Tomo Daidoji

H5N1 Avian Influenza Virus Induces Apoptotic Cell Death in Mammalian Airway Epithelial Cells

ABSTRACT In recent years, the highly pathogenic avian influenza virus H5N1 has raised serious worldwide concern about an influenza pandemic; however, the biology of H5N1 pathogenesis is largely unknown. To elucidate the mechanism of H5N1 pathogenesis, we prepared primary airway epithelial cells from alveolar tissues from 1-year-old pigs and measured the growth kinetics of three avian H5 influenza viruses (A/Crow/Kyoto/53/2004 [H5N1], A/Duck/Hong Kong/342/78 [H5N2], and A/Duck/Hong Kong/820/80 [H5N3]), the resultant cytopathicity, and possible associated mechanisms. H5N1, but not the other H5 viruses, strongly induced cell death in porcine alveolar epithelial cells (pAEpC), although all three viruses induced similar degrees of cytopathicity in chicken embryonic fibroblasts. Intracellular viral growth and the production of progeny viruses were comparable in pAEpC infected with each H5 virus. In contrast, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive cells were detected only in H5N1-infected pAEpC, and the activities of caspases 3, 8, and 9 were significantly elevated in pAEpC infected with H5N1, but not with H5N2 and H5N3. These results suggest that only H5N1 induces apoptosis in pAEpC. H5N1 cytopathicity was inhibited by adding the caspase inhibitor z-VAD-FMK; however, there were no significant differences in viral growth or release of progeny viruses. Further investigations using reverse genetics demonstrated that H5N1 hemagglutinin protein plays a critical role in inducing caspase-dependent apoptosis in infected pAEpC. H5N1-specific cytopathicity was also observed in human primary airway epithelial cells. Taken together, these data suggest that avian H5N1 influenza virus leads to substantial cell death in mammalian airway epithelial cells due to the induction of apoptosis.

Highly Pathogenic H5N1 Avian Influenza Virus Induces Extracellular Ca2+ Influx, Leading to Apoptosis in Avian Cells

ABSTRACT In this study, we show that the highly pathogenic H5N1 avian influenza virus (AIV) (A/crow/Kyoto/53/04 and A/chicken/Egypt/CL6/07) induced apoptosis in duck embryonic fibroblasts (DEF). In contrast, apoptosis was reduced among cells infected with low-pathogenic AIVs (A/duck/HK/342/78 [H5N2], A/duck/HK/820/80 [H5N3], A/wigeon/Osaka/1/01 [H7N7], and A/turkey/Wisconsin/1/66 [H9N2]). Thus, we investigated the molecular mechanisms of apoptosis induced by H5N1-AIV infection. Caspase-dependent and -independent pathways contributed to the cytopathic effects. We further showed that, in the induction of apoptosis, the hemagglutinin of H5N1-AIV played a major role and its cleavage sequence was not critical. We also observed outer membrane permeabilization and loss of the transmembrane potential of the mitochondria of infected DEF, indicating that mitochondrial dysfunction was caused by the H5N1-AIV infection. We then analyzed Ca2+ dynamics in the infected cells and demonstrated an increase in the concentration of Ca2+ in the cytosol ([Ca2+]i) and mitochondria ([Ca2+]m) after H5N1-AIV infection. Regardless, gene expression important for regulating Ca2+ efflux from the endoplasmic reticulum did not significantly change after H5N1-AIV infection. These results suggest that extracellular Ca2+ may enter H5N1-AIV-infected cells. Indeed, EGTA, which chelates extracellular free Ca2+, significantly reduced the [Ca2+]i, [Ca2+]m, and apoptosis induced by H5N1-AIV infection. In conclusion, we identified a novel mechanism for influenza A virus-mediated cell death, which involved the acceleration of extracellular Ca2+ influx, leading to mitochondrial dysfunction and apoptosis. These findings may be useful for understanding the pathogenesis of H5N1-AIV in avian species as well as the impact of Ca2+ homeostasis on influenza A virus infection.

Disposition of flavonoids via enteric recycling: UDP-glucuronosyltransferase (UGT) 1As deficiency in Gunn rats is compensated by increases in UGT2Bs activities.

Flavonoids have poor bioavailabilities largely because of metabolism via UDP-glucuronosyltransferases (UGTs). This study aims to further understand the functions of UGT in metabolizing genistein and apigenin, two compounds metabolized more extensively in the gut than in the liver. Because Gunn rats are deficient in UGT1As, we determined whether this deficiency would result in less flavonoid glucuronidation, using rat intestinal perfusion model and microsomes prepared from rat liver and intestine. In yeast-expressed rat UGT isoforms, rat UGT1A isoforms (especially UGT1A7) were mainly responsible for flavonoid metabolism. In perfusion studies, the two flavonoids were rapidly absorbed at comparable rates, but the intestinal excretions of glucuronides in Gunn rats compared with Wistar rats were not only comparable for genistein but also were higher (p < 0.05) for apigenin, suggesting up-regulation of UGT isoforms in Gunn rats. To determine the possible compensatory UGT isoforms, we first verified that UGT1A activities were significantly lower (p < 0.05) in Gunn rats by using UGT1A-specific probes 7-ethyl-10-hydroxycamptothecin (SN-38) and prunetin. We then demonstrated using UGT2B probes testosterone, ezetimibe, and indomethacin that UGT2B activities were usually significantly higher in Gunn rats. In addition, testosterone was metabolized much faster in liver microsomes than in intestinal microsomes, and in microsomes prepared from Gunn rats compared with Wistar rats. In conclusion, flavonoids are efficiently metabolized by UGT1A-deficient Gunn rats because of compensatory up-regulation of intestinal UGT2Bs and hepatic anion efflux transporters, which increases their disposition and limits their oral bioavailabilities.

UDP-GLUCURONOSYLTRANSFERASE ISOFORMS CATALYZING GLUCURONIDATION OF HYDROXY-POLYCHLORINATED BIPHENYLS IN RAT

Polychlorinated biphenyls (PCBs) are highly toxic environmental contaminants that can cause irreversible damage in humans and wildlife. The mechanism of toxicity is not clear, but biotransformation products such as hydroxy PCBs (OH-PCBs) are a major concern. Efforts to elucidate the metabolism of PCBs and their metabolites, however, have paid little attention to the structure of the compound to be eliminated. The objectives of this study were to clarify organ tissue distribution of the glucuronidation activities toward OH-PCBs and to determine the UDP-glucuronosyltranseferase (UGT) isoforms responsible for glucuronidation in relation to the OH-PCB structure. 2,4,6-Trichlorobiphenyl and 2,3,4,5-tetrachlorobiphenyl were incubated in primary culture of rat hepatocytes, and the metabolites were identified by HPLC. Organ tissue glucuronidation activities toward 10 OH-PCBs were investigated by reactions of microsomes prepared from brain, liver, small and large intestine, lung, kidney, and testis tissues. To determine substrate specificity of the isoforms toward the OH-PCBs, rat UGT isoforms UGT1A1, UGT1A3, UGT1A5, UGT1A6, UGT1A7, UGT2B1, UGT2B3, and UGT2B12 were expressed in yeast strain AH22. Glucuronidation of the PCBs was found to be contingent on their hydroxylation. The organ tissues had strong glucuronidation activities toward the OH-PCBs tested; and most OH-PCBs were glucuronidated by UGT1A1, UGT1A6, and UGT2B1, all of which were substrate-specific. In conclusion, glucuronidation activities of UGT1A1, UGT1A6, and UGT2B1 toward OH-PCBs is relative to expression of the isoforms in each tissue, and glucuronidation intensity of the isoforms is relative to the structure of the OH-PCB to be glucuronidated.

Maturation efficiency of viral glycoproteins in the ER impacts the production of influenza A virus

We have studied which steps are enhanced in the infectious cycle of influenza A virus in Madin-Darby canine kidney (MDCK) cells, a cell line investigated for use in the production of an influenza vaccine because of its ability to yield high levels of virus. We have confirmed that MDCK had the highest production levels of virions among several cell lines early in the infection. Influenza A virus showed similar levels of viral genomic RNA replication, mRNA transcription, and protein expression in A549 as in MDCK. Thus, we focused on the post-translational transport of viral glycoproteins from the endoplasmic reticulum (ER) to the plasma membrane. Comparative characterization revealed more efficient processing in the folding and maturation of hemagglutinin and neuraminidase in the ER in MDCK than in A549. Also, the subsequent transport of these glycoproteins to the plasma membrane occurred much earlier in MDCK. These results indicate that the folding and maturation efficiencies of viral glycoproteins in the ER impact the efficiency with which influenza A viral particles are produced.

Differential metabolism of 4-n- and 4-tert-octylphenols in perfused rat liver

Octylphenols, widely used in a variety of detergents and plastics, are known to exhibit estrogenicity in vivo. The details of their metabolism are needed to better understand the endocrine disruptions. We have previously shown that alkylphenols, having short alkyl chains, are glucuronidated and readily excreted into the bile from the liver, while 4-n-nonylphenol, having longer alkyl chains, remains as the alkylphenols glucuronide in the tissue. This study elucidated the dependence of the metabolism on the shape of the alkyl chains by comparing 4-n-octylphenol and 4-tert-octylphenols in a perfused rat liver. Both octylphenols were highly glucuronidated by the liver microsomal fractions. The Vmax value of 4-tert-octylphenol glucuronidation was twice as high as that of 4-n-octylphenol in the liver microsomes. On the other hand, the Km values, being measures of enzymatic activity against these chemicals, were similar. 4-n-Octylphenol and 4-tert-octylphenol were both glucuronidated by a UDP-glucuronosyltransferase isoform, UGT2B1, expressed in the liver. In the liver perfusion, almost all of the 4-n-octylphenol perfused was metabolized directly to the glucuronide, whereas a portion of 4-tert-octylphenol was hydroxylated and then glucuronidated. The glucuronide of 4-n-octylphenol accumulated in the liver tissue in the same manner as 4-n-nonylphenol, but 4-tert-octylphenol and the hydroxylated metabolites were excreted readily into the bile. Only a small amount of 4-n-octylphenol-glucuronide and glucuronides of 4-tert-octylphenol and its hydroxylated metabolites could be excreted into the bile of Eisai hyperbilirubinemic rats (EHBR). These animals are deficient in xenobiotic conjugate transporter, multidrug resistance-associated protein (MRP-2), indicating that the glucuronides of both octylphenols are transported by MRP-2. These results indicate that the differences in metabolism of these octylphenols are due to the shape of their alkyl chains, suggesting that the estrogenic activities of not only the parent chemicals but also these metabolites must be taken into consideration.

Slow elimination of nonylphenol from rat intestine

Nonylphenol, a possible endocrine disrupter, tends to persist in rat liver tissue after detoxification as a glucuronide conjugate by UDP-glucuronosyltransferase 2B1 expressed in the liver. In the intestine, however, the metabolism and dynamics of nonylphenol remain to be elucidated. The objectives of this study were to clarify the metabolism and excretion of nonylphenol having a long alkyl chain in the first barrier intestine and to estimate whether the nonylphenol alkyl chain governs the speed of excretion from intestinal tissue. Organ tissue glucuronidation activity toward alkylphenols (C2, C9) was investigated using microsomes prepared from intestinal tissue. To elucidate the elimination pathway of alkylphenols (C2, C4, C6, C9), a perfusion study was conducted on everted intestine. After oral administration (5 mg) of alkylphenols (C2, C9) to rats, gastrointestinal contents and related organ tissues (gastrointestinal tissue, liver, and kidney), blood, and urine were analyzed for alkylphenols (C2, C9) and glucuronides. The intestine showed strong glucuronidation activity toward alkylphenols (C2, C9). In everted intestinal assay, nonylphenol was glucuronidated within the intestinal wall, as was the case for other alkylphenols (C2, C4, C6), but nonylphenol-glucuronide was not excreted from intestinal tissue. Orally administered nonylphenol remained for long periods in gastrointestinal tissue as both the parent compound and glucuronide. The present study confirmed that intestinal tissue possesses an alkylphenol elimination system using UDP-glucuronosyltransferase; however, this system is impaired by the marginal transport of alkylphenol-glucuronide possessing long alkyl chain, such as nonylphenol.

Broad and potent anti-influenza virus spectrum of epigallocatechin-3-O-gallate-monopalmitate.

Numerous influenza pandemics in the last century resulted in the death of millions of people worldwide. With the current and future threats of influenza pandemics development of new antiviral compounds remains a great demand. However, currently only a limited number of drugs are available for the treatment and prophylaxis of influenza. A neuraminidase (NA) inhibitor, oseltamivir phosphate, is the most commonly used antiviral drug. However, a number of reports suggest the emergence of oseltamivir phosphate resistance in new seasonal influenza viruses and highly pathogenic avian influenza (H5N1) (for example, de Jong et al, 2005). (–)-Epigallocatechin-3-O-gallate (EGCG: 1), a major component of green tea plant (Camellia sinensis), has been recognized to possess antiviral properties. Reports on the anti-influenza activity of 1 found that it inhibited virus adsorption (Nakayama et al, 1993), as well as acidification of endosomes and lysosomes (Imanishi et al, 2002). Such virus inhibition activity is different from other current NA or proton pump inhibitors, suggesting that 1 can be developed into a new class of antiviral compounds that are effective against current drug resistant influenza strains. However, 1 has not been used as an antiviral compound because of its poor lipid membrane permeability and low chemical stability. It was previously reported that the introduction of long alkyl chain groups to 1 improved its lipid membrane permeability (Tanaka et al, 1998), while protection of its hydroxyl groups with acyl groups increased its chemical stability under physiological conditions (Lam et al, 2004). Recently, we reported a method to synthesize EGCG-fatty acid monoesters using lipase-catalyzed transesterification and demonstrated that EGCG-fatty acids monoesters possessed improved influenza virus inhibitory effect against influenza A/PR8/34/(H1N1) in an alkyl length dependent manner (Mori et al, 2008). Here, we investigated the spectrum of influenza virus inhibition activity of EGCG (1) and EGCG-C-16 (2). As shown in Figures 1a, 2 is a mixture of four regio-isomers and the ratio of each regio-isomer 2a:2b:2c:2d is 38:35:7:20, respectively. Because the B-ring-modified esters (2a and 2b) and D-ring-modified esters (2c and 2d) showed exactly the same antiviral activities (data not shown), we used the mixture of four regio-isomers (2a-d) in the following assays. Figure 1. A. Chemical structure of EGCG (1) and EGCG-C16 (2). 2 is a mixture of four regio-isomers (2a-d). 2a: R2=R3= R4=H, R1= CO(CH)14CH3, 2b: R1=R3=R4=H, R2= CO(CH)14CH3, 2c: R1=R2=R4=H, R3= CO(CH)14CH3, 2d: R1=R2=R3=H, R4= CO(CH)14CH3. B. Avian influenza virus ... A series of human influenza viruses, an experimental strain (A/Puerto Rico/8/34/(H1N1)), vaccine strains (A/Beijing/262/95/(H1N1), A/Panama/2007/99/(H3N2), and B/Yamanashi/166/98/), drug-resistant strains (Yokohama/77/2008/(H1N1) OPR: oseltamivir phosphate-resistant (OPR), Yokohama/63/2007/(H1N1) AR: amantadine-resistant (AR), A/Yokohama/91/2008/(H1N1) OPR/AR: (OPR/AR) and avian pathogenic influenza (A/Duck/Hong Kong/342/78/(H5N2)), were directly incubated with 1 or 2 for 30 min prior to inoculation into MDCK cells. The cells were inoculated with virus, with or without the compounds, for 1hr, and the direct virus inhibitory effects were assessed by a plaque formation assay at 54hr post-incubation. The plaque inhibition activity was calculated relative to no compound. The cytotoxicity of compounds on MDCK cells were evaluated by the MTT cell proliferation assay. Briefly, the cells were incubated with 1 or 2 for 24hr for the MTT assay. The results of the plaque inhibition assay and MTT assay are expressed as mean ± standard error of three independent experiments. We also investigated the virus inhibition activity of 1 and 2 on avian influenza A/Duck/Hong Kong/342/78 (H5N2) virus in ovo using 11-day-old chicken embryonated eggs (n=12) inoculated with compound-treated or untreated viruses. As shown in Table 1, both 1 and 2 showed broad virus inhibitory effects on MDCK cells. The EC50 values of 2 on these viruses were between 10 to 61nM, which were approximately 7.1 to 44-fold lower than those of 1. The CC50 value of 2 was 82μM, which was only 3.1-fold lower than that of 1 (255 μM). Thus, the SI value of 2 was improved 2.2 to 14-fold compared to 1. Table 1. Direct virus inhibitory effect of 1 and 2 based on the plaque formation assay With respect to the avian influenza virus inhibition assay in ovo, virus treated with 1, zanamivir, or oseltamivir phosphate showed a moderate viral inhibitory effect (Figures 1b). However, 2 almost completely inhibited the infection (Figures. 1b), and the efficacy was retained even at 0.1μM (data not shown). In summary, 2 inhibited human and avian influenza A and B viruses, including drug-resistant viruses. 2 was found to be more effective than neuraminidase inhibitors, and strongly inhibited the infection of avian influenza (H5N2) virus in chicken embryonated eggs. This unique viral inhibitory action has the potential to be utilized to effectively control a broad spectrum of influenza viruses.