Melatonin prevents the free radical and MADD metabolic profiles induced by antituberculosis drugs in an animal model Introduction Treatment of pulmonary tuberculosis (TB) consists of two phases, i.e. an intensive phase of treatment with four anti- TB drugs (2 months) and then a maintenance phase for the rest of the treatment period (4 months). TB treatment is carried out using a combined therapy of anti-TB drugs which include isoniazid, rifampicin, ethambutol and pyra- zinamide with less risk for development of drug resistance. Various combinations of anti-TB drugs, such as Rifater, are commercially available as a single administration, which constitutes a fixed combination of isoniazid, rif- ampicin and pyrazinamide and this three-drug combination is widely used in TB treatment. However, while these agents are effective antibiotics, TB patients treated with rifampicin, pyrazinamide, and isoniazid can experience hepatotoxicity resulting in a discontinuation of treatment in these patients [1,2]. Anti-TB chemotherapy can also be associated with a number of other side-effects which include myocardial damage, respiratory complications [3], hypoglycaemia [4], neurological complications [5–7], and acidosis [6]. At present, very little is known about the metabolism of anti-TB chemotherapeutics. It is however well known that the catalase enzyme of Mycobacterium tuberculosis is responsible for the conversion of isoniazid to its active bactericidal equivalent isonicotinic acid hydrazide as well as acetylisoniazid [8]. Superoxide is formed during isoniazid oxidation and is thought to be involved in the activation process [9]. Further isoniazid metabolites that have been detected in humans and rats include isonicotinic acid, isonicotinylglycine [10], acetylhydrazine, and diacetylhydr- azine [11]. This can also take place via host-specific enzymes, such as N-acetyltransferase [12]. Rifampicin is already in its active state at the stage of administration and can be converted into two inactive products, Rip- Ma 3-formyl-23-[O-(alpha-d-ribofuranosyl)] and Rip-Mb 23-[O-(alpha-d-ribofuranosyl)] by mycobacteria [13]. A number of rifampicin metabolites have also been identified in human plasma, urine and saliva [14, 15]. Pyrazinamide in turn is metabolized into a number of metabolites which include pyrazinoic acid, 5-hydroxypyrazinoic acid, 5-hy- droxypyrazinamide [16] and pyrazinuric acid [17]. Although a number of drug metabolites have been identified, their effects in vivo have not yet been fully established. The safety of anti-TB treatment is presently under scrutiny and various drug combinations and treatment times have been suggested to alleviate the burden of toxic side-effects [2]. Here we show that the anti-TB drug combination, Rifater, increases the oxidative stress in an animal model and affects the organic acid profiles. We also show that the application of the antioxidant, melatonin, Abstract: The objective was to determine the effect of combined antituberculosis (anti-TB) drug therapy and an antioxidant, melatonin, on the free radical and organic acid profiles in an experimental rat model. A combined anti-TB drug, Rifater, consisting of 12.0 mg rifampicin, 0.8 mg isoniazid, and 23.0 mg pyrazinamide and 18.56 lg melatonin/kg body weight per day (corresponding to average physiological human intake) were orally administered to Sprague–Dawley rats. Hydroxyl radical production was monitored by quantifying 2,3-dihydroxybenzoic acid produced after intraperitonial sodium salicylate injections. Organic acid extractions and gas chromatography-coupled mass spectrometry analyses were performed on collected urine samples. The results show hydroxyl radicals (P ¼ 0.0019) and organic acids (P-value range: 0.037 to <0.001), characteristic of a multiple acyl-CoA dehydrogenase defect (MADD), were elevated with Rifater treatment and these elevations were significantly lowered with melatonin pretreatment (P-value range: 0.031 to <0.001), probably because of its inherent antioxidant activity. We conclude that hydroxyl radical production and an increased organic acid profile induced by anti-TB medication indicates inhibition of the electron transport chain. We also conclude that free radicals leading to clinical symptoms associated with an MADD metabolic profile induced by anti-TB treatment could be alleviated by melatonin intervention. Du Toit Loots 1 , Ian J. Wiid 2 , Benedict J. Page 3 , Lodewyk J. Mienie 4 and Paul D. van Helden 2 1 Department of Nutrition, School for Physiology, Nutrition and Consumer Science, North-West University, Potchefstroom; 2 Department of Medical Biochemistry, MRC Centre for Molecular and Cellular Biology, and 3 Department of Anatomy, Faculty of Health Sciences, University of Stellenbosch, Tygerberg; 4 Department of Biochemistry, School of Chemistry and Biochemistry, North- West University, Potchefstroom, South Africa Key words: melatonin, organic acids, radicals, Rifater Address reprint requests to Ian Wiid, MRC Centre for Molecular and Cellular Biology, Department of Medical Biochemistry, Faculty of Health Sciences, University of Stellenbosch, PO Box 19063, Tygerberg, 7505, South Africa. E-mail: iw@sun.ac.za Received April 20, 2004; accepted August 20, 2004. J. Pineal Res. 2005; 38:100–106 Doi:10.1111/j.1600-079X.2004.00176.x Copyright Ó Blackwell Munksgaard, 2004 Journal of Pineal Research 100