Abstract
Altered expression of drug transporters and metabolic enzymes is known to occur in infection-induced inflammation. We hypothesize that in human immunodeficiency virus (HIV)–infected individuals, further alteration could occur as a result of augmented inflammation. The HIV-1 transgenic (Tg) rat is used to simulate HIV pathologies associated with the presence of HIV viral proteins. Therefore, the objective of this study was to examine the effect of endotoxin administration on the gene expression of drug transporters in the liver of HIV-Tg rats. Male and female HIV-Tg and wild-type (WT) littermates were injected with 5 mg/kg endotoxin or saline (n = 7–9/group). Eighteen hours later, rats were euthanized and tissues were collected. Quantitative real-time polymerase chain reaction and Western blot analysis were used to measure hepatic gene and protein expression, respectively, and enzyme-linked immunosorbent assay was used to measure serum cytokine levels. Although an augmented inflammatory response was seen in HIV-Tg rats, similar endotoxin- mediated downregulation of Abcb1a, Abcc2, Abcg2, Abcb11, Slco1a1, Slco1a2, Slco1b2, Slc10a1, Slc22a1, Cyp3a2, and Cyp3a9 gene expression was seen in the HIV-Tg and WT groups. A significantly greater endotoxin- mediated downregulation of Ent1/Slc29a1 was seen in female HIV-Tg rats. Basal expression of inflammatory mediators was not altered in the HIV-Tg rat; likewise, the basal expression of most transporters was not significantly different between HIV-Tg and WT rats. Our findings suggest that hepatobiliary clearances of endogenous and exogenous substrates are altered in the HIV-Tg rat after endotoxin exposure. This is of particular importance because HIV-infected individuals frequently present with bacterial or viral infections, which are a potential source for drug–disease interactions.
Introduction
Human immunodeficiency virus (HIV) is associated with chronic inflammation and persistent immune activation that ultimately leads to accelerated CD4+ T cell death, opportunistic infections, and AIDS (Kedzierska and Crowe, 2001; Hazenberg et al., 2003; Mogensen et al., 2010). It is believed that an important factor in immune activation during chronic HIV is the presence of HIV viral proteins and viral nucleic acid. Viral proteins gp120, Tat, and Nef are capable of activating lymphocytes and macrophages, causing the release of proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α and resulting in organ dysfunction (Lee et al., 2003; Swingler et al., 2003; Appay and Sauce, 2008; Yim et al., 2009; Mogensen et al., 2010). Although the use of combination antiretroviral therapy (ART) is highly effective in suppressing viral replication and improving overall immune function (Detels et al., 1998), chronic inflammation does not completely resolve, and elevated markers of inflammation such as C-reactive protein, IL-6, and TNF-α are seen in patients with HIV and are predictive of disease progression and mortality (Appay and Sauce, 2008; Deeks, 2009; Corbeau and Reynes, 2011). Other important factors include the presence of clinical or subclinical coinfections, which patients with HIV encounter due to dysregulation in their immune system (Brenchley et al., 2006; Douek et al., 2009; Gonzalez et al., 2009; Corbeau and Reynes, 2011; Chihara et al., 2012). Subclinical endotoxemia is also seen in patients with HIV due to mucosal immune dysfunction which causes systemic translocation of gut microflora from the intestinal lumen (Marchetti et al., 2008, 2013; Sandler and Douek, 2012). Lipopolysaccharide (LPS), a bacterial endotoxin that is part of Gram-negative bacterial cell wall, is considered a major immune-activating molecule in patients with HIV and is one of the main causes of subclinical endotoxemia as well as sepsis (Proctor, 2001; Brenchley et al., 2006; Douek, 2007; Silva and dos Santos, 2013). Indeed, although the use of ART is associated with decreased circulating levels of LPS, levels are still higher in patients with HIV than in noninfected individuals (Mehandru et al., 2006; Marchetti et al., 2008; d'Ettorre et al., 2011; Jenabian et al., 2015).
Infection-induced inflammation has been shown to alter the disposition of drugs. Proinflammatory cytokines including IL-6, TNF-α, and IL-1β have been frequently reported to decrease the expression and function of ATP-binding cassette (ABC) and solute carrier (SLC) transporters and cytochrome P450 (CYP or P450) metabolizing enzymes (Piquette-Miller et al., 1998; Tang et al., 2000; Kalitsky-Szirtes et al., 2004; Wang et al., 2005; Englund et al., 2007; Petrovic et al., 2008). Studies in HIV-infected individuals have also reported altered expression of drug-metabolizing enzymes and transporters. One study reported that HIV-infected individuals who are not taking ART (ART naïve) had lower CYP3A4 and CYP2D6 enzymatic activity compared with uninfected individuals, which correlated with an increased plasma cytokine level (Jones et al., 2010). More recently, ART- naïve patients with HIV were found to have significantly lower P-glycoprotein (P-gp/ABCB1) and multidrug resistance–associated protein 2 (MRP2/ABCC2) protein levels in the rectal-sigmoid colon compared with noninfected subjects (De Rosa et al., 2013). In addition, in vitro incubation of cells with the HIV gp120 viral protein was found to increase proinflammatory cytokines IL-6, IL-1β, and TNF-α as well as decrease the expression and function of P-gp in rat and human brain astrocytes (Ronaldson and Bendayan, 2006; Ashraf et al., 2011). Because of dysregulation of the immune system, it is plausible that infection-induced inflammatory responses may be potentiated in patients with HIV (da Silva et al., 1999; Nguyen and Biron, 1999; Lester et al., 2008, 2009; Bukh et al., 2011). We hypothesize that augmented inflammatory responses in response to coinfections could lead to further downregulation in the expression of transporters and drug-metabolizing enzymes. Because many of the antiretroviral agents are substrates of drug transporters and metabolizing enzymes, understanding their regulation in patients with HIV is important in maintaining therapeutic drug levels and predicting potential drug–disease interactions. Moreover, protease inhibitors, an integral part of combination ART, are potent inducers/inhibitors of drug transporters and metabolizing enzymes, which increases the risk of drug–drug interactions in these patients (Gutmann et al., 1999; Sulkowski et al., 2000; Kim, 2003; Klaassen and Aleksunes, 2010; Griffin et al., 2011). Indeed, the existence of coinfections and alterations in drug metabolism is known to increase the risk of ART-associated liver damage in patients with HIV (Sulkowski et al., 2000; Soriano et al., 2008; Puoti et al., 2009).
In this study, we examined the effect of endotoxin-mediated inflammation on the expression of drug transporters and metabolizing enzymes in HIV-1 transgenic (Tg) rats. The HIV-Tg rat model is a noninfectious small animal model of HIV, expressing all of the HIV-1 viral proteins except for the replication gag and pol genes (Reid et al., 2001; Hatziioannou and Evans, 2012). This makes the model useful for demonstrating effects of HIV viral proteins in the absence of viral replication, which shares similarity with patients with HIV given ART, because there is no viral replication yet there is persistent inflammation (Chang et al., 2007a,b; Homji et al., 2012). As the rats age, progressive illness that shares many similarities with HIV-infected humans becomes apparent. This includes pneumonitis, neurologic deficits, wasting, respiratory difficulty, cardiac abnormalities, and renal disease (Ray et al., 2003; Peng et al., 2010). Immune dysregulation is also evident, including impaired macrophage phagocytic function (Joshi and Guidot, 2011), defects in T helper 1 immune responses (Reid et al., 2001, 2004; Yadav et al., 2006; Royal et al., 2012), and defective leukocyte endothelial adhesion (Chang et al., 2007b). Increased cytokine levels have been found in liver and brain tissue lysates (Joshi and Guidot, 2011; Royal et al., 2012). In response to endotoxin administration, HIV-Tg rats have previously demonstrated augmented inflammatory response (Chang et al., 2007a,b), similar to that seen in HIV-positive individuals (Lester et al., 2008; Bukh et al., 2011).
Materials and Methods
Animals and Experimental Design.
Male Hsd–HIV-1 (F344) transgenic (HIV-Tg) rats and female F344/NHsd rats, purchased from Harlan Laboratories (Indianapolis, IN), were bred and HIV-Tg and control wild-type (WT) littermates were separated at weaning. Rats were housed in accordance with the University of Toronto Animal Care Committee and the Canadian Council on Animal Care and were provided with ad libitum food and water on a 12-hour light/dark schedule. Three-month-old male and female HIV-Tg or same-generation WT littermates were given an intraperitoneal injection of either 5 mg/kg LPS (Escherichia coli 055:B5; Sigma-Aldrich, Oakville, ON, Canada) or saline. Rats (n = 7–9 per group) were euthanized 18 hours after injection and serum and tissues were collected and snap-frozen with liquid nitrogen and stored at −80°C until analysis. We have previously observed significant endotoxin-mediated changes in the expression of numerous drug transporters in rats at various times between 6 and 24 hours after endotoxin administration (Wang et al., 2005; Petrovic et al., 2008). Because significant changes in mRNA with corresponding changes in protein expression were generally seen at 18 hours postinjection in previous studies, we therefore examined the effect of endotoxin in HIV-Tg and WT rats at 18 hours postinjection.
Serum and Tissue Analysis.
Serum cytokine concentrations were measured via commercially available rat-specific enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Minimum detectable levels for IL-6, IL-1β, TNF-α, and interferon-γ were 36, 5, 9, and 10 pg/ml, respectively. Total bile acid concentrations were measured in the serum and in 100 g liver homogenate using a Rat Total Bile Acids Assay Kit (Crystal Chem Inc., Downers Grove, IL) according to the manufacturer’s instructions and as previously described (Ghoneim et al., 2015). Alanine aminotransferase (ALT) activity in the serum was measured following the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO) and results were reported as milliunit/milliliter, where 1 mU ALT is defined as the amount of enzyme that generates 1 nmol pyruvate/min at 37°C.
Quantitative Real-Time Polymerase Chain Reaction.
Total RNA was extracted from approximately 50 mg frozen tissue using the TRIzol extraction method (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and 2 μg RNA was then treated with DNaseI (Invitrogen) and reverse transcribed with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Real-time quantitative polymerase chain reaction (PCR) was carried out for triplicate samples of genomic cDNA specific for each primer set (Supplemental Table 1) and was performed using the Power SYBR Green detection system (ABI HT 7900; Applied Biosystems, Streetsville, ON, Canada). A comparative threshold cycle method was used to calculate relative mRNA expression of each gene of interest normalized to housekeeping gene β-actin. Similar results were obtained using glyceraldehyde-3-phosphate dehydrogenase or cyclophilin as housekeeping genes.
Western Blot Analysis.
Isolation of crude membrane fractions was previously described (Petrovic and Piquette-Miller, 2010). Briefly, 0.3 g liver tissue was homogenized in lysis buffer containing 0.1 M Tris-HCL, 3μl/ml protease inhibitor, and 50μg/ml phenylmethylsulfonyl fluoride and was centrifuged (30,000g for 1 hour) and quantified using the Bradford assay. Forty micrograms of protein was separated by SDS-PAGE and transferred to polyvinylidene floride membranes (Bio-Rad Laboratories, Mississauga, ON, Canada). Membranes were blocked using 5% skim milk and were incubated with the following primary antibodies: P-gp/ABCB1 (C219, 1:500; Enzo Life Sciences, Farmingdale, NY), ABCC2 (M2 III-5, 1:50; Abcam Inc., Cambridge, MA), and equilibrative nucleoside transporter 1 (ENT1 /SLC29A1; F-12, 1:100; Santa Cruz Biotechnology, Dallas, Texas). After a series of washing, membranes were incubated with secondary anti-mouse antibody (1:3000; Jackson ImmunoResearch Laboratories, West Grove, PA). Immunodetectable protein was detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL) and band density was quantified using Alpha Ease FC imaging software (Alpha Innotech, Santa Clara, CA). β-actin (AC-15, 1:10,000; Sigma-Aldrich, Oakville, ON, Canada) was used to control for loading variability and a calibrator sample for each gender was used to control for different gels. Mouse heart tissue extract was used as a positive control for Ent1.
Genotyping.
Genotyping was performed by PCR amplification of viral proteins (gp120, tat, vif, nef) using 0.5 g spleen tissue and PCR products were separated by gel electrophoresis. Separation was carried on a 2% agarose gel in 1× Tris/borate/EDTA, 6× loading dye using Fast start DNA ladder 100 bp (Thermo Scientific) and bands were visualized by DNA safe SYBR 10,000× staining (Invitrogen).
Statistical Analysis.
Statistical analysis was performed with GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA) using a one-way analysis of variance, followed by Fisher’s least significant difference test to compare the effect of endotoxin administration in WT and HIV-Tg rats, in which male and female rats were analyzed separately and data were expressed as a fold change from WT saline-injected animals for each gender. One-way analysis of variance, followed by Fisher’s least significant difference test, was also used to determine differences in basal gene expression between male and female WT and HIV-Tg rats, in which results were compared relative to male WT saline-injected animals. A t test was used to compare cytokine levels after endotoxin administration between WT and HIV-Tg rats because serum levels were undetectable in saline-injected animals.
Results
Analysis of HIV-Tg Rat and Transgene Expression.
HIV-Tg rats appeared healthy at age 3 months with a 17% lower body weight compared with WT littermates (data not shown). Liver weight did not differ from WT once adjusted for body weight. HIV-Tg phenotypes develop cataracts by age 2 weeks, a finding that is absent in WT littermates. Genotyping confirmed the presence of the HIV transgene in the spleen of HIV-Tg rats. Gp 120, tat, vif, and nef were expressed in HIV-Tg rats but not in WT rats (Supplemental Fig. 1).
Basal Hepatic Gene Expression in the Liver.
The basal hepatic expression of most ABC and SLC transporters and nuclear receptors were not significantly different between HIV-Tg and WT rats (Table 1). Expression of Cyp3a2 was 62% lower in male HIV-Tg rats compared with WT rats, whereas expression of Cyp3a9, Slc10a1, and Abcb11 was lower (25%–54%) in female HIV-Tg rats compared with WT littermates. Hepatic mRNA levels of TNF-α and inducible nitric oxide synthase (iNOS) were slightly but significantly higher in saline-treated female HIV-Tg rats compared with WT rats (Table 1).
Basal hepatic gene expression in HIV-Tg and WT rats
Values are presented as means ± S.E.M. Results are reported relative to male WT, which was standardized to 1.
Gender Differences in Metabolic Enzymes and Drug Transporter Expression.
Gender differences were detected in both HIV-Tg and WT saline controls (Table 1). A 250-fold higher expression of the Cyp3a2 enzyme was detected in WT male rats compared with female rats and a 14-fold higher expression of the Cyp3a9 enzyme was detected in WT female rats compared with male rats. With regard to hepatic transporters, 1.5- to 4-fold higher mRNA levels of Abcb1a, Abcb11, Abcc3, Slco1b2, Slc10a1, and Slc22a1 were detected in female WT rats compared with male rats. Similar findings were seen in HIV-Tg rats.
Inflammatory Response to Endotoxin.
Proinflammatory cytokines were examined as a measure of systemic and local inflammatory responses to endotoxin administration. Although serum levels of the proinflammatory cytokines were below the detection limit in saline-treated HIV-Tg and WT rats, a significant endotoxin-mediated induction of IL-6, IL-1β, TNF-α, and interferon-γ was observed in the serum of both control WT and HIV-Tg rats (Table 2). Higher serum levels of cytokines were seen in the endotoxin-treated HIV-Tg rats. Differences reached significance for TNF-α in female rats and for IL-1β in both genders. The presence of an inflammatory response within the liver was confirmed by measuring the hepatic mRNA expression of IL-6, IL-1β, iNOS, and TNF-α (Fig. 1). Within the liver of female rats, HIV-Tg had a more pronounced endotoxin-mediated induction of cytokines, which also occurred in male rats to a lesser extent.
Serum cytokine concentrations in endotoxin-treated HIV-Tg and WT rats
Values are presented as means ± S.E.M.
Effect of endotoxin (LPS) on the mRNA expression of inflammatory mediators in male (A) and female (B) rats. Values represent the ratio of gene of interest mRNA to β-actin mRNA, as determined by quantitative real-time PCR and expressed as the percent change from saline control values. Values are presented as means ± S.E.M. (n = 7 to 8 rats except for female WT saline, where n = 4). *P < 0.05; **P < 0.01; ***P < 0.001; ##P < 0.01; ###P < 0.001 (one-way analysis of variance; asterisks indicate significantly different from saline controls, whereas pound signs indicate significantly different from endotoxin-treated WT).
Effect of Endotoxin on Hepatic Expression of Metabolic Enzymes and ABC and SLC Transporters in WT and HIV-Tg Rats.
After endotoxin administration, a significant downregulation in the expression of several ABC transporters (Abcb1a, Abcb11, Abcg2, and Abcc2) (Figs. 2–4), SLC transporters (Slco1a1, Slco1a2, Slco1b2, Slc22a1, Slc10a1, and Slc29a1) (Figs. 5 and 6), and metabolic enzymes (Cyp3a2, Cyp3a9, and Cyp7a1) (Fig. 7) was seen in both HIV-Tg and WT rats. Slc29a1 was significantly lower in female HIV-Tg rats compared with WT rats (Fig. 6A). In addition, a significant downregulation in the expression of Pxr, Car, and Fxr, which are nuclear receptors involved in the regulation of many of the aforementioned genes, was also seen in both HIV-Tg and WT rats (Fig. 8). Western blot analysis confirmed significant downregulation in the protein expression of Mrp2 in both HIV-Tg and WT rats after endotoxin administration (Fig. 3B). The protein expression of P-gp was significantly downregulated in endotoxin-treated HIV-Tg and WT male rats and in female HIV-Tg but not WT rats (Fig. 2B). Moreover, Ent1 protein levels were significantly lower only in HIV-Tg rats (Fig. 6B). A significant negative correlation (P < 0.05) was found between TNF-α and Abcg2, Abcc2, Abcb11, Cyp3a2, Slco1a1, Slco1a1, Slco1b2, Slc22a1, Slco29a1, and Slc10a1 as well as between IL-1β and Abcb1a, Abcg2, Abcc2, Abcb11, Cyp3a2, Slco1a1, Slco1a1, Slco1b2, Slc22a1, Slco29a1, and Slc10a1 and between iNOS and Abcb1b, Abcg2, Abcb11, Slco1a1, Slco1a1, Slco1b2, Slc22a1, and Slco29a1 (Supplemental Table 2).
Effect of endotoxin (LPS) on the mRNA (A) and protein expression (B) of ABCB1. Values represent the ratio of gene of interest mRNA to β-actin mRNA, as determined by quantitative real-time PCR and expressed as the percent change from saline control values, and protein expression of P-gp, as determined by Western blot analysis and expressed as a percentage of WT saline, normalized to β-actin level and a calibrator. Values are presented as means ± S.E.M. (n = 5–8). Representative gels are shown below. *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.05 (one-way analysis of variance; asterisks indicate values that are significantly different from saline controls, whereas pound signs indicate values that are significantly different from endotoxin-treated WT).
Effect of endotoxin on the mRNA (A) and protein (B) expression of ABCC2. Values represent the ratio of gene of interest mRNA to β -actin mRNA, as determined by quantitative real-time PCR and expressed as the percent change from saline control values and protein expression of Mrp2, as determined by Western blot analysis and expressed as a percentage of WT saline, normalized to β-actin level and a calibrator. Values are presented as means ± S.E.M. (n = 5–8). Representative gels are shown below. *P < 0.05; **P < 0.01; ***P < 0.001; ###P < 0.001 (one-way analysis of variance; asterisks indicate values that are significantly different from saline controls, whereas pound signs indicate values that are significantly different from saline-treated WT).
Effect of endotoxin (LPS) on the mRNA expression of ABC transporters in male (A) and female (B) rats. Values represent the ratio of gene of interest mRNA to β-actin mRNA, as determined by quantitative real-time PCR and expressed as the percent change from saline control values. Values are presented as means ± S.E.M. (n = 7 to 8 rats except for female WT saline, where n = 4). *P < 0.05; **P < 0.01; ***P < 0.001 (one-way analysis of variance; asterisks indicate significantly different from saline controls).
Effect of endotoxin (LPS) on the mRNA expression of SLC transporters in male (A) and female (B) rats. Values represent the ratio of gene of interest mRNA to β-actin mRNA, as determined by quantitative real-time PCR and expressed as the percent change from saline control values. Values are presented as means ± S.E.M. (n = 7 to 8 rats except for female WT saline, where n = 4). ***P < 0.001 (one-way analysis of variance; asterisks indicate significantly different from saline controls).
Effect of endotoxin (LPS) on the mRNA (A) and protein (B) expression of Slc29a1 (Ent1). Values represent the ratio of gene of interest mRNA to β -actin mRNA, as determined by quantitative real-time PCR and expressed as the percent change from saline control values, and protein expression of Ent1, as determined by Western blot analysis and expressed as a percentage of WT saline, normalized to β-actin level and a calibrator. Values are presented as means ± S.E.M. (n = 5–8). Representative gels are shown below. *P < 0.05; ***P < 0.001; #P < 0.05 (one-way analysis of variance; asterisks indicate values that are significantly different from saline controls, whereas pound signs indicate values that are significantly different from endotoxin-treated WT).
Effect of endotoxin (LPS) on the mRNA expression of metabolic enzymes in male (A) and female (B) rats. Values represent the ratio of gene of interest mRNA to β-actin mRNA, as determined by quantitative real-time PCR and expressed as the percent change from saline control values. Values are presented as means ± S.E.M. (n = 7 to 8 rats except for female WT saline, where n = 4). *P < 0.05; **P < 0.01; ***P < 0.001 (one-way analysis of variance; asterisks indicate significantly different from saline controls).
Effect of endotoxin on the mRNA expression of nuclear receptors in male (A) and female (B) rats. Values represent the ratio of gene of interest mRNA to β-actin mRNA, as determined by quantitative real-time PCR and expressed as the percent change from saline control values. Values are presented as means ± S.E.M. (n = 7 to 8 rats except for female WT saline, where n = 4). *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.05; ##P < 0.01 (one-way analysis of variance; asterisks indicate values that are significantly different from saline controls, whereas pound signs indicate values that are significantly different from endotoxin-treated WT).
Biochemical Changes in Response to Endotoxin.
Because the expression of several bile acid transporters as well as Cyp7a1 was altered in the endotoxin-treated groups, we examined the effect on serum and hepatic concentrations of total bile acids (Table 3). Endotoxin resulted in a significant increase in serum total bile acid concentrations in the HIV-Tg but not WT rats. Intrahepatic concentrations of total bile acids were significantly increased in endotoxin-treated HIV-Tg and WT male but not female rats. ALT activity, which is a measure of liver toxicity, was significantly increased in the serum of endotoxin-treated HIV-Tg groups but not in the WT groups (Table 3).
Biochemical changes in response to endotoxin administration
Values are presented as means ± S.E.M.
Discussion
Despite the use of ART, patients with HIV often encounter bacterial endotoxin infections either as opportunistic infections or from microbial translocation. This adds to the state of inflammation and immune activation (Hazenberg et al., 2003; Brenchley et al., 2006; Appay and Sauce, 2008; Mogensen et al., 2010). The HIV-Tg rat has demonstrated augmented inflammatory response to endotoxin similar to that observed in patients with HIV (Chang et al., 2007a,b; Lester et al., 2008; Bukh et al., 2011). In addition, HIV viral proteins have been shown to contribute to inflammation-altered expression and function of drug transporters. Because endotoxin-mediated effects on the expression of hepatic gene expression are commonly examined in rodents (Piquette-Miller et al., 1998; Tang et al., 2000; Cherrington et al., 2004; Kalitsky-Szirtes et al., 2004; Wang et al., 2005; Petrovic et al., 2008) and similar endotoxin-mediated effects on the expression and activity of drug-metabolizing enzymes are seen in humans and rodents (Shedlofsky et al., 1994, 1997), we examined whether immunologic changes associated with the expression and activity of HIV viral proteins would affect endotoxin-mediated effects in HIV-Tg rats.
At the basal level, the HIV-Tg rat demonstrated significantly lower gene expression of drug-metabolizing enzymes Cyp3a2 and Cyp3a9 in male and female rats, respectively. Decreased enzymatic activity of CYP3A4 and CYP2D6 was previously reported in HIV-infected individuals and correlated with an increased plasma cytokine level (Jones et al., 2010). Although serum concentrations and basal expression of the proinflammatory cytokines were relatively unchanged in the HIV-Tg rat, it is possible that other factors including oxidative stress may play a role. HIV viral proteins gp120 and tat are known to induce oxidative stress markers (Nicolini et al., 2001; Walsh et al., 2004; Price et al., 2005). Indeed, there was a significant elevation in the mRNA level of iNOS and TNF-α in female HIV rats compared with WT rats. This could partially explain the reduction in Cyp3a expression, because several studies suggest the involvement of nitric oxide in downregulating P450 enzymes (Carlson and Billings, 1996; Donato et al., 1997; Aitken et al., 2008). Likewise, Abcb11 and Slc10a1 were significantly lower in female HIV-Tg rats. In general, apart from these differences, HIV-Tg rats displayed similar basal expression of most hepatic drug transporters relative to their WT littermates. Changes in the expression of Abcb1a and Abcc1 have also been reported in 24-week-old but not 8-week-old HIV-Tg rats (Robillard et al., 2014). Because 5-fold higher mRNA levels of gp120 were seen in the livers of the 24-week-old rats compared with 8-week-old rats, it is likely that we did not observe similar changes because of the age difference in our study. Differences in HIV-Tg–associated inflammation are seen in the literature. Although we did not detect measurable levels of cytokines in the serum of our HIV-Tg rats, Chang et al. (2007a) observed a significant elevation of serum IL-1β in 5-month-old HIV-Tg rats. On the other hand, Homji et al. (2012) did not detect changes in serum levels of IL-1β and TNF-α in 5-month-old HIV-Tg rats.
One of the best-characterized models of infection and systemic inflammation is the bacterial endotoxin LPS model. LPS administration results in a profound increase in the level of proinflammatory cytokines IL-6, IL-1β, TNF-α, as well as IFN-γ, which we observed in both HIV-Tg and WT rats. Endotoxin administration was also associated with significant downregulation in the hepatic expression of almost all examined ABC and SLC transporters, metabolic enzymes, and nuclear receptors. Transcriptional regulation of hepatic transporters and P450 enzyme expression during endotoxin-induced inflammation occurs via cytokine-mediated activation of transcription factors and cell signaling pathways (Shedlofsky et al., 1997; Piquette-Miller et al., 1998; Cherrington et al., 2004; Aitken et al., 2006; Cressman et al., 2012). Reduced expression and activity of the pregnane X receptor has also been linked to these inflammation-mediated changes (Teng and Piquette-Miller, 2008). Although changes in transporter expression in response to endotoxin administration in our study are transient due to the acute nature of the inflammatory stimulus, more persistent changes are likely to occur if inflammation becomes chronic and is not successfully resolved.
Compared with WT rats, the administration of endotoxin to HIV-Tg rats was associated with a profound and augmented inflammatory response. This suggests that the immune response is altered in these animals despite the absence of physical signs of disease. Likewise, previous studies also reported exacerbated responses to endotoxin in older HIV-Tg rats (Chang et al., 2007a,b). Consistent with the observed inflammatory response, we detected a significant endotoxin-mediated downregulation in the hepatic expression of numerous ABC and SLC transporters in addition to metabolic enzymes in the HIV-Tg rats. Of note, although we observed a higher induction of inflammatory markers in the endotoxin-treated HIV-Tg rats, the degree of gene downregulation was generally similar in the HIV-Tg and WT groups. This is likely due to maximum inhibition occurring in both groups in response to the dose of LPS used. Indeed, an 80%–90% reduction in hepatic expression was seen for most genes in both endotoxin-treated groups. A maximal downregulation of hepatic transporter expression after cytokine exposure was previously reported. In vitro studies in human hepatocytes showed a nonlinear downregulation in the mRNA expression of numerous drug transporters after incubation with TNF-α over a 10- to 100-fold range of concentrations (Le Vee et al., 2009). Similar results were obtained after LPS and cytokine treatment in mice (Hartmann et al., 2001; Li et al., 2004).
Of particular interest was our finding that endotoxin administration imposed a more pronounced downregulation of Ent1 (Slc29a1) in the HIV-Tg rat. The nucleoside transporter Ent1 is the primary transporter for the nucleoside inhibitor ribavirin (Jarvis et al., 1998). Ribavirin is commonly prescribed in patients with hepatitis C virus (HCV) and is reported to be less effective in the presence of HIV/HCV coinfections (Carrat et al., 2004; Torriani et al., 2004; Price and Thio, 2010). Although the antiviral mechanism of ribavirin for the treatment of HCV is not clearly understood (Hofmann et al., 2008), it has been suggested that the therapeutic response to ribavirin is dependent on its uptake into hepatocytes (Ibarra and Pfeiffer, 2009; Iikura et al., 2012). Our results, which demonstrate a pronounced downregulation of this nucleoside transporter in an HIV model of bacterial coinfection, could provide some insight into the decreased effectiveness of ribavirin in patients with HIV/HCV. To this point, ENT1 genetic polymorphisms have been shown to influence the virological response to ribavirin therapy in patients with HIV/HCV and HCV, possibly due to modulated ribavirin uptake into hepatocytes (Morello et al., 2010; Iikura et al., 2012; Tsubota et al., 2012).
It is well documented that endotoxin administration can decrease bile acid–dependent and bile acid–independent flow in a process that is believed to occur due to inhibited transport of bile salts and organic anions on the sinusoidal and canalicular membranes of hepatocytes (Moseley et al., 1996; Bolder et al., 1997; Cherrington et al., 2004; Geier et al., 2007). Although the extent of change in the bile acid transporters after endotoxin exposure was not very different between WT and HIV-Tg rats, a dramatic increase in total bile acids was present in the serum of HIV-Tg rats after endotoxin. It is plausible that the augmented increase in serum bile acids in the HIV-Tg rats results from combined changes in multiple transporters rather than through a single transport mechanism. Moreover, intrahepatic bile concentrations were increased after endotoxin administration in both WT and HIV-Tg male rats. Although we did not detect differences in hepatic bile acid concentration solely as a result of HIV, the presence of coexisting infections is considered a frequently encountered cause of cholestasis in individuals with HIV (Te, 2004). Moreover, abnormal liver enzymes are common among HIV-infected individuals and are predisposed by medication toxicity from antiretroviral drug therapy as well as the presence of coinfections (Price and Thio, 2010). Administration of bacterial endotoxin clearly elevated ALT activity in the serum of HIV-Tg rats more so than in WT rats, which confirms that the presence of coinfections does increase the risk of hepatotoxicity in the presence of HIV viral proteins.
Gender differences in the expression of drug transporters and metabolic enzymes were detected in our study in both HIV-Tg and WT rats. We observed significantly higher hepatic expression of several transporters in female rats. A higher hepatic expression of Mdr1 in female rats has been previously reported (Piquette-Miller et al., 1998; Salphati and Benet, 1998), which is contrary to what is reported in humans (Schuetz et al., 1995; Yang et al., 2012). On the other hand, Cyp3a2 expression was dramatically higher in male rats, whereas Cyp3a9 expression was more predominant in female rats. This is in agreement with other studies that have described Cyp3a9 and Cyp3a62 as the predominant Cyp3a isoform in female rats (Salphati and Benet, 1998; Matsubara et al., 2004). Gender differences in responses to ART have been observed and it has been suggested that these differences may be linked to gender differences in drug transporter and/or P450 expression (Ofotokun, 2005). Although this has not been examined or confirmed in vivo, it is plausible that gender differences in gene regulation could contribute to reported differences in therapeutic response to ART.
In conclusion, we demonstrate that endotoxin imposes a pronounced downregulation of numerous hepatic transporters and metabolic enzymes in HIV-Tg rats. Our findings suggest that hepatobiliary clearances of endogenous and exogenous substrates are altered in the HIV-Tg rat after endotoxin exposure. This is particularly important for drugs that are highly dependent on both metabolism and transporter-mediated processes such as protease inhibitors (Choo et al., 2000; Huisman et al., 2000; Griffin et al., 2011). This is of particular relevance because HIV-infected individuals frequently present with bacterial or viral infections. Although the HIV-Tg rat model does not encounter all aspects of HIV infection, shared similarities with pathologies occurring in HIV may help us to predict and identify potential drug–disease interactions.
Acknowledgments
The authors thank Walaa Abualsunun and Navaz Karimianpour for technical assistance.
Authorship Contributions
Participated in research design: Ghoneim, Piquette-Miller.
Conducted experiments: Ghoneim.
Performed data analysis: Ghoneim.
Wrote or contributed to the writing of the manuscript: Ghoneim, Piquette-Miller.
Footnotes
- Received October 15, 2015.
- Accepted March 8, 2016.
This research was supported by the Canadian Institutes of Health Research [Operating Grant MOP 13346]. R.G. was a recipient of the King Abdulaziz University Scholarship for Postgraduate Studies.
This work was previously presented as a poster at the 24th Annual Canadian Conference on HIV/AIDS Research (CAHR-2015); 2015 Apr 30–May 3; Toronto, ON, Canada.
↵
This article has supplemental material available at dmd.aspetjournals.org.
Abbreviations
- ABC
- ATP-binding cassette
- ALT
- alanine aminotransferase
- ART
- antiretroviral therapy
- CYP or P450
- cytochrome P450
- HCV
- hepatitis C virus
- HIV
- human immunodeficiency virus
- IL
- interleukin
- iNOS
- inducible nitric oxide synthase
- LPS
- lipopolysaccharide
- P-gp
- P-glycoprotein
- PCR
- polymerase chain reaction
- SLC
- solute carrier
- Tg
- transgenic
- TNF
- tumor necrosis factor
- WT
- wild type
- Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics
References
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Effect of Endotoxin on Drug Transporters in HIV-Tg Rats
