Evaluation of efavirenz on neurochemical and oxidative stress markers and addictive-like behaviours in rats

Abstract

HIV positive patients treated with the antiretroviral (ARV) drug efavirenz have been observed to experience various neuropsychiatric symptoms including insomnia, dizziness, somnolence, vivid and abnormal dreams and thoughts and manic episodes, clearly indicating central nervous system (CNS) effects. Moreover, recent news reports in South Africa describe people recreationally using efavirenz by crushing and smoking this ARV drug in a mixture of drugs known as "Nyaope" or "Whoonga". Other than efavirenz as the main constituent, this mixture is known to contain marijuana, milk powder, rat poison, amongst others. The abuse of this drug places tremendous pressure on the Department of Health and endangers the lives of people in need of ARV treatment. A previous preclinical study suggests that the effects of efavirenz are in line with that of lysergic acid diethylamide (LSD), mediated by a partial agonist effect on the serotonin (5-HT) - 2A receptor subtype, the primary binding site for drugs of abuse known as the hallucinogens. Drugs of abuse are known to alter the behaviour and neurochemistry of the abuser and some studies also suggest alterations in the anti-oxidant system of the body, linked to monoamine alterations, in this instance, dopamine (DA), 5-HT and noradrenaline. Animal models enable research to screen for alterations in the above-mentioned indicators and to determine the underlying mechanism through which a drug may elicit its abuse potential and related addictive-like effects. A well-known behavioural test to assess rewarding properties of drugs is the conditioned place preference (CPP) test in which the motivational or aversive properties of a drug serves as an unconditioned stimulus that is continuously paired with an environment. Through Pavlovian principles, this leads to an association of the previously neutral environment with the properties of the drug. Rewarding or addictive drugs are known to significantly increase the time spent in the drug-paired compartment after conditioning. Various other behavioural screening tests such as the sucrose preference test (SPT) may indicate whether a drug interacts with the reward pathways under the control of DAergic signalling, leading to changes in sucrose consumption (as hedonic measure). Moreover, the psychomotor stimulant theory suggests that drugs of abuse induce hyper-locomotion (in the open field test (OFT)) in animals, which is considered a valuable test to screen for drugs of abuse. Furthermore, this test may be of value in supporting the outcome of other tests (such as the CPP test) in which locomotion plays an imperative role. This study aimed to assess the addictive-like properties of efavirenz after sub-acute (6 days) and sub-chronic (14 days) exposure in rats. The present study (ethical approval no: NWU - 00267- 16- S5) used a total of 84 male Sprague Dawley rats (randomly allocated to 7 groups of rats with 12 rats per drug exposure group), bred and housed in the DST/NWU PCDDP Vivarium. The sub-acute paradigm exposed 5 groups of rats to i.p injections of either 5, 10 or 20 mg/kg/d efavirenz, 1 mg/kg/d methamphetamine (MA) (as a positive control) or vehicle in order to establish the most rewarding dosage of efavirenz. CPP, sucrose preference and locomotor activity were assessed in the sub-chronic study, evaluating the effects of efavirenz on reward (CPP), anhedonic manifestations over time (SPT) and locomotion (OFT). In the sub-acute study, MA was compared to a vehicle in order to validate the CPP paradigm under our laboratory conditions. The sub-chronic study was conducted using the most rewarding dose of efavirenz as determined in the sub-acute phase (5mg/kg/d alternate days x 14 days). Furthermore, quantification of frontal cortical, striatal and hippocampal DA, 5-HT, and their respective metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-Hydroxyindoleacetic acid (5-HIAA) as well as NA was performed in the sub-chronic efavirenz exposure study, using a high-performance liquid chromatography system with electrochemical detection (HPLC-EC). Regional brain lipid peroxidation and plasma superoxide dismutase (SOD) levels were also assessed after sub-chronic exposure to efavirenz, utilizing ELISA kits. One-way ANOVA with Dunnett's post hoc test or unpaired student t-tests were applied for statistical analyses with p < 0.05 deemed significant. In the sub-acute study, 5 mg/kg efavirenz induced a significant increase in the time spent in the drug-paired compartment compared to the control group in the CPP test. These results were comparable to the rewarding effects of MA in the same test. Efavirenz at 10 mg/kg showed no changes, while 20 mg/kg showed a significant decrease in the time spent in the drug-paired compartment in comparison to the control group. In contrast to data described in the CPP test, no changes in locomotion or sucrose preference were observed. Neurochemical analyses in the sub-chronically exposed efavirenz animals indicated a significant increase in frontal cortical DA and 5-HT levels whilst 5 HIAA levels were significantly lower compared to vehicle exposed animals. DA and 5-HT turnover was significantly decreased in animals exposed to sub-chronic efavirenz compared to a vehicle control. Striatal DA, 5-HT and NA were increased while DOPAC levels and DA turnover was decreased in these animals compared to the vehicle control. Hippocampal DA, DOPAC, 5-HIAA and 5-HT turnover was significantly decreased whereas DA turnover rate was significantly increased in animals exposed to efavirenz compared to the vehicle control. Peripheral (plasma) SOD levels were significantly increased in efavirenz treated animals, while regional brain lipid peroxidation was significantly elevated in the DA rich areas (frontal cortex and striatum), compared to their vehicle control. The findings in the sub-acute and sub-chronic study demonstrate a significant dose dependant rewarding effect of efavirenz in rats (in a validated CPP paradigm under our laboratory conditions), with lower doses being most effective and higher doses eliciting aversive responses. The unaltered preference for sucrose after sub-chronic efavirenz exposure further supports the outcomes that the lower doses are rewarding since no anhedonic behaviour manifested over the period of treatment. Unaltered locomotion suggests that sub-chronic efavirenz does not elicit its effects through the same striatal-mediated mechanism as do many other drugs of abuse but supports the outcome in the CPP test. Moreover, this implies that performance in the CPP was not a result of undue effects of the drug or condition on the mobility of the animals. The findings obtained in the sub-chronic study confirm that efavirenz alters regional brain DA, 5-HT, NA and lipid peroxidation as well as peripheral SOD levels. This animal study has significant relevance in elucidating the possible mechanism through which efavirenz induces a rewarding effect, which in turn may underlie its abuse potential.