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Biochemical Pharmacology (v.70, #10)

Editorial Advisory Board (pp. iii).

Inactivators in competition by Jean-Marie Fr�re; Pierre Marchot (pp. 1417-1423).

A method is described to determine the values of the equilibrium ( K) and rate ( k₂) constants for enzyme inactivations which occur according to two-step pathways involving a first non-covalent complex and a covalent, irreversibly inactivated adduct.The method rests on a competition between a reference compound [R] for which the k₂ and K values are already known and another inactivator [C]. During the experiments, the disappearance of the reference compound or the appearance of the ER^* adduct is monitored. The analysis shows that under conditions where the k₂ and K values for the competing substrate can be determined, the measured apparent first-order rate constant for the disappearance of the reference compound is not the sum of the rate constants obtained for each inactivator in the absence of the other.The method can be used to determine the K and k₂ constants when an adequate reference compound is available, in particular, for the interactions between β-lactam antibiotics and penicillin-binding proteins. The precautions which must be taken to avoid large errors on the estimation of the parameters of the competing inactivator are discussed.Examples found in the literature are discussed where an erroneous simplified equation has been utilised, thus yielding incorrect values for k₂ and K. Interestingly, the correct values can be calculated on the basis of the published results which do not contain the raw experimental data. But some of the values should be considered with a lot of caution since the experiments have not been performed under optimal conditions.

Keywords: Enzyme inactivation; Competition; β-Lactams; Kinetics; Staphylococcus aureus; BlaR; Staphylococcus aureus; PBP2a

Modulation of MDR1 gene expression in multidrug resistant MCF7 cells by low concentrations of small interfering RNAs by V�r�ne Stierl�; Alain Laigle; B�atrice Joll�s (pp. 1424-1430).

MDR1 overexpression is one form of the multidrug resistance (MDR) phenotype, which can be acquired by patients initially responsive to chemotherapy. Because of the high toxicity of the inhibitors of P-glycoprotein (P-gp), the protein encoded by MDR1, attention has been focused on selective modulation of the MDR1 gene. Small interfering RNAs (siRNAs) were shown to be powerful tools for such a purpose, even when used at low concentrations (≤20nM) in order to avoid sequence nonspecific effects. Two siRNAs used at 20nM were shown to lead to efficient down-regulation of MDR1 at the protein level (only ca. 20% total P-gp expression remaining) in the doxorubicin selected MCF7-R human cell line. Cell surface expression of P-gp was inhibited, leading to reversal of the drug efflux phenotype (about 40% reversal with the most efficient siRNA) and enhancement of chemosensitivity (about 35%). At the mRNA level, the down-regulation of MDR1 obtained with the most efficient siRNA increased from about 50% (5nM siRNA) to 60% (10 or 20nM). The advantage of using a combination of siRNAs instead of a single one has been suggested.

Keywords: Multidrug resistance; Small interfering RNA; P-gp expression; MDR1; silencing; Low concentrations of siRNA; Combination of siRNAs

Effects of anthracycline derivatives on human leukemia K562 cell growth and differentiation by Malgorzata Czyz; Agata Szulawska; Andrzej K. Bednarek; Markus D�chler (pp. 1431-1442).

New derivatives of daunorubicin (DRB), doxorubicin (DOX), and epidoxorubicin (EDOX) with an amidine group bonded to C-3′ of daunosamine moiety with either morpholine or hexamethyleneimine ring attached to the amidine group are studied in this paper. We have shown that all of these newly synthesized anthracycline derivatives inhibit human leukemia K562 cell line proliferation but only some of them induce erythroid differentiation when used at subtoxic concentrations. Morpholine derivative of DOX has the greatest potential to inhibit proliferation and to induce differentiation in vitro. The correlation between these two cellular processes was also significant for other tested compounds. In cell cycle analysis, we have demonstrated that those anthracycline derivatives that exert the greatest cytostatic potential caused G₂/M arrest, which in turn, might contribute to the development of a differentiating phenotype. The concentrations of the compounds used in the study are pharmacologically relevant. These new potent inducers of differentiation might be exploited as anticancer drugs for treatment of leukemia by differentiation therapy.

Keywords: Abbreviations; ACLA; aclarubicin; CML; chronic myelogenous leukemia; DOX; doxorubicin; DOXH; hexamethyleneimine derivative of doxorubicin; DOXM; morpholine derivative of doxorubicin; DRB; daunorubicin; DRBH; hexamethyleneimine derivative of daunorubicin; DRBM; morpholine derivative of daunorubicin; EDOX; epidoxorubicin; EDOXM; morpholine derivative of epidoxorubicin; TPA; 12-; O; -tetradecanoyl-phorbol 13-acetateAnticancer drugs; Anthracycline derivatives; Erythroid differentiation; Growth arrest; K562 cells; Differentiation therapy

Honokiol inhibits TNF-α-stimulated NF-κB activation and NF-κB-regulated gene expression through suppression of IKK activation by Anfernee Kai-Wing Tse; Chi-Keung Wan; Xiao-Ling Shen; Mengsu Yang; Wang-Fun Fong (pp. 1443-1457).

Honokiol, a small molecular weight lignan originally isolated from Magnolia officinalis, shows anti-angiogenic, anti-invasive and anti-proliferative activities in a variety of cancers. In this study, we investigated whether honokiol affects the transcription factor nuclear factor-kappa B (NF-κB) which controls a large number of genes involved in angiogenesis, metastasis and cell survival. We observed that the tumor necrosis factor-alpha (TNF-α)-induced NF-κB activation was blocked by honokiol in four different cancer cell lines as evidenced by EMSA. Honokiol did not directly affect the NF-κB-DNA binding. Immunoblot experiments demonstrated that honokiol inhibited the TNF-α-stimulated phosphorylation and degradation of the cytosolic NF-κB inhibitor IκBα. Furthermore, honokiol suppressed the intrinsic and TNF-α-stimulated upstream IκB kinases (IKKs) activities measured by a non-radioactive kinase assay using immunoprecipitated IKKs, suggesting a critical role of honokiol in abrogating the phosphorylation and degradation of IκBα. In a HeLa cell NF-κB-dependent luciferase reporter system, honokiol suppressed luciferase expression stimulated by TNF-α and by the transient transfection and expression of NIK (NF-κB-inducing kinase), wild type IKKβ, constitutively active IKKα and IKKβ, or the p65 subunit. Honokiol was also found to inhibit the nuclear translocation and phosphorylation of p65 subunit of NF-κB. RT-PCR results showed that honokiol suppressed NF-κB-regulated inflammatory and carcinogenic gene products including MMP-9, TNF-α, IL-8, ICAM-1 and MCP-1. In line with the observation that NF-κB activation may up-regulate anti-apoptotic genes, it was shown that honokiol enhanced TNF-α-induced apoptotic cell death. In summary, our results demonstrate that honokiol suppresses NF-κB activation and NF-κB-regulated gene expression through the inhibition of IKKs, which provides a possible mechanism for its anti-tumor actions.

Keywords: Abbreviations; CHX; cycloheximide; EMSA; electrophoretic mobility shift assay; GST; glutathione; S; -transferase; ICAM-1; intercellular adhesion molecule-1; IL-8; interleukin-8; IκB; inhibitor subunit of NF-κB; IKK; IκB kinase; LPS; lipopolysaccharide; MCP-1; monocyte chemoattractant protein-1; MMP-9; matrix metalloproteinase-9; NF-κB; nuclear factor-kappa B; NIK; NF-κB-inducing kinase; PMA; phorbol myristate acetate; TNF-α; tumor necrosis factor-alpha; TUNEL; terminal deoxynucleotidyltransferase-mediated dUTP mick end labelingHonokiol; NF-κB; IκB; IKK; TNF-α; Cell signaling

Determination of apoptosis, uracil incorporation, DNA strand breaks, and sister chromatid exchanges under conditions of thymidylate deprivation in a model of BER deficiency by Li Li; Ellen E. Connor; Sondra H. Berger; Michael D. Wyatt (pp. 1458-1468).

Thymidylate synthase (TS) is an important target of several chemotherapeutic agents. During TS inhibition, dTTP levels decrease with a subsequent increase in dUTP. Uracil incorporated into the genome is removed by base excision repair (BER). BER has been hypothesized to play a role in the response to thymidylate deprivation, despite a lack of direct evidence. We previously found that β-pol null murine fibroblasts were ∼six-fold more resistant than wild-type cells to raltitrexed, a folate-based inhibitor specific for TS. In this study, a number of endpoints were determined to understand the influence of BER and β-pol during raltitrexed treatment. Raltitrexed induced apoptosis in wild-type cells to a greater extent than in β-pol null cells. A PARP inhibitor decreased the sensitivity to raltitrexed, although the extent was not different between wild-type and β-pol null cells. No evidence was seen for extensive strand break formation that preceded apoptosis, although raltitrexed induced more sister chromatid exchanges in wild-type cells. Increased levels of uracil in DNA were detected following treatment in wild-type and β-pol null cells. However, uracil levels were only ∼two-fold higher in DNA from treated cells compared to untreated. Uracil DNA glycosylase activity was slightly higher in β-pol null cells, although not sufficiently different to explain the difference in sensitivity to raltitrexed. Taken together, the data suggest that the sensitivity of the wild-type cells to raltitrexed is not associated with activation of PARP-1 dependent BER, extensive uracil incorporation into DNA and persistent strand breaks, but rather with changes suggestive of DNA recombination.

Keywords: Abbreviations; AP site; apurinic/apyrimidinic (abasic) site; 3-AB; 3-aminobenzamide; BER; base excision repair; 5-FU; 5-fluorouracil; FdUrd; 5-fluoro-2′-deoxyuridine; MEFs; murine embryonic fibroblasts; PARP; poly-ADP ribose polymerase; PFGE; pulsed-field gel electrophoresis; β-pol; DNA polymerase β; RTX; raltitrexed (Tomudex); SCE; sister chromatid exchange; TS; thymidylate synthase; UDG; uracil DNA glycosylaseRaltitrexed; DNA repair; Thymineless stress; Cancer chemotherapy; Base excision repair; DNA polymerase β

Inhibition of adipogenesis by RGD-dependent disintegrin by Yu-Ting Lin; Chih-Hsin Tang; Woei-Jer Chuang; Seu-Mei Wang; Tur-Fu Huang; Wen-Mei Fu (pp. 1469-1478).

Adipogenesis plays a central role in obesity development. The processes of adipogenesis include migration, adhesion, proliferation and survival of preadipocytes and differentiation to mature adipocytes. Many of these biological functions are related to integrins. Here, we found that snake venom-derived arginine–glycine–aspartic acid (RGD)-containing disintegrin inhibited adipogenesis. Rhodostomin but not rhodostomin RGD mutants (RGE-Rn and AKGDWN-Rn) caused the detachment of primary cultured preadipocyte. Furthermore, rhodostomin also inhibited focal adhesion of preadipocyte, including the inhibition of the expression of focal adhesion kinase (FAK) and FAK phosphorylation, assembly of vinculin and reorganization of actin cytoskeleton. Cell viability of preadipocytes was decreased after rhodostomin treatment in a concentration-dependent manner. The results of flow cytometric analysis showed that rhodostomin induced cell apoptosis. In addition, chromatin condensation was observed in DAPI staining. The increase of Bax expression and activation of capsase-3 was detected following rhodostomin treatment. Addition of dexamethasone, IBMX and insulin induced differentiation of preadipocytes into mature adipocytes and treatment of cells with rhodostomin during the initial 3 days showed less mature adipocytes following 9–10 days of differentiating period. The triglyceride content and gene expression of peroxisome proliferators-activated receptor gamma (PPARγ) and leptin also decreased in response to the treatment of rhodostomin. These results suggest that disintegrin inhibits processes of adipogenesis and may be developed to treat obesity.

Keywords: Adipocyte; Disintegrin; Integrin; Adipogenesis

Enhancement of ligand-dependent Vitamin D receptor transactivation by the cardiotonic steroid bufalin by Hiroyuki Nakano; Manabu Matsunawa; Atsutaka Yasui; Ryutaro Adachi; Katsuyoshi Kawana; Iichiro Shimomura; Makoto Makishima (pp. 1479-1486).

Bufalin, a bufadienolide type cardiotonic steroid that is one of the major components of the toad venom-prepared traditional Chinese medicine called Ch’an Su or Senso, exhibits a cardiotonic action by inhibiting the membranous Na⁺,K⁺-ATPase. Bufalin also induces differentiation of leukemia cells alone or in combination with other differentiation inducers including 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. In this study, we performed a transient cotransfection assay using a Vitamin D receptor (VDR) expression vector and a luciferase reporter and found that although bufalin did not transactivate the VDR, it effectively enhanced VDR activity induced by 1,25(OH)₂D₃. Bufalin also augmented VDR activation by bile acid ligands, such as lithocholic acid and 3-ketocholanic acid. Other cardiotonic steroids including ouabain, digitoxigenin and cinobufagin did not enhance VDR activation. Bufalin did not bind directly to VDR but did modulate the interaction of VDR and cofactors, such as steroid receptor coactivator-1 and nuclear receptor corepressor. Bufalin treatment significantly increased the expression of an endogenous VDR target gene, CYP24, in kidney- and monocyte-derived cell lines treated with 1,25(OH)₂D₃. The data indicate that bufalin-mediated cellular mechanisms such as interaction with Na⁺,K⁺-ATPase may affect VDR transcriptional activity. Bufalin may be a useful tool in the investigation of VDR regulation by membrane-originating cellular signals and of pathophysiological mechanisms linking VDR to cardiovascular dysfunction.

Keywords: Abbreviations; 1,25(OH); ₂; D; ₃; 1α,25-dihydroxyvitamin D; ₃; VDR; Vitamin D receptor; AF-2; activation function 2; RXR; retinoid X receptor; DR; direct repeat; ER; everted repeat; SRC-1; steroid receptor coactivator-1; N-CoR; nuclear receptor corepressor; HEK; human embryonic kidney; MAPK; mitogen-activated protein kinaseVitamin D receptor; Bufalin; Cardiotonic steroid; Transcription; Nuclear receptor; Active Vitamin D

Human adenosine A₃ receptor leads to intracellular Ca²⁺ mobilization but is insufficient to activate the signaling pathway via phosphoinositide 3-kinase γ in mice by Kazuya Yamano; Miho Inoue; Shigehiro Masaki; Mayumi Saki; Michio Ichimura; Mitsuo Satoh (pp. 1487-1496).

Selective antagonists for the adenosine A₃ receptor (A3AR), a member of the G protein-coupled receptors, have been indicated as potential drugs for anti-asthma or anti-inflammation. However, potent antagonists for the rodent A3AR have not been identified. To evaluate the pharmacological effects of human A3AR antagonists in mice, we here generated A3AR-humanized mice, in which the mouse A3AR gene was replaced by its human counterpart. The expression levels of human A3AR in the A3AR-humanized mice were equivalent to those of mouse A3AR in wild-type mice. Elevation of the intracellular Ca²⁺ concentration induced by an A3AR agonist was observed in bone marrow-derived mast cells from the A3AR-humanized mice and this Ca²⁺ mobilization was completely antagonized by a human A3AR antagonist. However, antigen-dependent degranulation was not potentiated by the A3AR agonist in the mast cells from A3AR-humanized mice. The agonist-stimulated human A3AR did not lead to the phosphorylation of either extracellular signal-regulated kinase 1/2 or protein kinase B in A3AR-humanized mice. The rate of human A3AR internalization in the mast cells was also markedly decreased compared with that of mouse A3AR in the mast cells. These results demonstrate that the human A3AR is insufficient to activate phosphoinositide 3-kinase γ-dependent signaling pathways in mice, probably due to the uncoupling of member(s) of the G proteins, which are capable of activating phosphoinositide 3-kinase γ, to the human A3AR, despite the mouse G protein(s) responsible for the Ca²⁺ elevation are coupled with the human A3AR.

Keywords: Abbreviations; A3AR; adenosine A; ₃; receptor; A3AR; ^h/h; mice; A3AR-humanized mice; BMMCs; bone marrow-derived mast cells; [Ca; ²⁺; ]; _i; intracellular Ca; ²⁺; concentration; Cl-IB-MECA; 2-chloro-; N; ⁶; -(3-iodobenzyl)adenosine-5′-; N; -methyluronamide; DT-A; diphtheria toxin A fragment; EPO; erythropoietin; ERK1/2; extracellular signal-regulated kinase 1/2; ES cells; embryonic stem cells; GPCR; G protein-coupled receptor; HAT; hypoxanthine/aminopterin/thymidine; HEK293 cells; human embryonic kidney 293 cells; HPRT; hypoxanthine phosphoribosyltransferase; [; ¹²⁵; I]AB-MECA; N; ⁶; -(4-amino-3-[; ¹²⁵; I]iodobenzyl)adenosine-5′-; N; -methyluronamide; KF26777; 2-(4-bromophenyl)-7,8-dihydro-4-propyl-1; H; -imidazo[2,1-; i; ]purin-5(4; H; )-one dihydrochloride; MAPK; mitogen-activated protein kinase; MIP-1α; macrophage inflammatory protein-1α; PCR; polymerase chain reaction; PI3Kγ; phosphoinositide 3-kinase γ; PKB; protein kinase B; PLCβ; phospholipase C β; (; R; )-PIA; (; R; )-; N; ⁶; -phenylisopropyladenosine; RANTES; regulated on activation of T cell expressed and secreted; RT-PCR; reverse transcription-PCR; TNP; 2,4,6-trinitrophenyl; anti-TNP IgE; monoclonal IgE antibody against 2,4,6-trinitrophenylAdenosine A; ₃; receptor; Phosphoinositide 3-kinase γ; G; _i/o; protein; G protein-coupled receptor; Humanized mice; Species difference

Ursolic acid promotes the release of macrophage migration inhibitory factor via ERK2 activation in resting mouse macrophages by Yasutaka Ikeda; Akira Murakami; Hajime Ohigashi (pp. 1497-1505).

Macrophage migration inhibitory factor (MIF) plays some pivotal roles in innate immunity and inflammation. Ursolic acid (UA), an anti-inflammatory triterpene carboxylic acid, was recently reported to induce the release of pro-inflammatory mediators in resting macrophages (Mϕ). We investigated the effects of UA on MIF protein release in resting RAW264.7 mouse Mϕ, and found that it decreased intracellular MIF protein levels and promoted the release of MIF into the culture media in dose- and time-dependent manners, without affecting mRNA levels. Further, the triterpene strikingly induced activation of mitogen-activated protein kinase kinase 1/2 (MEK1/2) and extracellular signal-regulated kinase 1/2 (ERK1/2) within 30min, whereas no phosphorylation of p38 MAPK or JNK protein was observed. In addition, UA-promoted MIF release was significantly inhibited by PD98059, a MEK1/2 inhibitor, while siRNA for ERK2, but not ERK1, significantly decreased the amount of MIF protein released. These results suggest that UA triggers the release of intracellular MIF protein through the ERK2 activation.

Keywords: Abbreviations; COX; cyclooxygenase; DMEM; dulbecco's modified eagle medium; DMSO; dimethylsulfoxide; ELISA; Enzyme-linked immunosorbent assay; ERK; extracellular signal-regulated kinase; FBS; fetal bovine serum; GAPDH; glyceraldehyde phosphate dehydrogenase; ICE; IL-1β-converting enzyme; IFN; interferon; IL; interleukin; iNOS; inducible nitric oxide synthase; JNK; c-Jun NH; ₂; -terminal kinase; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinase; MEK; mitogen-activated protein kinase kinase; MIF; macrophage migration inhibitory factor; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-κB; nuclear factor-kappa B; NO; nitric oxide; PMA; phorbol 12-myristate-13-acetate; RNAi; RNA interference; RT-PCR; reverse transcription-polymerase chain reaction; SDS; sodium dodecyl sulfate; siRNA; short small interfering RNA; TACE; TNF-α-converting enzyme; TLR; toll-like receptor; TNF; tumor necrosis factor; UA; ursolic acidMacrophage; Macrophage migration inhibitory factor; Ursolic acid; Extracellular signal-regulated kinase 2; Dietary factor

Inhibitory effects of dimethylacetyl-β-cyclodextrin on lipopolysaccharide-induced macrophage activation and endotoxin shock in mice by Hidetoshi Arima; Keiichi Motoyama; Akihiro Matsukawa; Yoji Nishimoto; Fumitoshi Hirayama; Kaneto Uekama (pp. 1506-1517).

The potential use of hydrophilic cyclodextrins (CyDs) as an inhibitor for lipopolysaccharide (LPS) was examined. Of the five CyDs used in this study, dimethylacetyl-β-cyclodextrin (DMA7-β-CyD) had greater inhibitory activity than other CyDs against the production of nitric oxide (NO) and various proinflammatory cytokines including tumor necrosis factor-α (TNF-α) in murine macrophages stimulated with two serotypes of LPS and lipid A. The inhibitory effect of DMA7-β-CyD on NO production was also observed in macrophages stimulated with lipoteichoic acid (LTA), but not peptidoglycan (PGN), polyinosinic–polycytidylic acid (poly I:C) or CpG oligonucleotide (CpG-ODN). Several studies have suggested that the inhibitory effects of DMA7-β-CyD could be ascribed to the interaction with LPS. Simultaneous administration of DMA7-β-CyD not only intraperitoneally but also intravenously and intraperitoneal injection of aqueous solution containing LPS andd-galactosamine in murine endotoxin shock model suppressed fatality. Also, DMA7-β-CyD decreased blood level of TNF-α as well as serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) in mice. In conclusion, DMA7-β-CyD may have promise as a new therapeutic agent for endotoxin shock induced by LPS.

Keywords: Cyclodextrin; Dimethylacetyl-β-cyclodextrin; Lipopolysaccharide; Sepsis; Macrophages; Antagonist

Effective prevention of lethal acute graft-versus-host disease by combined immunosuppressive therapy with prodigiosin and cyclosporine A by Sang-Bae Han; Chang Woo Lee; Yeo Dae Yoon; Jong Soon Kang; Ki Hoon Lee; Won Kee Yoon; Young Kook Kim; Kiho Lee; Song-Kyu Park; Hwan Mook Kim (pp. 1518-1526).

Prodigiosin (PDG), a bacterial metabolite, is a known T cell-specific immunosuppressant. Here, we compared its inhibitory potency and mode of action with cyclosporine A (CsA) in a mouse model. PDG efficiently inhibited T cell proliferation with an IC₅₀ of 3.37ng/ml, a similar dose to that of CsA (IC₅₀ of 2.71ng/ml). PDG inhibited only IL-2Rα expression, but not IL-2 expression, whereas CsA inhibited both. Exogenously added IL-2 reversed the suppressive activity of CsA, but not that of PDG. Moreover, although both PDG and CsA markedly reduced mortality rates in lethal acute graft-versus-host disease (GVHD), the combined treatment was more effective than either drug alone. These results demonstrate that PDG and CsA have similar inhibitory potencies, but different modes of action, and suggest that PDG has potential use as a supplementary immunosuppressant in combination with CsA for the treatment of GVHD.

Keywords: Prodigiosin; Cyclosporin A; Graft-versus-host disease

Evidence for induced microsomal bilirubin degradation by cytochrome P450 2A5 by A’edah Abu-Bakar; Michael R. Moore; Matti A. Lang (pp. 1527-1535).

Oxidative metabolism of bilirubin (BR) – a breakdown product of haem with cytoprotective and toxic properties – is an important route of detoxification in addition to glucuronidation. The major enzyme(s) involved in this oxidative degradation are not known. In this paper, we present evidence for a major role of the hepatic cytochrome P450 2A5 (Cyp2a5) in BR degradation during cadmium intoxication, where the BR levels are elevated following induction of haem oxygenase-1 (HO-1). Treatment of DBA/2J mice with CdCl₂ induced both the Cyp2a5 and HO-1, and increased the microsomal BR degradation activity. By contrast, the total cytochrome P450 (CYP) content and the expression of Cyp1a2 were down-regulated by the treatment. The induction of the HO-1 and Cyp2a5 was substantial at the mRNA, protein and enzyme activity levels. In each case, the up-regulation of HO-1 preceded that of Cyp2a5 with a 5–10h interval. BR totally inhibited the microsomal Cyp2a5-dependent coumarin hydroxylase activity, with an IC₅₀ approximately equal to the substrate concentration. The 7-methoxyresorufin 7- O-demethylase (MROD) activity, catalyzed mainly by the Cyp1a2, was inhibited up to 36% by BR. The microsomal BR degradation was inhibited by coumarin and a monoclonal antibody against the Cyp2a5 by about 90%. Furthermore, 7-methoxyresorufin, a substrate for the Cyp1a2, inhibited BR degradation activity by approximately 20%. In sum, the results strongly suggest a major role for Cyp2a5 in the oxidative degradation of BR. Secondly, the coordinated up-regulation of the HO-1 and Cyp2a5 during Cd-mediated injury implicates a network of enzyme systems in the maintenance of balancing BR production and elimination.

Keywords: Abbreviations; AhR; aryl hydrocarbon receptor; BNF; β-naphthoflavone; BR; bilirubin; Cd; cadmium; COH; 7-coumarin hydroxylase; CYP; cytochrome P450; EROD; 7-ethoxyresorufin 7-; O; -demethylase; HO-1; haem oxygenase-1; hmox-1; haem oxygenase-1 gene; 3-MC; 3- methylcholanthrene; MROD; 7-methoxyresorufin 7-; O; -demethylase; Nrf2; erythroid-related factor 2; ROS; reactive oxygen species; 3,4-TCB; 3,4,3′,4′-tetrachlorobiphenyl; TCDD; 2,3,7,8-tetrachlorodibenzo-; p; -dioxinCadmium; Cytochrome P450 2A5; Alternative pathway of bilirubin degradation; Haem oxygenase 1; Oxidative stress; Bilirubin

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Biochemical Pharmacology (v.70, #10)