induced VDRB1 transactivation was significantly lower than VDRA in Caco-2 (68%), but comparable to VDRA in HEK293 and COS1 cells. Ligand-dependence of VDRB1 differential transactivation was investigated using the secondary bile acid lithocholic acid (LCA). On the CYP24 promoter LCA-induced transactivation was similar for both isoforms in COS1, whereas in Caco-2 and HEK293 cells VDRB1 was less active. On the CYP3A4 promoter, LCA activation of VDRB1 was comparable to VDRA in all the cell lines tested. Mutational analysis indicated that both the 1,25(OH)(2)D-3 and LCA-regulated activities of both VDR isoforms required a functional ligand-dependent activation function (AF-2) domain. In gel shift assays VDR:DNA complex formation was stronger in the presence of 1,25(OH)(2)D-3 than with LCA. These results indicate that regulation of VDRB1 transactivation activity is dependent on cellular context, promoter, and the nature of the ligand. (c) 2005 Elsevier Inc. All rights reserved.

Hepatic pharmacokinetics of propranolol in rats with adjuvant-induced systemic inflammation

Systemic inflammation is known to affect drug disposition in the liver. This study sought to relate and quantitate changes in hepatic pharmacokinetics of propranolol with changes in hepatic architecture and physiology in adjuvant-treated rats. Transmission electron microscopy was used to assess morphological changes in mitochondria and lysosomes of adjuvant-treated rat livers. The disposition of propranolol was assessed in the perfused rat liver using the multiple indicator dilution technique. Hepatic extraction and mean transit time were determined from outflow-concentration profiles using a nonparametric method. Kinetic parameters were derived from a two-phase physiologically based organ pharmacokinetic model. Possible relationships were then explored between the changes in hepatic drug disposition and cytochrome P-450 activity and iron concentration. Adjuvant treatment induced the appearance of mitochondrial inclusions/tubularization and irregularly shaped lysosomes in rat livers. Livers from adjuvant-treated rats had (relative to normal) significantly higher alpha(1)-acid glycoprotein (orosomucoid) and iron tissue concentrations but lower cytochrome P-450 content. The hepatic extraction, metabolism, and ion trapping of propranolol were significantly impaired in adjuvant-treated rats and could be correlated with altered iron store and cytochrome P-450 activity. It is concluded that adjuvant-induced systemic inflammation alters hepatocellular morphology and biochemistry and consequently influences hepatic disposition of propranolol.

Reactions catalyzed by bacterial cytochromes P450

The cytochromes P450 are a large family of oxidative haemoproteins that are responsible for a wide variety of oxidative transformations in a variety of organisms. This review focuses upon the reactions catalyzed specifically by bacterial enzymes, which includes aliphatic hydroxylation, alkene epoxidation, aromatic hydroxylation, oxidative phenolic coupling, heteroatom oxidation and dealkylation, and multiple oxidations including C-C bond cleavage. The potential for the practical application of the oxidizing power of these enzymes is briefly discussed.

Crystal structure of P450cin in a complex with its substrate, 1,8-cineole, a close structural homologue to D-camphor, the substrate for P450cam

Cytochrome P450cin catalyzes the monooxygenation of 1,8-cineole, which is structurally very similar to D-camphor, the substrate for the most thoroughly investigated cytochrome P450, cytochrome P450cam. Both 1,8-cineole and D-camphor are C-10 monoterpenes containing a single oxygen atom with very similar molecular volumes. The cytochrome P450cin-substrate complex crystal structure has been solved to 1.7 Angstrom resolution and compared with that of cytochrome P450cam. Despite the similarity in substrates, the active site of cytochrome P450cin is substantially different from that of cytochrome P450cam in that the B' helix, essential for substrate binding in many cytochrome P450s including cytochrome P450cam, is replaced by an ordered loop that results in substantial changes in active site topography. In addition, cytochrome P450cin does not have the conserved threonine, Thr252 in cytochrome P450cam, which is generally considered as an integral part of the proton shuttle machinery required for oxygen activation. Instead, the analogous residue in cytochrome P450cin is Asn242, which provides the only direct protein H-bonding interaction with the substrate. Cytochrome P450cin uses a flavodoxin-like redox partner to reduce the heme iron rather than the more traditional ferredoxin-like Fe2S2 redox partner used by cytochrome P450cam and many other bacterial P450s. It thus might be expected that the redox partner docking site of cytochrome P450cin would resemble that of cytochrome P450BM3, which also uses a flavodoxin-like redox partner. Nevertheless, the putative docking site topography more closely resembles cytochrome P450cam than cytochrome P450BM3.

Steatosis in chronic hepatitis C: Association with increased messenger RNA expression of collagen I, tumor necrosis factor-α and cytochrome P450 2E1

Background: Increased levels of tumor necrosis factor (TNF)-alpha and oxidative stress have been implicated as factors contributing to hepatic injury in fatty liver diseases. As steatosis is associated with an accelerated progression of fibrosis in chronic hepatitis C (HCV), we hypothesized that the messenger (m)RNA expression of genes involved with the production of reactive oxygen species, inflammation and cellular injury would be increased in liver tissue from subjects with steatosis and chronic HCV. Methods: Real-time polymerase chain reaction was performed to determine relative mRNA expression levels of collagen I, TNF-alpha, cytochrome P450 2E1 (CYP 2E1), transforming growth factor-beta1 and CD14 in liver biopsies from 38 patients with chronic HCV. The mRNA expression levels were compared between subjects with and without steatosis, fibrosis, and inflammation. Results: Multivariate analysis demonstrated that collagen I mRNA expression was increased by 199% in steatosis (P = 0.02), 85% in moderate to severe fibrosis (P = 0.02) and 157% in inflammation (P = 0.03). Livers of patients with steatosis also had an increase in TNF-alpha mRNA expression by 50% (P = 0.03) and CYP 2E1 expression by 37% (P = 0.04) compared with non-steatotic livers. Tumor necrosis factor-alpha protein was localized to Kupffer cells, bile ducts and portal inflammatory cells by immunohistochemistry. Conclusion: Increased expression of TNF-alpha may be involved in the pathogenesis of liver injury and progression of fibrosis in individuals who have steatosis in association with chronic HCV. (C) 2003 Blackwell Publishing Asia Pty Ltd.

Molecular modelling of human CYP1B1 substrate interactions and investigation of allelic variant effects on metabolism

Molecular modelling of human CYP1B1 based on homology with the mammalian P450, CYP2C5, of known three-dimensional structure is reported. The enzyme model has been used to investigate the likely mode of binding for selected CYP1B1 substrates, particularly with regard to the possible effects of allelic variants of CYP1B1 on metabolism. In general, it appears that the CYP1B1 model is consistent with known substrate selectivity for the enzyme, and the sites of metabolism can be rationalized in terms of specific contacts with key amino acid residues within the CYP1B1 heme locus. Further-more, a mode of binding interaction for the inhibitor, a-naphthoflavone, is presented which accords with currently available information. The current paper shows that a combination of molecular modelling and experimental determinations on the substrate metabolism for CYP1B1 allelic variants can aid in the understanding of structure-function relationships within P450 enzymes. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Cytochrome c(551) from Starkeya novella - Characterization, spectroscopic properties, and phylogeny of a diheme protein of the SoxAX family

Cytochromes from the SoxAX family have a major role in thiosulfate oxidation via the thiosulfate-oxidizing multi-enzyme system (TOMES). Previously characterized SoxAX proteins from Rhodovulum sulficlophilum and Paracoccus pantotrophus contain three heme c groups, two of which are located on the SoxA subunit. In contrast, the SoxAX protein purified from Starkeya novella was found to contain only two heme groups. Mass spectrometry showed that a disulfide bond replaced the second heme group found in the diheme SoxA subunits. Apparent molecular masses of 27,229 +/- 10.3 Da and 20,258.6 +/- 1 Da were determined for SoxA and SoxX with an overall mass of 49.7 kDa, indicating a heterodimeric structure. Optical redox potentiometry found that the two heme cofactors are reduced at similar potentials (versus NHE) that are as follows: + 133 mV (pH 6.0); + 104 mV (pH 7.0); +49 (pH 7.9) and +10 mV (pH 8.7). EPR spectroscopy revealed that both ferric heme groups are in the low spin state, and the spectra were consistent with one heme having a His/Cys axial ligation and the other having a His/Met axial ligation. The His/Cys ligated heme is present in different conformational states and gives rise to three distinct signals. Amino acid sequencing was used to unambiguously assign the protein to the encoding genes, soxAX, which are part of a complete sox gene cluster found in S. novella. Phylogenetic analysis of soxA- and soxX-related gene sequences indicates a parallel development of SoxA and SoxY, with the diheme and monoheme SoxA sequences located on clearly separated branches of a phylogenetic tree.

Acute cadmium chloride administration induces hepatic and renal cytochrome P450 2A5 in the mouse

Modulation of the cytochrome P450 (CYP) monooxygenase system by cadmium was investigated in male, adult DBA/2J mice treated with a single dose (16 Amol/kg body weight, i.p.) of cadmium chloride (CdCl2) at various time points. The total CYP content of kidney microsomes started to decrease 4 hours earlier than in the liver (P < 0.05), with maximal decreases at 24 hours of 56% and 85% in the liver and kidney, respectively. In contrast, both hepatic and renal coumarin 7-hydroxylase (COH) activity (indicative of CYP2A5 activity) relative to total CYP content started to progressively increase at 8 hours, with renal activity 61 times higher than the hepatic activity. Maximum increases were observed, 15-fold in the liver and 64-fold in the kidney after 24 hours. Liver and kidney CYP2A5 mRNA levels increased maximally 12 and 4 hours after treatment, respectively and decreased to almost half 6 hours later. In contrast, kidney and liver CYP2A5 protein levels increased maximally at 18 and 24 hours. This study demonstrates that hepatic and renal CYP2A5 is upregulated by cadmium with a faster response in the kidney than in the liver. This observation is concordant with the fact that kidney is the target organ for cadmium toxicity. The observed increase in the mRNA but not in protein levels after maximal induction suggests involvement of post-transcriptional mechanisms in the regulation of CYP2A5 expression by cadmium.