Research Article | Open Access
Changes in IL-2 and IL-10 during Chronic Administration of Isoniazid, Nevirapine, and Paracetamol in Rats
The aim of this study was to illustrate the initial subclinical drug-induced liver injury and the associated adaptive immune response by monitoring for the changes in plasma IL-2, IL-10, and some cytochrome P450 activity during chronic administration of nevirapine (NVP), isoniazid (INH), and paracetamol (PAR) in rats without clinical hepatotoxicity. Male Sprague-Dawley (SD) rats were divided into four groups (saline (S), NVP, INH, and PAR) of 25 animals each. The drugs were administered daily for 42 days at therapeutic doses (NVP 200 mg/kg, PAR 500 mg/kg, and INH 20 mg/kg) to the respective groups by oral gavage and five rats per group were sacrificed weekly. All the three drugs induced a subclinical liver injury in the first 2-3 weeks followed by healing, indicating adaption. The liver injury was pathologically similar and was associated with immune stimulation and increased cytochrome P450 activity. NVP- and PAR-induced liver injury lasted up to 14 days while that for INH lasted for 28 days. NVP-induced liver injury was associated with increased IL-2, CD4 count, and CYP3A2 activity, followed by increased IL-10 during the healing phase. In conclusion, the initial drug-induced subclinical liver injury, its spontaneous healing, and the associated adaptive immune response have been demonstrated.
Drug-induced liver injury is a major contributor to adverse drug reactions that has restricted the use of efficacious drugs such as isoniazid (INH) and nevirapine (NVP), while paracetamol (PAR) overdose is associated with fatal drug-induced liver injury. Although several mechanisms regarding INH, NVP, and PAR-induced hepatotoxicity have been postulated, the immune system has been implicated as a mediator and major determinant for progression of the liver injury [1–4]. It was proposed that metabolic activation of these drugs leads to the formation of reactive metabolites, which attack cellular proteins and result in the formation of metabolite-protein adducts, some of which are antigenic [5–9]. As a result, the immune system is activated and starts a process to eliminate hepatocytes expressing these immunogenic adducts [10–13]. It was then explained that most patients do not develop hepatotoxicity because their counter mechanisms are able to efficiently eliminate the antigenic adducts and/or to counter the proinflammatory response [14–17]. The elimination process is mediated by proinflammatory cytokines such as tumour necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), IL-2, and IL-4, while the counter mechanisms are mediated by anti-inflammatory cytokines such as IL-6, IL-10, and IL-13. However, in a few individuals, there is failure to counter the proinflammatory response, and this leads to progressive destruction of hepatocytes and overt drug-induced hepatotoxicity ensues [16, 17]. Indeed, several reports have described increased levels of some proinflammatory cytokines during hepatotoxicity by INH , PAR , and NVP .
Unfortunately, this explanation of initial hepatic injury and recovery, though plausible, has never been illustrated. It was envisaged that an experimental illustration of the initial hepatic injury and recovery is critical to promoting further research on preventive strategies for drug-induced hepatotoxicity such as the development of biomarkers for early prediction of patients who are likely to progress to overt hepatotoxicity. Therefore, the aim of this study was to illustrate the initial drug-induced subclinical liver injury and associated changes in the plasma profiles of IL-2, a proinflammatory cytokine, and IL-10, an anti-inflammatory cytokine, as well as the activity of the relevant cytochrome P450 isoforms (CYP3A2, CYP1A2, and CYP2E1) during chronic administration of NVP, INH, and PAR in rats.
2.1. Materials and Reagents
NVP (Viramune) oral suspension of 50 mg/5 ml and tablets 200 mg (Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT, USA) were purchased from a local pharmacy while INH and PAR were from Sigma-Aldrich Inc. (St. Louis, MO, USA). ELISA kits for rat interleukin-2 (IL-2) and interleukin-10 (IL-10) were purchased from RayBio (RayBiotech Inc., Norcross, GA, USA) and Invitrogen (Invitrogen Corporation, Camarillo, CA, USA), respectively.
2.2. Animal Care
Ethical approval was obtained from the Interfaculty Animal Ethics Committee of the University of the Free State. Male Sprague-Dawley (SD) rats weighing 200–250 g were used. Animals were housed at the Animal House of the University of the Free State, where they were fed and looked after by qualified staff. Standard rat chow and water were available to the animals ad libitum. Drug administration was done in a side room at the Animal House, and animals were inspected for skin lesions and other visible adverse events every day.
2.3. Experimental Design
Rats were divided into four groups of 25 animals each, that is, saline (S; control group), NVP, INH, and PAR. The drugs were administered daily at therapeutic doses to the respective groups by oral gavage (S, NVP 200 mg/kg, INH 20 mg/kg, and PARA 500 mg/kg), and five rats per group were sacrificed after 2, 7, 14, 28, and 42 days of treatment. The respective doses were used in previous studies to produce a concentration range similar to the therapeutic range in humans: NVP , INH, and PAR . This because small animals have a higher metabolic rate than humans. The doses were adjusted according to weight every week. A separate group of five rats were not treated with any drug and were used for baseline data.
2.4. Blood Collection and Surgical Procedure
Under isoflurane anaesthesia, blood (8 ml) was drawn by cardiac puncture and immediately aliquoted to the appropriate test tubes. Thereafter, the abdomen was opened through a vertical incision to expose the liver. A piece of liver (10 g) was cut and stored in 10% formalin and sent for histopathology. The remainder of the liver was quickly dissected out, washed in a 1.5% potassium chloride solution, frozen with liquid nitrogen, and stored at −85°C. Thereafter, the animals were sacrificed by exsanguination while being still under anaesthesia. Blood or plasma was analysed for full blood count, CD4 and CD8 counts, cytokines, drug concentrations, and liver and renal function.
The liver and renal function tests were analysed in the Toxicology Laboratory of the Department of Pharmacology, while the full blood count (FBC) was done at the National Health Laboratory Service (NHLS), Bloemfontein, South Africa. The CD4 and CD8 counts were analysed by cytometry at the Department of Haematology and Cell Biology, University of the Free State, and histopathology of the livers was performed and reported by an independent veterinary pathologist (Idexx Laboratories, Johannesburg, South Africa).
2.5. Analysis of Cytokines
The serum cytokines (IL-2 and IL-10) were detected by enzyme linked immune-assay (ELISA) technique using a Multiskan Ascent UV spectrophotometer with a 96-well microplate reader (Thermo Electric Corp., Shanghai, China), according to the manufacturer’s instructions. The ELISA well plates were already precoated with the specific capture antibody of the respective cytokine to be analysed. The appropriate volumes of the incubation buffer, respective standards, and samples as well as the biotin-conjugate secondary antibody were added to the relevant wells and then incubated for 2 hours at room temperature. Thereafter, streptavidin-horseradish peroxidase was added and the plate was incubated for 30 minutes at room temperature. Lastly, a chromogen substrate was added and the plate was further incubated in the dark for 30 minutes at room temperature. The reaction was stopped by addition of acid and absorption was measured at 450 nm with a microplate reader. A standard curve was prepared for each cytokine in pg/ml, and for IL-2, the standard curve concentration ranged from 23 to 1500 pg/ml, while for IL-10 it was 15–1000 pg/ml.
2.6. Drug Analysis
The plasma concentrations of NVP, INH, and PAR were measured using a high performance liquid chromatography (HPLC) method for simultaneous determination of the three drugs. Briefly, to 100 μL of plasma, 20 μL of sulfapyridine (internal standard) was added and vortexed for 15 seconds. Thereafter, 50 μL of 15% zinc sulphate and 50 μL of methanol were added to precipitate the proteins. The sample was vortexed for 30 seconds and then centrifuged at 7026 ×g for 15 minutes. The supernatant was further purified by solid phase extraction; a C18 solid phase extraction cartridge (1 ml) was conditioned with 2 ml HPLC grade methanol, 2 ml deionised water, and 2 ml 0.05 M potassium phosphate buffer (pH 4.5), respectively. The supernatant was then placed on the column and allowed to elute. Thereafter, the column was washed with 100 μL of potassium phosphate buffer. The compounds were eluted with 100 μL HPLC grade acetonitrile followed by 100 μL of methanol of which 50 μL was injected in the HPLC. The analyte was eluted with a gradient mobile phase of 0.06% trifluoroacetic acid (A) and acetonitrile (B) over a C18 (4.60 × 250 mm) 5 μm analytical column at 1 ml/min and was detected by UV at 260 nm. The respective retention times for INH, PAR, IS, and NVP were 3.1, 9.8, 10.4, and 11.6 minutes. The 5-day calibration curves for INH (1–10 μg/ml), NVP (1–10 μg/ml), and PAR (1–20 μg/ml) were linear with correlation coefficients () of 0.9954, 0.9968, and 0.9997 and accuracy above 98%, 94%, and 97%, respectively.
2.7. Microsomal Preparation and CYP450 Activity
A small piece (1-2 g) of liver was homogenised and microsomes were prepared by differential density ultracentrifugation, and the microsomal pellets were resuspended in 0.1 M potassium phosphate buffer containing 20% glycerol and stored at −85°C until use as reported by Von Moltke et al. (1993) . Total protein concentration was determined by the Biuret method, while the microsomal enzyme activities for CYP3A2, CYP2E1, and CYP1A2 were measured by HPLC using a midazolam, chlorzoxazone, and 7-ethoxyresorufin as the respective enzyme markers.
2.7.1. CYP3A2 Activity
This was a modification of the procedure described by Von Moltke (1996) . To 260 μL of 0.1 M sodium phosphate buffer (pH 7.4) were added final concentrations of 0.8 mg/ml microsomal protein, 4 mM magnesium chloride, and 61.3 nmol midazolam. After preincubation for 5 minutes at 37°C, the reaction was started by the addition of the NADP regenerating system and incubated for 10 minutes at 37°C, after which the reaction was stopped by the addition of 250 ml of cold HPLC grade acetonitrile and 50 μL carbamazepine (internal standard). Thereafter, the sample was alkalinised with sodium hydroxide and extracted with diethyl ether by liquid-liquid extraction. The organic phase evaporated under nitrogen, and the residue was reconstituted with 150 μL of mobile phase of which 100 μL was injected into the HPLC for analysis of midazolam, 1-hydroxymidazolam, and the internal standard. The compounds were analysed on a reversed phase Ultrasphere C18 analytical column (5 μm, 250 mm × 4.6 mm ID) (Beckman, USA) with a mobile phase of 45% acetonitrile in sodium acetate buffer (pH 4.0) at 1 ml/min, and detection was by UV at 220 nm.
2.7.2. CYP2E1 Activity
This was a modification of the procedure described by Peter et al. (1990) . To 250 μL of 0.1 M sodium phosphate buffer (pH 7.4) were added final concentrations of 0.2 mg/ml microsomal protein and 505 nmol chlorzoxazone. After preincubation for 5 minutes at 37°C, the reaction was started by the addition of the NADP regenerating system and was incubated for 10 minutes at 37°C after which the reaction was stopped by the addition of 40 μL of 0.1 M hydrochloric acid and 15 μL paracetamol (internal standard). Thereafter, the samples were analysed by HPLC for chlorzoxazone and its metabolite, 6-hydroxychlorzoxazone. The sample was purified by solid phase extraction on a 6 cc Bond elut C18 column catridge, after which 30 μL of the eluent was analysed on a RP Ultrasphere C18 analytical column (5 μm, 250 mm × 4.6 mm ID) (Beckman, USA) using a gradient solvent system for a mobile phase consisting of acetonitrile (solvent A) and phosphate buffer (pH 4.5; solvent B) over 15 min. Detection was by UV at 280 nm.
2.7.3. CYP1A2 Activity
This was a modification of the procedure described by Burke and Mayer (1974) . To 210 μL of 0.1 M HEPES potassium salt buffer (pH 7.4) were added final concentrations of 0.2 mg/ml microsomal protein, 60 μM EDTA, 5 mM magnesium sulphate, and 20 nmol ethoxyresorufin. After preincubation for 5 minutes at 37°C, the reaction was started by the addition of the NADP regenerating system and incubated for 10 minutes at 37°C after which the reaction was stopped by the addition of 2.5 ml cold acetonitrile. The sample was transferred to a quarts cuvette and detection was read by fluorescence at wavelengths for resorufin of excitation of 560 nm and emission of 585 nm.
2.8. Data Analysis
Data was analysed by nonparametric methods using the GraphPad Instat statistical program and the Mann–Whitney U test was used for data comparison with the level of significance set at .
Over the treatment period, there were no signs of abnormalities or deaths. All groups exhibited a progressive increase in body weight as expected with growth (Table 1). Likewise, in Table 2, the progressive increase in red cell count, haemoglobin, and mean corpuscular haemoglobin concentrations (MCHC) over the 42 days of treatment, versus a decreased mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH), was also observed in the control group, implying that it was also due to normal growth and development.
|S: saline; INH: isoniazid; NVP: nevirapine; PAR: paracetamol; .|