Get Plastic Out Of Your Diet our foods—plastics, pesticides, growth hormones, irradiation, and microwave. This is clear from the mass of expert and citizen testimony against such technologies that In the interests of a healthy population, we regulatory agencies bend over backwards and have decided to re-print this well jump through flaming hoops to please their researched and irr
Hepatitiscentral.netTing Zhang,1,2 Yuan Li,1 Jian-Ping Lai,1 Steven D. Douglas,1 David S. Metzger,3 Charles P. O’Brien,3 and Wen-Zhe Ho1 Alcohol consumption accelerates liver damage and diminishes the anti– hepatitis C virus
(HCV) effect of interferon alfa (IFN-␣) in patients with HCV infection. It is unknown,
however, whether alcohol enhances HCV replication and promotes HCV disease progres-
sion. The availability of the HCV replicon containing hepatic cells has provided a unique
opportunity to investigate the interaction between alcohol and HCV replicon expression.
We determined whether alcohol enhances HCV RNA expression in the replicon containing
hepatic cells. Alcohol, in a concentration-dependent fashion, significantly increased HCV
replicon expression. Alcohol also compromised the anti-HCV effect of IFN-␣. Investigation
of the mechanism(s) responsible for the alcohol action on HCV replicon indicated that
alcohol activated nuclear factor B (NF-B) promoter. Caffeic acid phenethyl ester (CAPE),
a specific inhibitor of the activation of NF-B, abolished alcohol-induced HCV RNA ex-
pression. In addition, naltrexone, an opiate receptor antagonist, abrogated the enhancing
effect of alcohol on HCV replicon expression. In conclusion, alcohol, probably through the
activation of NF-B and the endogenous opioid system, enhances HCV replicon expression
and compromises the anti-HCV effect of IFN-␣. Thus, alcohol may play an important role
in vivo as a cofactor in HCV disease progression and compromise IFN-␣– based therapy
against HCV infection. (HEPATOLOGY 2003;38:57-65.)
HepatitisCvirus(HCV)isresponsibleforthe cipientsoflivertransplantation.HCV,firstmolecularly vast majority of cases of transfusion-associated cloned in 1989,1 is a positive-strand RNA virus of the and community-acquired non-A, non-B hepa- flavivirus family with a genome size of ϳ10 kb, which titis1,2 and infects an estimated 170 million people world- encodes a number of structural (core, E1, E2, and p7) and wide.3 The seroprevalence of anti-HCV antibody in the nonstructural (NS2, NS3, NS4A, NS4B, NS5A and United States has been estimated at 1.8%, which corre- NS5B) proteins.6 HCV has at least 6 distinct but related sponds to approximately 4 million people.4 HCV is the genotypes with more than 50 subtypes, and genotype 1 is leading cause of chronic viral hepatitis in the United the most common in the United States, Europe, and most States,5 and HCV-infected individuals are the major re- parts of Asia. HCV typically escapes clearance by thehost’s immune system and leads to the establishment of apersistent infection in approximately 70% of infected in- Abbreviations: HCV, hepatitis C virus; IFN-␣, interferon alfa; CAPE, caffeic dividuals.7 The consequences of a subset of patients with acid phenethyl ester; 4-MP, 4-methylpyrazole; IgG, immunoglobulin G; RT-PCR, chronic HCV infection are cirrhosis, liver failure, and reverse-transcription polymerase chain reaction; mRNA, messenger RNA; NF-B,nuclear factor kappa B; 5Ј-NCR, 5Ј-noncoding region; MB, molecular beacon. hepatocellular carcinoma.8,9 Treatment of HCV with in- From the 1Division of Allergy and Immunology, Joseph Stokes Jr. Research Insti- terferon alfa (IFN-␣) and ribavirin is associated with a tute at The Children’s Hospital of Philadelphia, Department of Pediatrics, Univer- sustained response rate of less than 50%.7,10,11 These lim- sity of Pennsylvania School of Medicine, Philadelphia, PA; the 2Departmentof Infectious Diseases, The Children’s Hospital of Fudan University, Shanghai, ited therapeutic efficacies and the absence of an effective China; and the 3Department of Psychiatry, The Center for Studies of Addiction, HCV vaccine underscore the importance of research on University of Pennsylvania School of Medicine, Philadelphia, PA. factors that enhance HCV infection and compromise Received November 20, 2002; accepted April 24, 2003. Supported by grants from the National Institutes of Health (DA12815 and DA16022 to W.-Z. Ho; MH49981 and AA13547 to S.D. Douglas). Alcohol is the most commonly used and abused drug Address reprint requests to: Dr. Wen-Zhe Ho, Division of Allergy and Immunol- in the United States. Alcohol abuse significantly affects ogy, The Children’s Hospital of Philadelphia, University of Pennsylvania School ofMedicine, 34th St. and Civic Center Blvd., Philadelphia, PA 19104. E-mail: morbidity and mortality from infectious diseases.12 Alco- email@example.com; fax: 215-590-2025. hol consumption accelerates liver damage, diminishes Copyright 2003 by the American Association for the Study of Liver Diseases. hepatocellular carcinoma in patients with chronic HCV infection.13-18 Alcohol added in vivo and in vitro also im- Southern blots are negative for replicon nucleic acid inte- pairs liver parenchymal cells and various functions of im- grated into the host chromosome in Huh.8 cells.21 Using mune cells, including monocytes, T cells, and natural a real-time reverse-transcription polymerase chain reac- killer cells, which contribute to hepatocyte damage in tion (RT-PCR) assay that we have recently developed,22 chronic HCV infection.12,19 Alcohol consumption and we were able to detect ϳ2,500 to 5,000 copies of HCV viral hepatitis infection, both recognized as major causes messenger RNA (mRNA) per Huh.8 cell. Huh.2 is a cell of liver disease worldwide, frequently coexist in patients clone containing a G418 selectable HCV replicon with with chronic liver disease.13,17,18 Alcohol and HCV most HCV NS5A mutation (S1172P).21 FCA-1 cells contain a likely act synergistically to promote the development and subgenomic replicon from a known infectious HCV progression of liver damage.17,18 There is little direct in- clone with several consensus mutations in NS3 as well as formation available concerning the effects of alcohol in NS5A (NS3: E177G; NS5A: D1229E, I1299V).20 abuse on HCV replication in hepatic cells. This lack of The HCV replicon containing cells and Huh7 cells were knowledge about the impact of alcohol abuse on HCV is maintained in Dulbecco Modified Eagle’s Medium a major barrier to fundamental understanding of HCV- (Gibco-BRL, Grand Island, NY) supplemented with related morbidity and mortality in alcohol abusers with 10% (vol/vol) fetal bovine serum (HyClone, Logan, UT), HCV infection. Thus, it is critical to investigate the im- 100 U/mL penicillin, 100 g/mL streptomycin, 2 pact of alcohol abuse on HCV replication in the target mmol/L L-glutamine, and 0.1 mmol/L minimum essen- cells such as hepatic cells. We investigated whether alco- tial medium nonessential amino acids in a humidified hol enhances HCV RNA expression in HCV replicon containing cell lines.20,21 We also studied whether the in medium for Huh.8, Huh.2, and FCA-1 cells contained vitro addition of alcohol to these cells compromises the G418 (750 g/mL). Cells were passaged every 2 or 3 days and seeded in 24-well plates at a density of 105 cells/well.
Alcohol Treatment. The HCV replicon containing
Materials and Methods
cells (Huh.8, Huh.2, and FCA-1) and Huh7 cells platedin 24-well plates were incubated with or without alcohol Reagents. Alcohol was purchased from Aaper Alcohol
(ϳ10 to 100 mmol/L) for up to 96 hours. For multiple and Chemical Company (Shelbyville, KY). Recombinant time-point treatment, the cells were treated with alcohol IFN-␣ was obtained from R&D Systems Inc. (Minneap- daily. To study whether alcohol interferes with the anti- olis, MN). The opiate receptor antagonist naltrexone was HCV effect of IFN-␣, Huh.8 cells were incubated with obtained from Sigma (St. Louis, MO). Caffeic acid phen- alcohol (100 mmol/L) and/or IFN-␣ (100 U/mL).
ethyl ester (CAPE) was purchased from Calbiochem-No- IFN-␣ was added to the cell cultures for 30 minutes be- vabiochem Corp. (San Diego, CA). 4-Methylpyrazole (4- fore the addition of alcohol. We selected IFN-␣ at the MP) and cyanamide were obtained from Sigma. Mouse concentration that was tested to have an anti-HCV effect monoclonal anti-HCV NS5 antibody was a gift obtainedfrom Dr. Bill Sun (Thomas Jefferson University, Phila- in HCV replicon containing cells.20,21 In the experiments delphia, PA). Horseradish peroxidase-conjugated goat to investigate whether CAPE, a specific inhibitor of the anti-mouse immunoglobulin G (IgG) was purchased activation of nuclear factor B (NF-B), suppresses the from Jackson Immune Research Labs (West Grove, PA) enhancing effect of alcohol on HCV RNA expression, Cell Lines. Huh.8 and Huh7 cell lines were obtained
Huh.8 cells were incubated with CAPE for 30 minutes from Dr. Charles Rice (The Rockefeller University, New before the addition of alcohol. The selected concentration York, NY, and Apath, L.L.C., St. Louis, MO). Huh.2 of CAPE (20 g/mL) was based on our pilot experiments, cells were obtained from the National Institutes of Health which showed that CAPE at the concentrations of 20 to AIDS Research and Reference Reagent Program. FCA-1 30 g/mL resulted in a maximum inhibitory effect on cells were obtained from Dr. Christoph Seeger (Fox HCV RNA expression without cytotoxic effect (trypan Chase Cancer Center, Philadelphia, PA). Huh7, a human blue dye staining) on Huh.8 cells (data not shown). In hepatoma cell line, is the parental cell line of Huh.8, addition, our pilot experiments showed that there was no Huh.2, and FCA-1. Huh.8 is a cell clone containing a cytotoxic effect of alcohol at the concentrations of 100 G418 selectable HCV RNA replicon with wild-type mmol/L23 or less on the HCV replicon containing cells as HCV nonstructural protein NS5A sequence.21 The rep- shown by trypan blue dye staining. In some experiments, licon in Huh.8 cells contains both the 5Ј nontranslating Huh.8 cells were preincubated with either 4-MP (0.1 region and 3Ј nontranslating region as well as the open mol/L) or cyanamide (0.2 mol/L) or naltrexone (10Ϫ8 reading frame of the nonstructural proteins NS3-5B.21 mol/L) for 30 minutes before the addition of alcohol.
To minimize alcohol evaporation that diminishes alco- The PCR buffer contained 5-carboxy-X-rhodamine (500 hol concentration in the plates, we maintained alcohol- nmol/L) as the reference dye for normalization of the reac- treated cells in the plates sealed with PARAFILM tions. Any possible fluctuations in 5-carboxy-X-rhodamine (American National Can, Greenwich, CT). Furthermore, signal are used to correct the sample signal. All amplification to avoid evaporated alcohol contamination of control cul- reactions were performed in duplicate. A standard curve was ture plates, alcohol-treated and control plates were main- generated with 10-fold dilutions of HCV 5Ј-NCR RNA control22 prequantitated by a spectrophotometer (Eppen- RNA Extraction. Total RNA (1 g) was extracted
dorf Scientific, Inc., Westbury, NY).
from HCV replicon containing cells as well as from Huh7 Immunoblot Assay. Total cell lysates were prepared
cells using Tri-Reagent (Molecular Research Center, Cin- from Huh.8 cells (105 cells per well in a 24-well plate) cinnati, OH). In brief, total RNA was extracted by a incubated with or without alcohol for 72 hours using a single-step guanidinium thiocyanate-phenol-chloroform lysis buffer (Promega, Madison, WI). The protein con- extraction. After centrifugation at 13,000g for 15 min- centration was determined by DC protein assay kit (Bio- utes, the RNA-containing aqueous phase was precipitated Rad, Hercules, CA). The immunoblot analysis of HCV in same volume isopropanol. RNA precipitates were then NS5 protein was performed using a Bio-Dot SF apparatus washed once in 75% alcohol and resuspended in 20 L of as described by the manufacturer. Briefly, total protein (0.5 g) extracted from Huh.8 cells incubated with or Real-Time RT-PCR for HCV RNA Quantification.
without alcohol (100 mmol/L) was applied onto a nitro- We used the real-time RT-PCR assay newly developed in cellulose membrane. After the blockage with phosphate our laboratory for the quantification of HCV RNA.22 The buffered saline containing 5% nonfat dry milk for 1 hour real-time PCR was performed with 1/10th of cDNA de- at room temperature, the membrane was incubated with a rived from RNA (1 g) extracted from the cells using ABI mouse monoclonal anti-HCV NS5 antibody at 4°C over- Prism 7700 Sequence Detection System (Applied Biosys- night. After washing 3 times with Tween phosphate-buff- tems, Foster City, CA). We designed the primer pair ered saline, the nitrocellulose membrane was incubated (S130/AS311) that is specific for the recognition of highly with horseradish peroxidase-conjugated goat anti-mouse conservative 5Ј-noncoding region (5Ј-NCR) in the HCV IgG for 1 hour. Bound antibody was visualized by devel- genome.22 Their sequences are as follows: 5Ј-CGGGAG- oping the membrane in SuperSignal West Pico chemilu- AGCCATAGTGGTCTGCG-3Ј (S130) and 5Ј-CTC- minescent substrate kit (Pierce, Rockford, IL). The results were recorded on film (Eastman Kodak, Rochester, NY).
probe (molecular beacon [MB]) sequence for HCV 5Ј-NCR NF-B Promoter Activation Assay. The plasmid
was selected within the primer pair of S130 and AS311, (pNF-B-Luc) containing NF-B promoter linked with which was designed to be perfectly complementary to the a luciferase gene was generated by Dr. Daniel Petrak.24 target sequence in 5Ј-NCR of HCV genome.22 The follow- Two copies of the mouse light chain enhancer25 were ing is the sequence of the MB: 5Ј-FAM- GCGAGCCAC- cloned into pBLCAT3 vector,26 and then the construct CGGAATTGCCAGGACGACCGCTCGC-DABCYL-3Ј. was modified by replacing the chloramphenicol acetyl-The stem sequence (underlined) of MB does not comple- transferase reporter with the luciferase gene obtained ment the sequences within the loop region. The MB was from pGEM-Luc.24 Plasmid DNA was prepared by Mini- labeled at the 5Ј end with 6-carboxyfluorescein (6-FAM) and prep techniques, according to the manufacturer’s instruc- the quencher 4-(4Ј-dimethylaminophenylaso) benzoic acid tions (Wizard Plus Minipreps, Promega, Madison, MI) (DABCYL) at the 3Ј end. Both primers and MB were sus- and used in transfection experiments. For transfection pended in Tris-EDTA buffer and stored at Ϫ30°C. Thermal experiments, Huh.8 and Huh7 cells were seeded in a cycling conditions were designed as follows: initial denatur- 6-well tissue culture plate at the density of 3 ϫ 105 cells ation at 95°C for 10 minutes, followed by 40 cycles of 95°C per well 1 day before the transfection. The cells were for 15 seconds and 60°C for 60 seconds. Fluorescence mea- transfected with the pNF-B-Luc using FuGENE 6 surements were recorded during each annealing step. At the Transfection Reagent (Roche Diagnostic Corporation, termination of each PCR run, the data were automatically Indianapolis, IN) with a ratio of FuGENE 6: plasmid 6:1 analyzed by the system and amplification plots were gener- (L:g). Six hours after the transient transfection, the ated. For each PCR reaction, 2 L of cDNA template was cells were incubated with or without alcohol (100 added to 48 L of PCR master mixture (5 L of 1ϫ PCR mmol/L) for 24 hours. At the termination of the experi- buffer II, 5 mmol/L MgCl2, 300 nmol/L dNTP, 400 ments, the cells were harvested and washed twice with nmol/L of each primer, 1.5 U of AmpliTaq Gold DNA phosphate buffered saline by centrifugation at 3,300g for polymerase, 400 nmol/L of MBs, and 24.7 L of water).
3 minutes at room temperature. The cell pellets were lysed Effect of Alcohol on HCV Replicon. To evaluate the
effect of alcohol on HCV RNA expression in HCV rep-licon containing cells, we incubated Huh.8, Huh.2, andFCA-1 cells with or without alcohol. The alcohol concen-trations selected for the study were based on our pilotexperiments, which showed that alcohol at the concentra-tion of 100 mmol/L or lower had no cytotoxic effect onHuh.8 cells (data not shown). Total cellular RNA ex-tracted from the cell cultures was subjected to the real-time RT-PCR for the quantification of HCV RNA levels72 hours after alcohol treatment. The addition of alcoholto Huh.8 cell cultures increased HCV RNA expression ina concentration-dependent fashion (Fig. 1A). To deter-mine whether the effect of alcohol on HCV RNA expres-sion is time dependent, we incubated Huh.8 cells withalcohol (100 mmol/L) and harvested cells at 4 differenttime points (24 hours, 48 hours, 72 hours, and 96 hours)after alcohol treatment for HCV RNA quantification.
The maximum HCV RNA expression in alcohol-treatedHuh.8 cells was observed at 72 hours post-treatment (Fig.
1B). Because NS5 protein plays a critical role in HCV Fig. 1. Effect of alcohol on HCV RNA expression in Huh.8 cells. (A) replication,21,27 we also examined whether alcohol alters Effect of alcohol on HCV RNA expression in Huh.8 cells at different HCV NS5 protein expression in Huh.8 cells using the concentrations. Huh.8 cells (105 cells/mL/well) plated in a 24-well platewere incubated with or without alcohol at indicated concentrations for 72 immunoblot assay. In comparison with untreated Huh.8 hours. (B) Effect of alcohol on HCV RNA expression in Huh.8 cells at cells, alcohol (100 mmol/L)-treated cells express higher different time points. Huh.8 cells were incubated with or without alcohol levels of NS5 protein as shown by the enhanced intensity (100 mmol/L). Total cellular RNA extracted from the cell cultures was of protein band (Fig. 2). To determine whether the en- subjected to real-time RT-PCR for HCV and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA quantification. The data are expressed as hancing effect of alcohol on HCV replicon is not cell HCV RNA copy numbers per reaction, which was normalized based on the clone dependent, we examined the effect of alcohol on ratio of HCV RNA/GAPDH mRNA that was amplified in the same plate by HCV replicon in 2 additional HCV replicon containing the real-time RT-PCR. The data shown are the mean Ϯ SD of triplicatecultures, and the experiment was repeated 5 times with similar results cell clones (Huh.2 and FCA-1). Similar to the findings (*P Ͻ .05; **P Ͻ .01).
observed in Huh.8 cells, alcohol increased HCV RNAexpression in both Huh.2 and FCA-1 cells (Fig. 3).
Alcohol Inhibits the Anti-HCV Effect of IFN-␣.
with 0.25 mL of 1ϫ Reporter Lysis Buffer (Promega) and Alcohol consumption impairs the therapeutic response to a cycle of freezing and thawing in dry ice. Cell-free lysates IFN-␣ in patients with HCV infection.14-16 Because were obtained by centrifugation at 10,000g for 30 secondsat room temperature. The effects of alcohol on the acti-vation of NF-B promoter in these transiently transfectedcells were determined by NF-B promoter-driven lucif-erase activity. Luciferase activity in cell lysate (50 L persample) was quantitated using a Luciferase Assay System(Promega) and a luminometer. The results are presentedas relative light units.
Statistical Analysis. All variables were tested in trip-
Fig. 2. Effect of alcohol on HCV NS5 protein expression in Huh.8 cells.
Huh.8 cells (105 cells/mL/well) plated in a 24-well plate were incubated licate, and experiments were repeated at least 3 times.
with or without alcohol (100 mmol/L) for 72 hours. Cell lysates were Triplicate wells had variability of less than 15%. One-way quantified by DC protein assay kit. Equal amounts (0.5 g) of protein ANOVA was used to test for the difference in means and extracted from alcohol-treated and untreated (control) Huh.8 cells wereapplied onto a nitrocellulose membrane for immunoblot assay. The post-hoc t test was used for comparisons. The differences results were recorded on the film (2-minute exposure). One representa- were considered significant if P Ͻ .05.
Fig. 3. Effect of alcohol on HCV RNA expression in different HCV replicon cell clones. Huh.8, Huh.2, and FCA-1 cells (105 cells/mL/well)plated in a 24-well plate were incubated with or without alcohol (100mmol/L) for 72 hours. Total cellular RNA extracted from the cell cultureswas subjected to real-time RT-PCR for HCV and GAPDH RNA quantifica-tion. The data are expressed as HCV RNA copy numbers per reaction,which was normalized based on the ratio of HCV RNA/GAPDH mRNA thatwas amplified in the same plate by the real-time RT-PCR. The resultsshown are the mean Ϯ SD of triplicate cultures, and the experiment wasrepeated 3 times with similar results (*P Ͻ .05; **P Ͻ .01).
IFN-␣ inhibited HCV RNA expression in Huh.8cells,20,21 we examined whether alcohol has a negative Fig. 5. The role of NF-B in alcohol-induced HCV RNA expression in Huh7 and Huh.8 cells. (A) Effect of alcohol on NF-B promoter in impact on the anti-HCV effect of IFN-␣ in HCV repli- pNF-B-Luc-transfected Huh7 and Huh.8 cells. Huh7 and Huh.8 cells con containing cells. As expected, IFN-␣, when added to (3 ϫ 105 cells/2 mL/well) plated in a 6-well plate were transfected with Huh.8 cell cultures, significantly inhibited HCV RNA pNF-B-Luc for 24 hours and then incubated with or without alcohol expression (up to 90%) in Huh.8 cells (Fig. 4). This anti- (100 mmol/L) and/or CAPE (20 g/mL) for 24 hours. NF-B promoter-directed luciferase activity quantitated from the cell-free lysates were HCV effect of IFN-␣ on HCV, however, was attenuated normalized using the total protein of the cell-free lysates. The data are means Ϯ SD of triplicate cultures, and the experiment was repeated 3 Alcohol Activates NF-B. To investigate the possible
times with similar results (*P Ͻ .05; **P Ͻ .01). (B) Effect of CAPE onalcohol-enhanced HCV RNA expression in Huh.8 cells. Huh.8 cells (105 mechanism(s) responsible for alcohol-mediated upregula- cells/mL/well) plated in a 24-well plate were incubated with or without tion of HCV RNA expression, we examined whether al- alcohol (100 mmol/L) and/or CAPE (20 g/mL) for 72 hours. Totalcellular RNA was extracted from the cell cultures and was then subjectedto real-time RT-PCR for HCV and GAPDH RNA quantification. The data areexpressed as HCV RNA copy numbers per reaction, which was normalizedbased on the ratio of HCV RNA/GAPDH mRNA that was amplified in thesame plate by the real-time RT-PCR. The results shown are the mean ϮSD of triplicate cultures, and the experiment was repeated 5 times withsimilar results (**P Ͻ .01).
cohol activates NF-B, an important transcriptionalnuclear factor that controls viral replication and cytokineproduction.28-30 Because HCV replicon expression inHuh.8 cells may activate NF-B, we transfected bothHuh7 cells, the parental cell line of Huh.8, and Huh.8cells with the plasmid (pNF-B-Luc) that contains Fig. 4. Effect of alcohol on anti-HCV effect of IFN-␣ in Huh.8 cells.
NF-B promoter-linked to a luciferase gene. The trans- Huh.8 cells (105 cells/mL/well) plated in a 24-well plate were incubated fected cells (24 hours post-transfection) were then incu- with or without alcohol (100 mmol/L) and/or IFN-␣ (100 U/mL) for 72hours. Total cellular RNA extracted from the cell cultures were subjected bated with or without alcohol. Alcohol enhanced the to real-time RT-PCR for HCV and GAPDH RNA quantification. The data are NF-B promoter-directed luciferase activity in both expressed as the percentage reduction in HCV RNA relative to the Huh7 and Huh.8 cells (Fig. 5A). The basal levels of untreated control levels (percent control). The results shown are the mean Ϯ SD of triplicate cultures, and the experiment was repeated 5 B promoter-directed luciferase activity in Huh.8 times with similar results (**P Ͻ .01).
cells are higher than those in Huh7 cells (Fig. 5A). CAPE, a specific inhibitor of the activation of NF-B, abrogatedalcohol-induced NF-B promoter activation (Fig. 5A) inboth Huh7 and Huh.8 cells. To further determinewhether NF-B activation is directly involved in alcohol-induced HCV RNA expression, we examined whetherCAPE abrogates the enhancing effect of alcohol in Huh.8cells. CAPE, when added to Huh.8 cell cultures, com-pletely blocked the enhancing effect of alcohol on HCVRNA expression (P Ͻ .01) (Fig. 5B).
Naltrexone Abrogates Alcohol Action. Several lines
of evidence indicate that alcohol interferes with the en-dogenous opioid system.31-33 Morphine has the ability topromote HCV RNA expression in Huh.8 cells, which isblocked by naltrexone (Li Y, unpublished data). In addi-tion, we have documented that both Huh7 and Huh.8cells express -opioid receptor (Li Y, unpublished data).
We therefore hypothesized that alcohol may activate theendogenous opioid system, which plays a role in alcohol-induced HCV RNA expression. To test this hypothesis,we first incubated Huh.8 cells with or without naltrexone(an opiate receptor antogonist) for 30 minutes before theaddition of alcohol to the Huh.8 cell cultures. Naltrexonenot only reversed the enhancing effect of alcohol on HCVRNA expression (Fig. 6A), but also inhibited the alcohol- Fig. 6. Antagonizing effect of naltrexone on alcohol-induced NF-B induced NF-B promoter activation (Fig. 6B).
promoter activation and HCV RNA expression in Huh.8 cells. (A) Effect of 4-MP and Cyanamide Inhibit Alcohol-Induced
naltrexone on alcohol-enhanced HCV RNA expression in Huh.8 cells.
HCV RNA Expression. To determine whether the en-
Huh.8 cells (105 cells/mL/well) plated in a 24-well plate were incubatedwith naltrexone (10Ϫ8 mol/L) for 30 minutes before the addition of hancing effect of alcohol on HCV RNA expression in alcohol (100 mmol/L). Total cellular RNA extracted from the cell cultures Huh.8 cells is specifically through the alcohol metabolite was then subjected to real-time RT-PCR for HCV RNA quantification 72 acetaldehyde, we examined whether 4-MP, an inhibitor hours after alcohol treatment. The data are expressed as HCV RNA copynumbers per reaction, which was normalized based on the ratio of HCV of alcohol dehydrogenase, or cyanamide, an inhibitor of RNA/GAPDH mRNA that was amplified in the same plate by the real-time acetaldehyde dehydrogenase, blocks the enhancing effect RT-PCR. The results shown are the mean Ϯ SD of triplicate cultures, and of alcohol on HCV RNA expression in these cells. Pre- the experiment was repeated 5 times with similar results (**P Ͻ .01).
treatment of Huh.8 cells with 4-MP or cyanamide atten- (B) Effect of naltrexone on alcohol-induced NF-B promoter activation.
Huh.8 cells (3 ϫ 105 cells/2 mL/well) plated in a 6-well plate were uated the alcohol-induced increase of HCV RNA transfected with pNF-B-Luc for 24 hours and then incubated with or expression (Fig. 7), whereas the addition of 4-MP or cy- without alcohol (100 mmol/L) and/or naltrexone (10Ϫ8 mol/L) for 24 anamide alone to the cell cultures had little impact on hours. NF-B promoter-directed luciferase activity was normalized usingthe total protein of the cell-free lysates. The data are means Ϯ SD of triplicate cultures, and the experiment was repeated 3 times with similarresults (**P Ͻ .01).
Alcohol abuse is a major cofactor in the development of HCV associated liver disease.16-18 Chronic alcohol number of responders in IFN therapy decreased as alcohol abuse mediates liver damage as a result of increase in pro- intake increased.40 These in vivo data strongly support the duction of proinflammatory cytokines.34-37 In the setting hypothesis that alcohol plays a role as a cofactor in pro- of chronic HCV infection, alcohol ingestion has an addi- moting HCV RNA expression. To directly link the action tional effect of diminishing immune clearance and in- of alcohol with HCV expression, we used recently devel- creasing viral burden to hasten the onset of cirrhosis and oped HCV replicon containing cell lines for this study.
hepatocellular carcinoma.38 Serum HCV RNA levels The establishment of the subgenomic replicon system were significantly higher in habitual alcohol drinkers with represents major progress in the molecular biology of chronic HCV infection than in infrequent alcohol drink- HCV replication,21,27,41 which provides the first effective ers with chronic HCV.39 HCV RNA levels were signifi- cellular system available for the study of the dynamics of cantly higher in alcohol drinkers than abstainers, and the virus replication. Although the HCV replicon system B. This finding is in agreement with a recent studyshowing that HCV NS5A alone or in the context of all theHCV nonstructural proteins is capable of inducing reac-tive oxygen species and activates NF-B.43 Interestingly,alcohol also has the ability to induce oxidative stress,which has been shown to activate NF-B.44 Thus, wehypothesized that the activation of NF-B is one of themechanisms by which alcohol enhances HCV replication.
We showed that CAPE, a specific inhibitor of the activa-tion of NF-B,45 blocked alcohol-induced NF-B pro-moter activation (Fig. 5A), suggesting that the activationof NF-B may be involved in alcohol-mediated upregu- Fig. 7. Effect of 4-MP, cyanamide on HCV RNA expression. Huh.8 cells (105 cells/mL/well) plated in a 24-well plate were incubated with 4-MP lation of HCV RNA expression. To confirm this specu- (0.1 mol/L) or cyanamide (0.2 mol/L) for 30 minutes before the lation, we examined whether CAPE could block the addition of alcohol (100 mmol/L). Total cellular RNA extracted from the enhancing effect of alcohol on HCV RNA expression.
cell cultures was then subjected to real-time RT-PCR for HCV and GAPDHRNA quantification 72 hours after alcohol treatment. The data are Our data showed that CAPE, when added to Huh.8 cell expressed as HCV RNA copy numbers per reaction, which was normalized cultures, abolished the enhancing effect of alcohol on based on the ratio of HCV RNA/GAPDH mRNA that was amplified in the HCV RNA expression (Fig. 5B). These data indicate that same plate by the real-time RT-PCR. The results shown are the mean Ϯ SD of triplicate cultures, and the experiment was repeated 3 times with similar results (**P Ͻ .01).
responsible for its action on HCV RNA expression.
Several lines of evidence indicate that alcohol interferes with the endogenous opioid system,31-33 suggesting that mimics only some aspects of HCV replication, this cell the endogenous opioid system may be involved in alcohol system provides an important means of investigating viral action. The endogenous opioids play a key role in the RNA and protein synthesis21,27,41 and of characterizing rewarding (addictive) properties of alcohol.46,47 Pretreat- those factor(s) that regulate HCV RNA expression. Using ment with opiate receptor antagonists (e.g., Naltrexone) this system, we have for the first time shown that alcohol decreases alcohol consumption in humans.48,49 Further- enhanced HCV replicon expression at both mRNA and more, treatment with naltrexone, an opiate antagonist, protein levels (Figs. 1 through 3). This enhancing effect of decreases ethanol consumption by alcoholic subjects.47-50 alcohol is mediated specifically through its metabolite ac- Opioid content, biosynthesis, and release are altered after etaldehyde, because 4-MP or cyanamide, the specific in- alcohol treatment, although there is considerable dis- hibitors of alcohol metabolism, blocks alcohol action on agreement regarding the direction of changes in the activ- HCV RNA expression (Fig. 7). These data indicate that ity of the endogenous opioid system by alcohol.46 We Huh.8 cells are able to metabolize alcohol, probably recently showed that Huh.8 cells express -opioid recep- through alcohol dehydrogenase, the main enzyme respon- tor and that morphine enhances HCV RNA expression in these cells (Li Y, unpublished data). Thus, we hypothesize The underlying mechanisms of interaction between that alcohol may activate the endogenous opioid system, alcohol and HCV remain largely unknown. In the present through which it induces HCV RNA expression. We study, we also investigated the possible mechanism(s) re- showed that naltrexone, an opiate receptor antagonist, sponsible for alcohol-mediated upregulation of HCV not only blocked the promoting effect of alcohol on HCV RNA expression. Because NF-B is a critical nuclear tran- RNA expression in Huh.8 cells (Fig. 6A), but also dimin- scription factor involved in the activation of viral replica- ished the enhancing effect of alcohol on NF-B promoter tion and cytokine production28-30 and alcohol enhances activation in these cells (Fig. 6B). These data strongly NF-B activation in different cell systems, such as suggest that the activation of the endogenous opioid sys- HepG2 cells,42 we investigated whether alcohol, through tem is also implicated in alcohol-induced HCV RNA ex- the activation of NF-B, enhances HCV expression in Huh7 and Huh.8 cells. We showed that alcohol enhanced Clinical trials indicate a therapeutic benefit of IFN-␣ NF-B promoter-directed luciferase activity in both treatment in chronic HCV infection.13,18 Currently avail- Huh7 and Huh.8 cells (Fig. 5A). Because the basal levels able combination therapy with IFN-␣ and ribavirin is, of NF-B promoter-directed luciferase activity in Huh.8 however, effective in less than 50% of treated sub- cells are higher than those in Huh7 cells, it seems that jects.7,10,11 Although a history of alcohol abuse is not a HCV replicon expression in Huh.8 cells can activate NF- contraindication to clinical therapy, continued alcohol use during therapy adversely affects response to HCV References
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Assessment of dna damage in coal open-cast mining workers using the cytokinesis-blocked micronucleus test and the comet assay
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i to t e n vAssessment of DNA damage in coal open-cast mining workers using thecytokinesis-blocked micronucleus test and the comet assayGrethel León-Mejía Lyda Espitia-Pérez , Luz Stella Hoyos-Giraldo , Juliana Da Silva ,Andreas Hartmann , João Antônio Pêgas Henriques ,, Milton Quintana a Laboratorio de In