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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

A portion of human immunodeficiency virus (HIV)-infected patients undergoing protease inhibitor (PI) therapy concomitantly consume or abuse alcohol leading to hepatic injury. The underling mechanisms are not known. We hypothesize that HIV PIs aggravate alcohol-induced liver injury through an endoplasmic reticulum (ER) stress mechanism. To address this, we treated mice, primary mouse hepatocytes (PMHs), and primary human hepatocytes (PHHs) with alcohol and the HIV PIs ritonavir (RIT) and lopinavir (LOP). In mice, RIT and LOP induced mild ER stress and inhibition of sarco/ER calcium-ATPase (SERCA) without significant increase in serum alanine aminotransferase (ALT) levels. However, a single dose of alcohol plus the two HIV PIs caused a more than five-fold increase in serum ALT, a synergistic increase in alcohol-induced liver lipid accumulation and ER stress response, and a decrease of SERCA. Mice treated with chronic HIV PIs and alcohol developed moderate liver fibrosis. In PMHs, the HIV drugs plus alcohol also inhibited SERCA expression and increased expression of glucose-regulated protein 78, C/EBP homologous protein, sterol regulatory element-binding protein 1c, and phosphorylated c-Jun N-terminal kinase 2, which were accompanied by a synergistic increase in cell death compared with alcohol or the HIV drugs alone. In PHHs, treatment with RIT and LOP or alcohol alone increased messenger RNA of spliced X box-binding protein 1 and decreased SERCA, which were accompanied by reduced levels of intracellular calcium. Alcohol combined with the HIV drugs significantly reduced intracellular calcium levels and potentiated cell death, which was comparable to the cell death caused by the SERCA inhibitor thapsigargin. Conclusion: Our findings suggest the possibility that HIV PIs potentiate alcohol-induced ER stress and injury through modulation of SERCA and maintaining calcium homeostasis could be a therapeutic aim for better care of HIV patients. (HEPATOLOGY 2012;)

Excessive alcohol consumption continues to be a leading cause of chronic liver disease worldwide.1, 2 Chronic alcohol-induced liver disease includes a spectrum of liver diseases, from fatty liver or simple steatosis, to alcoholic hepatitis, to hepatic fibrosis or cirrhosis.3 A growing list of primary and secondary risk factors for alcohol-induced liver disease has been identified. Primary factors can be alcohol metabolite-acetalaldehyde,4 oxidative stress from mitochondrial malfunction and cytochrome P450IIE1 (CYP2E1),5 increased endotoxin and inflammatory tumor necrosis factor α,6 centrilobular hypoxia,7 impaired one carbon metabolism,8, 9 impaired innate and adaptive immunity,3 and epigenetic alterations.8, 10 Secondary factors can be malnutrition or complications with diabetes, obesity, sex difference, smoking, or coinfection with hepatitis C virus and human immunodeficiency virus (HIV).11-13 Alcohol-induced unfolded protein response in the endoplasmic reticulum (ER) has evolved as an important factor contributing to alcoholic fatty liver and injuries.14-20 Potential causes for alcohol-induced ER stress are directly or indirectly related to alcohol metabolism, which include but may not be limited to: toxic acetaldehyde that forms protein adducts, increased homocysteine/homocysteine thiolactone that modifies proteins, oxidative stress that disturbs oxidative protein folding, alterations of S-adenosyl methionine to S-adenosyl homocysteine ratio that causes epigenetic modifications of ER stress response components, and disturbance of calcium homeostasis.14-20 In addition, interactions between the primary factors and secondary factors may determine severity of liver injury from alcohol-induced ER stress. For instance, the sarco/ER calcium-ATPase (SERCA), which regulates calcium store and ER homeostasis, has recently been identified to be a key factor involved in the complex obesity-induced ER stress and fatty liver injury.21, 22 It is not known whether SERCA also plays a role in interactions between the primary and secondary risk factors for alcohol-induced stress and liver injury.

HIV protease inhibitors (PIs) have been used in highly active antiretroviral combination therapy (HAART), which dramatically decreases the mortality rate of HIV-infected patients in western countries.23, 24 However, HIV PI–induced hepatotoxicity or lipodystrophy has emerged as an important potential complication of HAART. Lipid dysregulation in hepatocytes and macrophages has been associated with HIV PIs, most commonly with a single administration of full doses.24-27 Mechanisms that contribute to the side effects by HIV PIs in the liver are not well understood. Recent evidence suggests that HIV PIs induce ER stress response and promote liver injury.28-31 For instance, at therapeutic concentrations (i.e., single PI at 5-50 μM), most HIV PIs, individually or combined, were found to increase the levels of ER stress markers such as active sterol regulatory element-binding proteins (SREBPs), X box-binding protein 1 (Xbp1), activating transcription factor 4 (ATF-4), C/EBP homologous protein (CHOP) and caspase-12, and increase apoptosis in macrophages and rat hepatocytes.28-30 A few possible cellular stress events, such as depletion of ER calcium store and impaired lipid synthesis in the ER by HIV PIs are proposed to activate the unfolded protein response.28-30 In addition, other ER stress–causing factors such as alcohol or hepatitis C and B virus may aggravate HIV PI–induced ER stress and liver injury. Indeed, a portion of HIV-infected patients consume or abuse alcohol,12, 13, 32 which complicates the outcome of HAART. Thus, in order to provide new strategies for better care of HIV-infected patients, it is necessary to investigate possible synergism between HIV PIs and alcohol with respect to ER stress and liver injury. In this study, we examined ER stress response and cell death in mice and primary mouse hepatocytes (PMHs) and primary human hepatocytes (PHHs) and found that calcium homeostasis is a potential target of the interaction between alcohol and HIV drugs that worsens liver injury.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Animal Experiments.

Male C57BL/6 mice from The Jackson Laboratory (Bar Harbor, ME) were used. For acute alcohol feeding, the animals were fasted for 6 hours and gavaged with a liquid diet containing alcohol (Dyets, Inc., Bethlehem, PA) at a dose of 5 g/kg, or with an isocaloric control diet. In some experiments, the HIV protease inhibitor ritonavir (RIT) (15 mg/kg) or RIT combined with lopinavir (LOP) (15 mg/kg) were mixed with the alcohol liquid diet and delivered into the mice through gavage. Mice were sacrificed 20–24 hours after alcohol and/or drug administrations. For chronic treatment, mice were fed an alcohol diet at 3 g/kg for the first week and 5 g/kg for the next 4 weeks. RIT plus LOP (15 mg/kg each) were injected intraperitoneally into mice every other day from the second week to the fifth week. All animals were treated in accordance with the Guide for Care and Use of Laboratory Animals, and the study was approved by the local animal care and use committee.

Liver Pathological Parameters.

Serum alanine aminotransferase (ALT) and liver lipid extraction and analysis have been described.14, 30, 33 For Oil Red O staining, liver tissues were embedded in Tissue Freezing Medium Tissue-Tek® O.C.T. Compound, Sakura Finetek, snap-frozen, sectioned at 5 μm, and mounted on glass slides. The tissues on the slides were fixed in 10% formalin and stained with an Oil Red O isopropanol solution (Electron Microscopy Sciences, Hatfield, PA).

High-Performance Liquid Chromatography Detection of HIV PIs.

RIT and LOP in mouse plasma were extracted using the solid phase Sep-Pak Plus C18 cartridge from Waters (Waters, Milford, MD). The cartridge was conditioned with 1 mL of methanol followed by 1 mL of water, loaded with an aliquot of plasma (250 μL), and incubated at room temperature for 30 minutes. The cartridge was then washed with 1 mL of water, followed by 1 mL of methanol-water solution at 30:70 (vol/vol). The drugs were eluted with 1 mL of methanol, dried by blowing with nitrogen gas, and then dissolved in a mobile solution (250 μL) containing acetonitrile mixed with NaH2PO4 (20 mM [pH 6]) at 60:40 (vol/vol). The mixture contained 0.025% triethylamine. An aliquot of the drug extracts (50 μL) with 1 μg of RIT or LOP (added as internal control) was injected onto a C18 reverse-phase high-performance liquid chromatography (HPLC) column. The drugs were monitored spectrophotometrically at 210 nm. The drug quantity was calculated based on total area under the curve of HPLC spectrum for each drug.

In Vitro Studies with PMHs.

PMHs were provided by the Cell Culture Core (USC Research Center for Liver Disease), and the cell culturing has been described.33 The hepatocytes were treated with 15 μg/mL of RIT and/or LOP with or without alcohol (85 mM) for 24 hours. Vehicle (dimethyl sulfoxide [DMSO], <0.5%, vol/vol) was used as control. The treated hepatocytes were washed with cold phosphate-buffered saline (PBS) and were either stained for cell death or scratched off for protein extraction. The cells were doubly stained with Sytox green (Molecular Probes, Eugene) and Hoechst 33258 dye (Sigma) for 30 minutes at 37°C. The cell death count (combination of necrosis and apoptosis) has been described.30, 33

In Vitro Studies with PHHs.

PHHs were freshly isolated from alcohol- or drug-free patients and were cold-preserved in 24-well plates overlaid with collagen or in suspension, which were shipped by CellzDirect to Shelly Lu, who allocated enough PHHs for each set of experiments. Upon arrival, the original media were replaced with a fresh DMEN medium containing gentamicin (0.1%, vol/vol), dexamethasone (0.01%, vol/vol), fetal bovine serum (5.5%, vol/vol), L-glutamine (1%, vol/vol), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) (15 mM), and insulin (0.1%, vol/vol). The PHHs in the fresh medium were incubated in a humidified incubator at 37°C for 8-12 hours to acclimatize, after which the medium was replaced with a serum-free DMEN medium containing penicillin-streptomycin (0.5%, vol/vol), dexamethasone (10 ppm), ITS (1%, vol/vol), L-glutamine (1%, vol/vol), and HEPES (15 mM). The PHHs were then treated with RIT (5-15 μg/mL) and LOP (10-15 μg/mL) and/or alcohol (35-85 mM), tunicamycin (3 μg/mL), thapsigargin (6 μg/mL), or homocysteine thiolactone (1 mM) for 24 hours. DMSO was used as control. After the treatments, the PHHs were washed with cold PBS and were either scratched off for protein and RNA extraction or stained for cell death. A portion of the treated PHHs was used for evaluation of intracellular Ca2+ using Fluo-4 (Molecular Probe) according to the manufacturer's protocol. The intracellular Ca2+ levels were monitored with Microplate Fluorescence Reader (Molecular Devices Corp.) after addition of thapsigargin (0.5 μM).

Immunoblotting and Reverse-Transcription Polymerase Chain Reaction.

Methods for protein extraction and analysis and for messenger RNA (mRNA) extraction and reverse-transcription polymerase chain reaction, antibodies for ER stress markers, and the specific primers for spliced Xbp1 have been reported.14, 30, 33 Antibodies for SERCA were from Santa Cruz (sc-271669). The intensity of protein bands on the Western blots were quantified with the NIH software, ImageJ, after the blots of protein samples were scanned into TIF files.

Confocal Fluorescence Microscopy.

The hepatocytes were fixed in cold acetone for 10 minutes and washed in PBS three times. The fixed cells were blocked with PBS containing 5% BSA and 2% serum for secondary antibodies for 30 minutes, washed three times in PBS, incubated with anti–glucose-regulated protein 78 (GRP78) antibodies in PBS containing 1.5% BSA for 60 minutes, and washed three times with PBS. Antibody binding was detected with FITC-conjugated secondary antibodies (Southern Biotechnology Associates, Birmingham, AL). Microfilaments of the cells were stained with rhodamine-phalloidin (1:100 dilutions, 30 minutes). The stained cells were examined by using a Nikon PCM2000 confocal laser-scanning microscope.

Statistical Analysis.

Values are expressed as the mean ± SEM unless indicated otherwise. Statistical analyses were performed using the Student t test or analysis of variance for comparison of multiple groups. P < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

HIV PI– and Alcohol-Induced ER Stress and Liver Injury.

Single-dose alcohol administration increased serum ALT levels by two-fold in mice (Fig. 1A). Neither RIT alone nor RIT combined with LOP or with alcohol had significant effects on ALT levels. However, a more than five-fold increase in ALT was detected in mice treated with alcohol plus the two HIV drugs. The acute alcohol treatment induced a moderate increase in liver triglycerides and a slight increase in liver cholesterol (Fig. 1B,C). Similar to their effects on ALT, RIT and LOP synergistically increased alcohol-induced lipid accumulation (Fig. 1B,C), which was confirmed histologically with Oil Red O staining (Fig. 1D). To know whether alcohol affected the metabolism or bioavailability of the two HIV drugs, we extracted RIT and LOP from plasma of the treated mice, and detected the drugs with HPLC. The retention times for RIT and LOP were 17.5 ± 0.2 and 19.6 ± 0.2 minutes, respectively (Fig. 2). Alcohol did not affect the plasma RIT level significantly. Interestingly, alcohol increased the LOP level, which was further increased in response to an increased alcohol dose (Fig. 2D).

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Figure 1. Liver injury in mice gavaged with alcohol and/or HIV PIs. (A) Serum ALT. (B) Liver triglycerides. (C) Liver cholesterol. *P < 0.05, **P < 0.01, ***P < 0.005 versus control (n = 5). (D) Synergistic effects of HIV protease inhibitors on alcohol-induced fat accumulation in the liver of mice gavaged with a liquid alcohol diet alone or mixed with RIT and LOP. The red lipid droplets in liver tissue were revealed by Oil Red O staining (magnification ×200).

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Figure 2. Effect of alcohol on HIV PI levels in mouse plasma. (A-C) HPLC spectra of RIT and LOP from plasma of mice gavaged with control diet plus RIT and LOP (A), half dose of alcohol diet plus RIT and LOP (B), and alcohol diet plus RIT and LOP (C). (D) Quantitation of the HIV drugs in mouse plasma. HPLC analysis was run twice, and the representative spectra were shown.

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Previous studies demonstrated that either HIV PIs or alcohol feeding at high concentrations induced ER stress in cell and animal models.14, 15, 30, 33 To know whether ER stress was induced in response to acute treatments with the HIV drugs and/or alcohol at reduced doses, we examined expression of major ER stress markers in the livers of treated mice. Compared with the control, RIT combined with LOP induced moderate ER stress response as indicated by increased expression of GRP78 and GRP94, IRE1, and ATF6 in mouse livers (Fig. 3A,B). Single doses of alcohol gavage (5 g/kg) did not induce apparent ER stress response. In alcohol plus RIT and LOP, alcohol synergistically increased the HIV drug-induced GRP78 and ratio of spliced Xbp1 (sXbp1) to Xbp1 (Fig. 3A,B). In addition, RIT and LOP inhibited protein expression of the SERCA, which was further inhibited in the presence of alcohol. CYP2E1 was not significantly affected in response to any of these acute treatments (Fig. 3A,B).

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Figure 3. HIV PIs and/or alcohol induce ER stress and injury in mouse liver. (A) Immunoblots of proteins from liver. ATF-6, activating transcription factor 6; IRE1α, inositol requiring enzyme 1; PDI, protein disulfide isomerase. (B) Protein quantity normalized with β-actin. *P < 0.05 and **P < 0.01 versus control. #P < 0.05 compared between RIT+LOP and alcohol+RIT+LOP. (C) Liver tissues stained with hematoxylin and eosin (magnification ×100). (D) Fibrotic changes in liver tissues stained with sirius red (magnification ×200). (E) Immunoblots of liver proteins. (F) Serum ALT values. C, pair-fed control; E, alcohol feeding; R+L, injected with RIP and LOP; E+R+L, alcohol feeding plus RIT and LOP injection. **P < 0.01.

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Chronic HIV drugs worsened alcoholic fatty liver injury (Fig. 3C) and induced moderate liver fibrotic changes, which were confirmed by sirius red staining and immunoblotting with anti–collagenase I antibodies (Fig. 3D,E). The chronic treatment induced ER stress (Fig. 3E) and increased ALT by 15-fold compared with control (Fig. 3F).

HIV PI– and Alcohol-Induced ER Stress and Death in PMHs.

To further investigate the synergistic effects of alcohol on the stress and injury induced by the two HIV drugs, PMHs were isolated. Figure 4A demonstrates that the RIT and LOP treatment increased GRP78 expression in the hepatocytes, but alcohol alone did not increase GRP78. However, using more sensitive in situ immunoblotting with fluorescent antibodies, we were able to detect enhanced GRP78 expression in the PMHs in response to the HIV drugs as well as to alcohol (Supporting Fig. 1). The GRP78 expression was remarkably increased in the combination treatment with RIT, LOP, and alcohol (Supporting Fig. 1 and Fig. 4B). Consistent with the in vivo study with mice, expression of SERCA in PMHs was also inhibited by RIT and LOP, and alcohol alone did not affect SERCA significantly (Fig. 4B). The mature SREBP1 that regulates triglyceride synthesis was increased in the presence of RIT, LOP, and alcohol despite the fact that neither the HIV drugs nor acute alcohol alone increased SREBP1 activation (Fig. 4B). CHOP and phosphorylated c-Jun N-terminal kinase 2 that mediate ER stress-caused cell death were induced by the HIV drugs and alcohol. Correspondingly, only moderate cell death was observed in the hepatocytes treated with either alcohol or the two HIV drugs (Fig. 4C,D). A synergistic increase of cell death was detected in the hepatocytes in response to the drug and alcohol combination treatment.

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Figure 4. ER stress and cell death in PMHs treated with HIV PIs and/or alcohol. (A) Immunoblots of proteins. JNK, c-Jun N-terminal protein kinase; nSREBP1, mature SREBP1 (68 kD); pJNK, phosphorylated JNK; pSREPB, SREBP1 precursor. (B) Protein quantity normalized with β-actin. *P < 0.05 and **P < 0.01 versus control. #P < 0.05 and ##P < 0.01 compared between RIT+LOP and alcohol+RIT+LOP. (C) Cell morphological changes under fluorescence microscopy after double-stained with Sytox green and Hoechst blue. Green spots indicate dead cells and blue spots indicate live cells. (D) Quantitation of cell death relative to control. *P < 0.05, ***P < 0.001.

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Effects of HIV PIs on Alcohol-Induced ER Stress in PHHs.

The above observed effects of HIV drugs on alcohol-induced ER stress and injury in animals and animal liver cells prompted us to ask whether similar effects occur in PHHs. In PHHs, tunicamycin increased GRP78 expression from 8 hours to 24 hours after treatment, indicative of a positive ER stress response (Fig. 5A,B). Neither alcohol nor RIT and LOP treatments induced a significant increase of GRP78. However, GRP78 was increased significantly in response to alcohol and the HIV drugs. To confirm the ER stress response in PHHs, we measured the unconventional splicing of Xbp1 at more treatment combinations: tunicamycin as positive control, low and high doses of RIT and LOP, low and high doses of alcohol, and homocysteine thiolactone that homocysteinylates proteins and mediates chronic alcohol-induced ER stress,14, 33 the HIV drugs plus alcohol, or the HIV drugs plus homocysteine thiolactone (Fig. 5C). As expected, tunicamycin increased the ratio of sXbp1 to Xbp1 mRNA, indicating an increased expression of sXbp1. Homocysteine thiolactone nearly eliminated the Xbp1 precursor and induced robust expression of sXbp1. RIT and LOP induced moderate Xbp1 splicing at low or high doses. Alcohol alone at low or high doses did not induce significant Xbp1 splicing. However, alcohol at 85 mM significantly increased RIT and LOP-induced Xbp1 splicing (Fig. 5C).

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Figure 5. Effects of HIV PIs and alcohol on ER stress response in PHHs. (A) GRP78 expression in response to the HIV drug and/or alcohol. C, control; E, alcohol; ERL, alcohol+RL; RL, RIT+LOP; T, tunicamycin (3 μg/mL used as ER stress positive control). (B) Quantitation of GRP78 expression after normalized with β-actin; *P < 0.05 and ***P < 0.005 versus control. (C) Reverse-transcription polymerase chain reaction of mRNA showing shifting from Xbp1 to sXbp1 upon ER stress. ERL1 and ERL2, RIT+LOP (10 μg/mL) plus alcohol at 85 mM and 35 mM, respectively; HcyL, homocystein thiolactone (1 mM); HRL, homocystein thiolactone (1 mM) plus RL at 5 μg/mL; RL, RIT+LOP at 15 μg/mL (RL1) and 15 μg/mL (RL2). Quantitation of ratio of sXbp1 to Xbp1 is shown under the gels. Comparisons were made between control and treatments (P = 1) or between RL1 and ERL1 or ERL2 (P = 2). (D) Immunoblots of ER stress markers. Detection of cyclin D indicated that the cells were viable in the presence of HcyL.

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Alcohol-induced ER stress has been linked to impaired lipogenesis in animal models.14, 15 To determine whether the linkage occurs in PHHs, we examined expression of SREBP1c as well as peroxisome proliferator–activated receptor-γ coactivator-1 (PGC1) that regulates lipid oxidation in the liver (Fig. 5D and Supporting Table 1). RIT and LOP but not alcohol or tunicamycin increased expression of the mature SREBP1. PGC1 was not affected by RIT and LOP. However, PGC1 was inhibited by alcohol or tunicamycin and was inhibited to a greater extent in response to alcohol plus the two HIV drugs. Interestingly, homocysteine thiolactone inhibits both SREBP1 and PGC1, indicating that PHHs may be more sensitive to protein homocysteienylation than PMHs. In addition, CYP2E1 was inhibited by the HIV drugs, and the inhibition was reduced in the presence of alcohol in PHHs. Derlin that regulates ER stress–associated protein degradation (ERAD) was not affected by the drugs but was inhibited by tunicamycin, homocysteine thiolactone, or alcohol (Fig. 5D and Supporting Table 1).

Inhibition of SERCA and Cell Death Promotion by HIV PIs and Alcohol in PHHs.

SERCA was inhibited by tunicamycin and to a greater extent by homocysteine thiolactone (Fig. 5D and Supporting Table 1). RIT and LOP but not alcohol alone significantly inhibited SERCA. SERCA remained inhibited by the combined treatment with the two HIV drugs and alcohol. It is known that SERCA sequesters cytosolic Ca2+ in the ER and inhibition of SERCA leads to discharges of ER calcium.34 To test this, we incubated the human hepatocytes with vehicle control, alcohol, RIT, and LOP, or alcohol combined with RIT and LOP, and then measured ER calcium discharges upon stimulation by the specific SERCA inhibitor thapsigargin. Figure 6 demonstrates that intracellular calcium levels in the human cells were reduced slightly by alcohol treatment and significantly by RIT and LOP. The preincubation with alcohol and the two HIV drugs remarkably reduced the intracellular calcium. Correspondingly, alcohol or the HIV drug–induced disturbance of intracellular calcium homeostasis promoted cell death (Fig. 7). The cell death was synergistically increased in the PHHs treated with alcohol plus the two HIV drugs, which was comparable to the cell death induced by thapsigargin, suggesting a specific involvement of impaired calcium homeostasis.

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Figure 6. Effects of alcohol and HIV PIs on thapsigargin-stimulated discharges of intracellular Ca2+ stores in PHHs. See Materials and Methods for details. Calcium levels are expressed as % of vehicle control at time zero.

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Figure 7. Synergistic effects of HIV PIs and alcohol on cell death in PHHs. PHHs were doubly stained with Sytox green and Hoechst blue dyes after incubation with vehicle DMSO (C), RIT and LOP (RIT+LOP), alcohol (EtOH), or thapsigargin (TG) (6 μg/mL) for 24 hours. Dead cells (combination of apoptosis and necrosis) were stained green. The graph depicts the cell death rate. *P < 0.05 and ***P < 0.005 versus control.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In this study, we tested the effects of HIV protease inhibitors and alcohol on hepatic injury in animals and in both animal and human liver cells. Acute alcohol administration by gavage caused moderate liver injury, which is consistent with other studies.30 Alcohol synergistically increased hepatotoxicity in the presence of RIT and LOP. Our results are clinically relevant for several reasons. First, the HIV drugs induced little injury in PMHs and PHHs. This implies that the drugs themselves, at proper doses and without concomitant alcohol consumption, may not be a significant problem, considering their dramatic curing potential for HIV-infected patients under HAART. Second, RIT is usually used to boost the efficacy of other HIV PIs.23 To comply with this practice, we tested the injurious effects by using a combination of RIT and LOP, which is the most popular regimen applied to the patients.23, 35 Third, the amount of drugs used in this study is compatible with the concentrations that restrain HIV effectively and have been used by others in both animal experiments and clinical practices. A minimum dose of RIT at 2.1 μg/mL has been shown to inhibit different strains of HIV-1 in the human T cell line MT4 system.23, 36, 37 Considering that the liver is the primary site for drug metabolism and is usually exposed to high concentrations of drugs, it is reasonable to use higher doses in liver cells than in peripheral cells in drug toxicity tests. In fact, a range of concentrations between 0 and 100 μM have been used in other studies.28, 29 For the in vivo studies, we adopted a safety conversion factor 10 in converting the known minimum dose in humans to an equivalent dose in mice. The equivalent dose of RIT for mice was calculated as 12.5 mg/kg. Thus, the single dose of HIV PI at 15 mg/kg was well justified for the in vivo experiments and is clinically relevant.

Alcohol plus the two HIV drugs induced a synergistic increase in serum ALT levels, which indicated an aggravated hepatic injury. The underlying mechanisms are likely multifold. The metabolisms of the HIV drugs and alcohol might mutually be affected, because both HIV drugs and alcohol are metabolized in the liver by the cytochrome P450 enzyme system.4, 12, 23, 28, 32 On one hand, alcohol induced moderate CYP2E1 expression (Fig. 5D), which is consistent with the results of other studies.5 On the other hand, the drugs slightly inhibited CYP2E1 expression in mice (Fig. 3A) and significantly reduced CYP2E1 expression in PHHs (Fig. 5D). In the presence of alcohol, the amount of LOP in mouse plasma was nearly doubled in the drug combination treatment (Fig. 2). This may be a result of drug-drug or drug-alcohol interactions, because in a regimen of drug combination, RIT boosts the bioavailability of other drugs,23, 38, 39 and alcohol and the drugs appeared to have opposite effects on CYP2E1 expression. In addition, because the increase of lopinair was also dependent on alcohol dose, the drug-drug interactions may not be the only cause. Conceivably, alcohol may potentiate the boosting effects of RIT on LOP and increased drug exposure and subsequent hepatotoxicity.

Our results support that the ER stress mediates the potentiation effects by alcohol. The ER is an essential organelle for protein and lipid synthesis and modifications, drug metabolism, and Ca2+ homeostasis. General attenuation of protein synthesis upon ER stress will affect the efficiency of enzymes metabolizing alcohol and drugs. Accumulating evidence indicates that either excessive alcohol consumption or high doses of HIV PIs induce ER stress and injury in cell and animal models.14, 15, 28-33 In the present study, especially for the first time with PHHs, we demonstrated an occurrence of ER stress in response to alcohol combined with the HIV drugs at practical concentrations. First, although increased expression of GRP78 was not readily detected in response to either alcohol or the HIV drugs alone, alcohol combined with the drugs induced significant GRP78 increase. Second, ER stress has been associated with impaired lipid metabolism in both alcoholic and nonalcoholic liver disease,14, 15, 40 and we detected an association of lipid accumulation with an increased expression of SREBP1c and a decreased expression of PGC1 in the presence of alcohol and HIV drugs. Third, alcohol-induced hyperhomocysteinemia is a well-established phenomenon in animals and patients.8, 14, 41 We observed a robust unconventional splicing of Xbp1 in the human cells challenged with homocysteine thiolactone that is interchangeable with homocysteine. With respect to what ER stress components are involved, we propose that disturbance of Ca2+ homeostasis is a major mechanism for the activation of ER stress by HIV PIs and alcohol. Previously, a number of possible cellular stress signals, such as increased cholesterol in ER membranes, deprivation of glucose, inhibition of ERAD, and depletion of ER calcium store were suggested to initiate the unfolded protein response by the HIV drugs.28-30 For instance, in macrophages, RIT significantly up-regulates CD36 and low-density lipoprotein receptor (LDLR) expression, promoting foam cell formation by disrupting ABCA1-mediated cholesterol efflux.28, 29, 42 The glucose transporter isoform GluT4 was also selectively inhibited by HIV PIs in macrophage.43 However, in hepatocytes treated with alcohol and the HIV drugs, we did not detect any apparent changes of LDLR and GluT2 (data not shown), excluding the involvement of cholesterol or glucose in activating the ER stress. With respect to ERAD, it was reported that RIT inhibited chymotrypsin-like activity but enhanced trypsin-like activity in T cells.44 However, in the human hepatocytes in this study, Derlin that regulates ERAD was not changed (Fig. 5D, lane ERL), ruling out the involvement of ERAD in the ER stress activation by alcohol and the drugs. Interestingly, SERCA that mainly regulates ER Ca2+ store was inhibited by RIT and LOP in mice as well as in PMHs and PHHs. Although we have not examined expression of SERCA mRNA in this study, the inhibition of SERCA protein was quite specific in the PHHs, since the inhibition was similar to the pharmacological ER stress-induced agent-thapsigargin (Fig. 5D). Further, the intracellular calcium in the PHHs was reduced after incubation with the two HIV drugs or with alcohol (albeit to a less extent). The calcium level was reduced further after incubation with both alcohol and the drugs. Parallel to the calcium reduction, cell death due to the ER stress in the human liver cells was synergistically increased in response to alcohol and the HIV drugs. Therefore, the HIV drugs may interact with SERCA, disturbing calcium homeostasis and causing ER stress and injury. The drugs may also affect the function of CYP2E1, delaying alcohol degradation. An increased alcohol exposure induces oxidative stress and/or hyperhomocysteinemia, which disturbs SERCA further and exacerbates the HIV drug-induced ER stress leading to hepatic steatosis and fibrosis (Fig. 8).

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Figure 8. Interplay between alcohol and HIV PIs on hepatic ER stress and injury. Cyto Ca2+, cytoplasmic calcium; ER Ca2+, calcium within the ER; Hcy, homocysteine; ROS, reactive oxygen species. An upward arrow ([UPWARDS ARROW]) indicates that the level is up; a downward arrow ([DOWNWARDS ARROW]) indicates that the level is down. Solid lines indicate potential direct effects; dotted lines indicate potential indirect effects.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank CellzDirect and Shelly Lu for providing primary human hepatocytes and the USC Research Center for Liver Diseases for technical support. Min Feng, MD., is a postgraduate medical research trainee at Keck School of Medicine of USC.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_25702_sm_SuppFig1.tif3946KSupporting Information Figure 1.
HEP_25702_sm_SuppTab1.tif560KSupporting Information Table 1.

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