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Biochemical Pharmacology (v.82, #3)

Editorial Advisory Board (pp. iii).

PPAR-γ and AMPK – Advantageous targets for myocardial ischemia/reperfusion therapy by Alex Morrison; Ji Li (pp. 195-200).

Ischemic heart disease stands as the number one leading cause of death in the United States. Current interventions rely on the immediate restoration of blood flow to the ischemic area; however, this in turn may trigger a series of undesirable events that are further injurious to the myocardium, termed ischemia/reperfusion (I/R) injury. Therefore, there is a need for novel therapeutic strategies aimed at limiting the extent of myocardial injury. Yet, the molecular mechanisms responsible for I/R injury remain largely indefinable. Research efforts are currently investigating various signaling mechanisms to be used for potential targets limiting cardiac injury due to such cardiovascular events. In this review, we highlight two potential molecular targets, PPAR-γ and AMPK, which have been extensively reported to have various cardioprotective capabilities against I/R injury. Although functionally different, the pathways these proteins mediate seem to intersect and possibly act synergistically potentiating a cardioprotective response.

Keywords: Peroxisome proliferator-activated receptor γ (PPARγ); AMP-activated protein kinase (AMPK); Ischemia/reperfusion (I/R); Cardioprotection

Acquired and intrinsic BRAF inhibitor resistance in BRAF V600E mutant melanoma by Inna V. Fedorenko; Kim H.T. Paraiso; Keiran S.M. Smalley (pp. 201-209).

The discovery of activating BRAF V600E mutations in 50% of all cutaneous melanomas has revolutionized the understanding of melanoma biology and provided new strategies for the therapeutic management of this deadly disease. Highly potent small molecule inhibitors of BRAF are now showing great promise as a novel therapeutic strategy for melanomas harboring activating BRAF V600E mutations and are associated with high levels of response. This commentary article discusses the latest data on the role of mutated BRAF in the development and progression of melanoma as the basis for understanding the mechanism of action of BRAF inhibitors in the preclinical and clinical settings. We further address the issue of BRAF inhibitor resistance and outline the latest insights into the mechanisms of therapeutic escape as well as describing approaches to prevent and abrogate the onset of both intrinsic and acquired drug resistance. It is likely that our evolving understanding of melanoma genetics and signaling will allow for the further personalization of melanoma therapy with the goal of improving clinical responses.

Keywords: BRAF; Melanoma; Vemurafenib; MEK; Resistance; Cot; MAPK

Identification of genes associated to 2′,2′-difluorodeoxycytidine resistance in HeLa cells with a lentiviral short-hairpin RNA library by Yunjian Xu; Anna Karlsson; Magnus Johansson (pp. 210-215).

Resistance to the cytotoxic nucleoside analog 2′,2′-diflurodeoxycytidine (dFdC) used in cancer chemotherapy is a frequent cause of treatment failure. Although several molecular mechanisms that cause resistance to dFdC have been identified, many cells acquire dFdC resistance by unknown mechanisms. We have used a short-hairpin RNA (shRNA) library in a lentiviral vector that contains ≈5000 shRNAs designed against genes encoding kinases, phosphatases, tumor suppressors and DNA binding proteins to perform a loss-of-function screen to identify genes causing dFdC resistance in HeLa cells when their expression is decreased. 155 cell lines with shRNA expression were isolated from the screen and several of these cell lines were in repeated experiments verified to show resistance to dFdC compared to wild-type cells. DNA sequencing of the shRNA vector integrated in the cellular genome was used to determine the shRNA expressed in the cells and the putative target genes were identified by sequence analysis. 16 cell lines with putative target genes previously not associated to dFdC resistance were identified. Chemically synthesized short-interfering RNAs (siRNAs) directed against the target genes were used to verify that the decreased expression of the identified genes caused dFdC resistance. Using these techniques we identified two splicing factor proteins, serine/arginine-rich splicing factor 3 (SRSF3) and splicing factor proline/glutamine-rich (SFPQ), that induced resistance to dFdC as well as other pyrimidine nucleoside analogs when their expression was decreased in HeLa cells.

Keywords: Abbreviations; araC; 1-β-arabinofuranocylcytosine; dCK; deoxycytidine kinase; dFdC; 2′,2′-difluorodeoxycytidine; 5-FdUrd; 5-fluorodeoxyuridine; RNAi; RNA interference; shRNA; short-hairpin RNA; siRNA; short-interfering RNA; SFPQ; splicing factor proline/glutamine-rich; SRSF3; serine/arginine-rich splicing factor 3Nucleoside analog; Short-hairpin RNA; Drug resistance; Chemotherapy; Small-interfering RNA; RNA interference

Sorafenib induces apoptotic cell death in human non-small cell lung cancer cells by down-regulating mammalian target of rapamycin (mTOR)-dependent survivin expression by Young-Sun Kim; Hyeon-Ok Jin; Sung-Keum Seo; Sang Hyeok Woo; Tae-Boo Choe; Sungkwan An; Seok-Il Hong; Su-Jae Lee; Kee-Ho Lee; In-Chul Park (pp. 216-226).

Sorafenib, a multikinase inhibitor, is emerging as a promising targeted agent that may possess antitumor activity against a broad range of cancers. The mechanism by which sorafenib induces lung cancer cell death and apoptosis, however, is not understood. In the present study, we provide evidence that sorafenib acts through inhibition of mammalian target of rapamycin (mTOR) to down-regulate survivin and promote apoptotic cell death in human non-small cell lung cancer (NSCLC) cells. Sorafenib induced ATF4-mediated Redd1 expression, leading to mTOR inhibition—the upstream signal for down-regulation of survivin. Overexpression of survivin reduced sorafenib-induced apoptosis, whereas silencing survivin using small interfering RNA (siRNA) enhanced it, supporting the interpretation that down-regulation of survivin is involved in sorafenib-induced cell death in human NSCLC cells. Furthermore, sorafenib abolished the induction of survivin that normally accompanies IGF-1-stimulated mTOR activation. We further found that Redd1-induced mTOR down-regulation and ATF4/CHOP-induced expression of the TRAIL receptor DR5 associated with sorafenib treatment helped sensitize cells to TRAIL-induced apoptosis. Our study suggests that sorafenib mediates apoptotic cell death in human NSCLC cells through Redd1-induced inhibition of mTOR and subsequent down-regulation of survivin, events that are associated with sensitization to TRAIL-induced apoptotic cell death.

Keywords: Abbreviations; mTOR; mammalian target of rapamycin; NSCLC; non-small cell lung cancer; 4E-BP1; eukaryotic initiation factor 4E binding protein 1; ATF4; activating transcription factor 4; TRAIL; TNF-related apoptosis-inducing ligandmTOR; Non-small cell lung cancer; Redd1; Sorafenib; Survivin

DNA interactions of 2-pyrrolinodoxorubicin, a distinctively more potent daunosamine-modified analogue of doxorubicin by Jana Stepankova; Martin Studenovsky; Jaroslav Malina; Jana Kasparkova; Barbora Liskova; Olga Novakova; Karel Ulbrich; Viktor Brabec (pp. 227-235).

It was shown earlier that 2-pyrrolinodoxorubicin was 500–1000 times more active towards human and mouse cancer cells in vitro than parental doxorubicin. However, the biochemical factors responsible for such a large difference in potency between doxorubicin and 2-pyrrolinodoxorubicin are not clear at the molecular level. To provide this information, we have investigated in cell-free media by biochemical and biophysical methods interactions of both anthracyclines with DNA, effects of these interactions on activity of human topoisomerase II, human Bloom's syndrome helicase and prokaryotic T7 RNA polymerase, and the capability of these drugs to form DNA interstrand cross-links in formaldehyde-free medium. Experiments aimed at understanding the properties of double-helical DNA in the presence of doxorubicin and 2-pyrrolinodoxorubicin revealed only small differences in DNA modifications by these anthracyclines and resulting conformational alterations in DNA. Similarly, the ability of 2-pyrrolinodoxorubicin modifications of DNA to inhibit catalytic activity of topoisomerase II does not differ significantly from that of doxorubicin. On the other hand, we demonstrate that an important factor responsible for the markedly higher antiproliferative potency of DNA modifications by 2-pyrrolinodoxorubicin is capability of these modifications to inhibit downstream cellular processes which process DNA damaged by this drug and involve separation of complementary strands of DNA, such as DNA unwinding by helicases or RNA polymerases. In addition, the results are also consistent with the hypothesis that in particular the capability of 2-pyrrolinodoxorubicin to readily form DNA interstrand cross-links is responsible for inhibition of these processes in the cells treated with this analogue of doxorubicin.

Keywords: Abbreviations; bp; base pair; CL; crosslink; CD; circular dichroism; CT; calf-thymus; DOX; doxorubicin; DTT; dithiothreitol; EtBr; ethidium bromide; KF; Klenow fragment of DNA polymerase I; LD; linear dichroism; PAA; polyacrylamide; p-DOX; 2-pyrrolinodoxorubicin; r; the molar ratio of a drug to nucleotide-phosphates at the onset of incubation with DNA; topoII; topoisomerase IIDoxorubicin; 2-Pyrrolinodoxorubicin; DNA; Topoisomerase II; Helicase; Interstrand cross-links

Effects of highly active novel artemisinin–chloroquinoline hybrid compounds on β-hematin formation, parasite morphology and endocytosis in Plasmodium falciparum by Tzu-Shean Feng; Eric M. Guantai; Margo Nell; Constance E.J. van Rensburg; Kanyile Ncokazi; Timothy J. Egan; Heinrich C. Hoppe; Kelly Chibale (pp. 236-247).

4-Aminoquinolines were hybridized with artemisinin and 1,4-naphthoquinone derivatives via the Ugi-four-component condensation reaction, and their biological activities investigated. The artemisinin-containing compounds6a–c and its salt6c-citrate were the most active target compounds in the antiplasmodial assays. However, despite the potent in vitro activities, they also displayed cytotoxicity against a mammalian cell-line, and had lower therapeutic indices than chloroquine. Morphological changes in parasites treated with these artemisinin-containing hybrid compounds were similar to those observed after addition of artemisinin. These hybrid compounds appeared to share mechanism(s) of action with both chloroquine and artemisinin: they exhibited potent β-hematin inhibitory activities; they caused an increase in accumulation of hemoglobin within the parasites that was intermediate between the increase observed with artesunate and chloroquine; and they also appeared to inhibit endocytosis as suggested by the decrease in the number of transport vesicles in the parasites. No cross-resistance with chloroquine was observed for these hybrid compounds, despite the fact that they contained the chloroquinoline moiety. The hybridization strategy therefore appeared to be borrowing the best from both classes of antimalarials.

Keywords: Artemisinin; β-Hematin inhibition; Chloroquinoline; Endocytosis; Parasite morphology; Plasmodium falciparum

Formation of new bioactive organic nitrites and their identification with gas chromatography–mass spectrometry and liquid chromatography coupled to nitrite reduction by Kristofer F. Nilsson; Michael Lundgren; Per Agvald; L. Christofer Adding; Dag Linnarsson; Lars E. Gustafsson (pp. 248-259).

Nitric oxide (NO) donors, notably organic nitrates and nitrites are used therapeutically but tolerance develops rapidly, making the use of e.g. nitroglycerin difficult. NO donation in the pulmonary vascular bed might be useful in critically ill patients. Organic nitrites are not associated with tachyphylaxis but may induce methaemoglobinemia and systemic hypotension which might hamper their use. We hypothesised that new lung-selective NO donors can be identified by utilizing exhaled NO as measure for pulmonary NO donation and systemic arterial pressure to monitor hypotension and tolerance development. Solutions of alcohols and carbohydrates were reacted with NO gas and administered to ventilated rabbits for evaluation of in vivo NO donation. Chemical characterization was made by liquid chromatography with on-line nitrite reduction (LC-NO) and by gas chromatography–mass spectrometry (GC–MS). In vivo experiments showed that the hydroxyl-containing compounds treated with NO gas yielded potent NO donors, via nitrosylation to organic nitrites. Analyses by LC-NO showed that the reaction products were able to release NO in vitro. In GC–MS the reaction products were determined to be the organic nitrites, where some are new chemical entities. Non-polar donors preferentially increased exhaled NO with less effect on systemic blood pressure whereas more polar molecules had larger effects on systemic blood pressure and less on exhaled NO. We conclude that new organic nitrites suitable for intravenous administration are produced by reacting NO gas and certain hydroxyl-containing compounds in aqueous solutions. Selectivity of different organic nitrites towards the pulmonary and systemic circulation, respectively, may be determined by molecular polarity.

Keywords: Nitric oxide donors; Nitrites; Pulmonary circulation; Pulmonary hypertension; Chromatography, High pressure liquid

Mitochondria-dependent apoptosis of activated T lymphocytes induced by astin C, a plant cyclopeptide, for preventing murine experimental colitis by Yan Shen; Qiong Luo; Huimin Xu; Fangyuan Gong; Xiaobin Zhou; Yang Sun; Xuefeng Wu; Wen Liu; Guangzhi Zeng; Ninghua Tan; Qiang Xu (pp. 260-268).

Facilitating T-cell apoptosis is implicated as an effective therapeutic strategy for treatment of T cell-mediated disease, including inflammatory bowel disease. Here, we report that astin C, a plant cyclopeptide isolated from the roots of Aster tataricus (Compositae), induced apoptosis of activated T cells in a mitochondria-dependent but Fas-independent manner in that such activity was still observed in T cells from Fas-mutated MRL^lpr/lpr mice. Although caspase 8 was not activated, astin C treatment led to the cleavage of caspase 9 and caspase 3, the upregulation of Bad protein expression as well as release of cytochrome c in activated T cells. Astin C did not induce the expression of GRP78 and GADD153, excluding involvement of endoplasmic reticulum stress-mediated pathway. Moreover, oral administration of astin C protected mice against TNBS-induced colonic inflammation, as assessed by a reduced colonic weight/length ratio and histological scoring. Administering astin C significantly decreased serum levels of TNF-α, IL-4 and IL-17, accompanied with the induction of apoptosis in activated T cells in vivo. The results demonstrate, for the first time, the ability of astin C to induce apoptosis in activated T cells and its potential use in the treatment of colonic inflammation.

Keywords: Astin C; T lymphocyte; Apoptosis; Mitochondria; Colitis

Role of peripheral polyamines in the development of inflammatory pain by Mariane A. Silva; Jonatas Z. Klafke; Mateus F. Rossato; Camila Gewehr; Gustavo P. Guerra; Maribel A. Rubin; Juliano Ferreira (pp. 269-277).

Polyamines (putrescine, spermidine and spermine) are aliphatic amines that are produced by the action of ornithine decarboxylase (ODC) in a rate-limiting and protein kinase C (PKC)-regulated step. Because high levels of polyamines are found in the synovial fluid of arthritic patients, the aim of the present study was to identify the role of peripherally produced polyamines in a model of inflammatory pain induced by adjuvant. The subcutaneous injection of Complete Freund's adjuvant (CFA, 50μL/paw) caused the development of mechanical allodynia and edema. Moreover, it increased ODC expression and activity and PKC activation. Administration of the selective ODC inhibitor DFMO (10μmol/paw) attenuated the development of allodynia and edema and decreased ODC activity in both control and CFA-treated animals. Furthermore, administration of the PKC inhibitor GF109203X (1nmol/paw) reduced allodynia and ODC activity in animals injected with CFA. A subcutaneous injection of putrescine (10μmol/paw), spermidine (3–10μmol/paw) or spermine (0.3–3μmol/paw) into the rat paw also caused mechanical allodynia and edema. The present results suggest that endogenously synthesized polyamines are involved in the development of nociception and edema caused by an adjuvant. Moreover, polyamine production in inflammatory sites seems to be related to an increase in ODC activity stimulated by PKC activation. Thus, controlling polyamine synthesis and action could be a method of controlling inflammatory pain.

Keywords: DFMO; Edema; Inflammation; ODC; Polyamine

Ginsenoside Rb1 and its metabolite compound K inhibit IRAK-1 activation—The key step of inflammation by Eun-Ha Joh; In-Ah Lee; Il-Hoon Jung; Dong-Hyun Kim (pp. 278-286).

In the preliminary study, ginsenoside Rb1, a main constituent of the root of Panax ginseng (family Araliaceae), and its metabolite compound K inhibited a key factor of inflammation, nuclear transcription factor κB (NF-κB) activation, in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages. When ginsenoside Rb1 or compound K were orally administered to 2,4,6-trinitrobenzene sulfuric acid (TNBS)-induced colitic mice, these agents inhibited colon shortening, macroscopic score, and colonic thickening. Furthermore, treatment with ginsenoside Rb1 or compound K at 20mg/kg inhibited colonic myeloperoxidase activity by 84% and 88%, respectively, as compared with TNBS alone ( p<0.05), and also potently inhibited the expression of tumor necrosis factor-α, interleukin (IL)-1β and IL-6, but increased the expression of IL-10. Both ginsenoside Rb1 and compound K blocked the TNBS-induced expressions of COX-2 and iNOS and the activation of NF-κB in mice. When ginsenoside Rb1 or compound K was treated in LPS-induced murine peritoneal macrophages, these agents potently inhibited the expression of the proinflammatory cytokines. Ginsenoside Rb1 and compound K also significantly inhibited the activation of interleukin-1 receptor-associated kinase-1 (IRAK-1), IKK-β, NF-κB, and MAP kinases (ERK, JNK, and p-38); however, interaction between LPS and Toll-like receptor-4, IRAK-4 activation and IRAK-2 activation were unaffected. Furthermore, compound K inhibited the production of proinflammatory cytokines more potently than did those of ginsenoside Rb1. On the basis of these findings, ginsenosides, particularly compounds K, could be used to treat inflammatory diseases, such as colitis, by targeting IRAK-1 activation.

Keywords: Compound K; Ginsenoside Rb1; Interleukin-1 receptor-associated kinase-1

The effect of sphingomyelin synthase 2 (SMS2) deficiency on the expression of drug transporters in mouse brain by Yu Zhang; Jibin Dong; Xingang Zhu; Weirong Wang; Qing Yang (pp. 287-294).

Sphingomyelin synthase (SMS), the last enzyme involved in the biosynthesis of sphingomyelin (SM), plays a critical role in the constitution of cell membrane and has impact on the expression of membrane proteins. SMS2, one of two SMS enzymes, is predominantly located in the plasma membrane, and is mainly expressed in the brain. Therefore, it is conceivable that SMS2 deficiency may have impact on expression of some membrane proteins, such as membrane-bound drug transporters. Using SMS2 gene deficient mouse brain tissues, we studied the gene and protein expression profiles of drug transporters, ERM proteins (ezrin/radixin/moesin) and the cytoskeleton protein, β-actin, in mouse brain by RT-PCR, western blot and immunohistochemistry analysis. We found that the mRNA expression of Mdr1 rather than the other drug transporters was significantly decreased in the SMS2 deficient brain. Accordingly, the expression and the function of Pgp (Mdr1/P-glycoprotein) were significantly downregulated in brain. In addition, the substantially downregulated expression of ezrin and β-actin was also observed in the SMS2 deficient brain. The immunohistochemistry analysis further revealed the suppressed expression of Pgp, ezrin and β-actin in both cortex and paraventricular areas of SMS2 knockout mice. Furthermore, both Pgp and β-actin were found to be co-immunoprecipitated with ezrin from the total brain lysate, suggesting the association between Pgp, ezrin and β-actin in the brain. These results indicate that SMS2 participates in the expression regulation of drug transporters, particularly Pgp, and suggest that SMS2 may be a potential target for enhancing drug access to the brain.

Keywords: Sphingomyelin synthase 2; Drug transporters; Brain; ERM proteins

Metabolism of bupropion by baboon hepatic and placental microsomes by Xiaoming Wang; Doaa R. Abdelrahman; Valentina M. Fokina; Gary D.V. Hankins; Mahmoud S. Ahmed; Tatiana N. Nanovskaya (pp. 295-303).

In baboons, the major enzyme responsible for hydroxylation of bupropion belongs to the CYP 2B subfamily of enzymes. The formation of the reduced metabolites is catalyzed by carbonyl reducing enzymes.The aim of this investigation was to determine the biotransformation of bupropion by baboon hepatic and placental microsomes, identify the enzyme(s) catalyzing the reaction(s) and determine its kinetics. Bupropion was metabolized by baboon hepatic and placental microsomes to hydroxybupropion (OH-BUP), threo- (TB) and erythrohydrobupropion (EB). OH-bupropion was the major metabolite formed by hepatic microsomes ( K_m 36±6μM, V_max 258±32pmolmgprotein⁻¹min⁻¹), however the formation of OH-BUP by placental microsomes was below the limit of quantification. The apparent K_m values of bupropion for the formation of TB and EB by hepatic and placental microsomes were similar. The selective inhibitors of CYP2B6 (ticlopidine and phencyclidine) and monoclonal antibodies raised against human CYP2B6 isozyme caused 80% inhibition of OH-BUP formation by baboon hepatic microsomes. The chemical inhibitors of aldo-keto reductases (flufenamic acid), carbonyl reductases (menadione), and 11β-hydroxysteroid dehydrogenases (18β-glycyrrhetinic acid) significantly decreased the formation of TB and EB by hepatic and placental microsomes. Data indicate that CYP2B of baboon hepatic microsomes is responsible for biotransformation of bupropion to OH-BUP, while hepatic and placental short chain dehydrogenases/reductases and to a lesser extent aldo-keto reductases are responsible for the reduction of bupropion to TB and EB.

Keywords: Abbreviations; V; _max; maximum reaction velocity; K; _m; substrate concentration causing 50% of the reaction velocity; P450; cytochrome P450; IC; ₅₀; concentration of compound causing 50% inhibition of the reaction; OH-BUP; hydroxybupropion; TB; threohydrobupropion; EB; erythrohydrobupropionBupropion; Metabolism; Baboon; Liver; Placenta

Regulation of drug transporter expression by oncostatin M in human hepatocytes by Marc Le Vee; Elodie Jouan; Bruno Stieger; Valérie Lecureur; Olivier Fardel (pp. 304-311).

The cytokine oncostatin M (OSM) is a member of the interleukin (IL)-6 family, known to down-regulate expression of drug metabolizing cytochromes P-450 in human hepatocytes. The present study was designed to determine whether OSM may also impair expression of sinusoidal and canalicular drug transporters, which constitute important determinants of drug hepatic clearance. Exposure of primary human hepatocytes to OSM down-regulated mRNA levels of major sinusoidal solute carrier (SLC) influx transporters, including sodium-taurocholate co-transporting polypeptide (NTCP), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1, organic cation transporter 1 and organic anion transporter 2. OSM also repressed mRNA expressions of ATP binding cassette (ABC) efflux transporters such as multidrug resistance protein (MRP) 2/ABCC2 and breast cancer resistance protein/ABCG2, without however impairing those of multidrug resistance gene 1/P-glycoprotein/ABCB1, MRP3/ABCC3, MRP4/ABCC4 and bile salt export pump/ABCB11. The cytokine concomitantly reduced NTCP, OATP1B1, OATP2B1 and ABCG2 protein expression and NTCP and OATP transport activities. OSM effects towards transporters were found to be dose-dependent and highly correlated with those of IL-6, but not with those of other inflammatory cytokines such as tumor necrosis factor-α or interferon-γ. In addition, OSM-mediated repression of some transporters such as NTCP, OATP1B1 and OATP2B1, was counteracted by knocking-down expression of the type II OSM receptor subunits through siRNA transfection. This OSM-mediated down-regulation of drug SLC transporters and ABCG2 in human hepatocytes may contribute to alterations of pharmacokinetics in patients suffering from diseases associated with increased production of OSM.

Keywords: Drug transporters; Human hepatocytes; Inflammation; Oncostatin-M; Biliary elimination

Selective role for tumor necrosis factor-α, but not interleukin-1 or Kupffer cells, in down-regulation of CYP3A11 and CYP3A25 in livers of mice infected with a noninvasive intestinal pathogen by Ryan D. Kinloch; Choon-Myung Lee; Nico van Rooijen; Edward T. Morgan (pp. 312-321).

Hepatic cytochrome P450 (P450) gene and protein expression are modulated during inflammation and infection. Oral infection of C57BL/6 mice with Citrobacter rodentium produces mild clinical symptoms while selectively regulating hepatic P450 expression and elevating levels of proinflammatory cytokines. Here, we explored the role of cytokines in the regulation of hepatic P450 expression by orally infecting tumor necrosis factor-α (TNFα) receptor 1 null mice (TNFR1−/−), interleukin-1 (IL1) receptor null mice (IL1R1−/−), and Kupffer cell depleted mice with C. rodentium. CYP4A mRNA and protein levels and flavin monooxygenase (FMO)3 mRNA expression levels were down-regulated, while CYP2D9 and CYP4F18 mRNAs remained elevated during infection in wild-type, receptor knockout, and Kupffer cell depleted mice. CYPs 3A11 and 3A25 mRNA levels were down-regulated during infection in wild-type mice but not in TNFR1−/− mice. Consistent with this observation, CYPs 3A11 and 3A25 were potently down-regulated in mouse hepatocytes treated with TNFα. Oral infection of IL1R1−/− mice and studies with mouse hepatocytes indicated that IL1 does not directly regulate CYP3A11 or CYP3A25 expression. Uninfected mice injected with clodronate liposomes had a significantly reduced number of Kupffer cells in their livers. Infection increased the Kupffer cell count, which was attenuated by clodronate treatment. The P450 mRNA and cytokine levels in infected Kupffer cell depleted mice were comparable to those in infected mice receiving no clodronate. The results indicate that TNFα is involved in the regulation of CYPs 3A11 and 3A25, but IL1β and Kupffer cells may not be relevant to hepatic P450 regulation in oral C. rodentium infection.

Keywords: Cytochrome P450; Inflammation; Kupffer cells; Interleukin-1; TNFα

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Biochemical Pharmacology (v.82, #3)