Experimental studies on the involvement of brain oxidative and nitrosative stress in methylxanthine induced neurobehavioral toxicity

K Gulati, A Ray. V.P. Chest Institute, University of Delhi, Delhi, Department of Pharmacology, 110007, India

Methylxanthines like theophylline have reemrged as effective adjunct therapies in respiratory disorders like bronchial asthma and COPD and newer aspects of their pharmacodynamics have been proposed. Theophylline has a narrow therapeutic index which restricts its rational and effective use by making it prone to adverse effects like cardiotoxicity and neurotoxicity. The drug is known to act by both peripheral and central mechanisms and while cardiac adverse effects are due to peripheral mechanisms, neurotoxicity is centrally mediated. Anxiety and seizures are amongst the two most common neurobehavioral manifestations of this methylxanthine and studies to explore conventional mechanisms have proved inconclusive. The brain is particularly sensitive to oxidative damage by free radicals and the present study investigated the possible involvement of oxidative stress during theophylline induced neurobehavioral toxicity in rats. Anxiety was induced by aminophylline, a water soluble salt of theophylline (100mg/kg) in rats and assessed by the elevated plus maze (EPM) test. Aminophylline induced anxiogenic behavior in rats in the EPM and the number of open arm entries and time spent in the open arms were markedly reduced as compared to the vehicle treated controls. 2-chloroadenosine (adenosine agonist) and the IBMX (phosphodiesterase inhibitor) showed inconsistent effects in the EPM test. Pretreatment with α-tocopherol (antioxidant, 20 and 40 mg/kg) or l-arginine (NO precursor, 100 and 500mg/kg) attenuated the neurobehavioral suppression induced by the methylxanthine in a dose related manner. Sub-threshold doses of theophylline (50mg/kg) in combination with restraint stress (RS) induced anxiogenic behavior which were also attenuated by pretreatment with α-tocopherol or L-arginine in separate groups. Aminophylline induced anxiety was accompanied by elevations in malondialdehyde (MDA) and reductions in reduced glutathione (GSH) and NO metabolite (NOx) levels in brain homogenates, which were also reversed after antioxidant and l-arginine pretreatments. In the next set of experiments, seizures were induced by aminophylline (250mg/kg) both alone and in combination with subthreshold electroshock (15mA for 0.2 sec) in mice. Aminophylline induced seizures and mortality, and these were not significantly attenuated by phenytoin, 2-chloroadenosine or IBMX. However, pretreatments with α-tocopherol attenuated such response, whereas, l-arginine produced opposite effect and aggravated the seizures and mortality. The NO synthase inhibitor, L-NAME (3 and 10 mg/kg) on the other hand, attenuated aminophylline seizures. Similar drug induced modulations were seen in the aminophylline+ECS model of seizures. Aminophylline seizures were associated with elevations of MDA levels and suppression of GSH levels in brain homogenates, whereas, brain NOx levels were enhanced. These biochemical changes were reversed by antioxidant and L-NAME pretreatments. The results suggest that reactive oxygen species and their complex interactions with reactive nitrogen species could be involved during aminophylline induced anxiety and seizures and antioxidants and NO modulators could be viewed as potential agents against such theophylline induced neurobehavioral toxicity.