The therapeutic effect of PEMF on reproduction Kumar S et al. executors of apoptosis.13 The caspase 3 effector acts on two pathways: the mitochondrial pathway (through the caspase 9 initiator) and the death receptor pathway (through the caspase 8 initiator).14 Creatine kinase is a marker of sperm maturity, and its elevated level is associated with an increased rate of functional abnormalities and increased cytoplasmic reten- tion.15 Creatine kinase (E.C.2.7.3.2) catalyses the reversible phosphorylation of ADP to ATP or creatine to creatine phosphate, thus maintaining an immediately accessible energy reservoir in the cell.16 Creatine phosphokinase, which is a key enzyme in transport and energy synthesis, supplies ATP to the sperm.17 Sperm are frequently associated with elevated activities of certain key enzymes, including creatine kinase, which indicate male infertility.18-20 In most cases of infertility, high concentrations of reactive oxygen species (ROS) have been reported.21 Kumar et al7 also reported an increased level of ROS due to microwave exposure. Oxidative stress is a cellular condition of increased ROS concentrations that causes molecular damage and leads to various diseases.22,23 However, ROS are neutralized by melatonin and its stimulatory effects on antioxidants. Melatonin is synthesized from serotonin in the pineal gland, and its level increases during the dark period and decreases during the light period. It has been reported that melatonin synthesis is suppressed by low-frequency EMF.24 Brainard et al25 have shown that melatonin synthesis and secretion from the pineal gland are under the control of visible light. In this study, a pulsed electromagnetic field (PEMF) was used to relieve the effects caused by microwaves. Following the experiments of DiCarlo et al26 on chick embryos exposed to radiation, the protection conferred by the magnetic fields appears to influence free radical scavenging. PEMF has a variety of biological effects, such as its effects on bone healing,27,28 pain relief,29 and the balance of the neuroendo- crine system (including hormone production and melatonin levels).30 PEMF has frequencies at the lower end of the electromagnetic spectrum (from 6 Hz to 500 Hz). In this study, we demonstrate that PEMF can be used as healing agent that activates the normal physiology of the body. PEMF activates normal metabolic processes, indirectly acts through the endocrine system, and controls the main cause of stress in the exposed system. We treated rats that had previously been exposed to 2.45 GHz radiation with PEMF. As biomarkers of this treatment, we chose the following parameters: melato- nin, creatine kinase activity, caspase assay, and testosterone. MATERIALS AND METHODS Animals Seventy-day-old male Wister rats (190¡10 g) were used in this study. The animals were maintained in accordance with national guidelines and protocols. The study was approved by the Institutional Animal Ethics Committee (IAEC-JNU/ 83/675-687; code no. 12/2008). A flowchart of the entire methodology is shown in Figure 1. The animals were housed in clean polypropylene cages and maintained at a controlled temperature with a constant 12-h/12-h light/dark schedule. The animals were fed a standardized normal diet (Tetragon Cheime Private Limited, Bangalore) and water ad libitum. Animal Exposure The rats were divided into five groups (Figure 1): Group I, microwave (MW) sham; Group II, MW exposure (2.45 GHz); 1238 CLINICS 2011;66(7):1237-1245 Group III, PEMF sham; Group IV, PEMF (100 Hz) exposure; Group V, MW/PEMF exposure. Each group contained three animals, and the experiments were repeated in triplicate. The rats were placed into a Plexiglas cage ventilated with one-cm-diameter holes; the exposure cage was fabricated such that the animals were comfortably housed. For the 2.45-GHz exposure, the chamber was lined with radar- absorbing material (attenuation, 40 dB) to minimize the reflection of the scattered beam. The exposure cage was placed vertically such that all of the animals were irradiated homogeneously at a single power level. The rats were exposed to a 2.45-GHz radiation source at a 50-Hz modulation frequency (input, 1080 W; output, 700 W). A microwave oven (Haier India Co. Ltd, made by PRC (China); model no. HR-18MS1) was used as the source of exposure. Exposure was performed through the horn antenna for 2 h per day for 60 days (Figure 2). Specific Absorption Rate (SAR) Calculation The emitted power of the microwaves was measured by a power meter, which is a peak-sensitive device (RF power sensor (6900 series) and IFR 6960 B RF power meter; made by Aeroflex, Inc., Wichita, Kansas, USA). Every day, the cage was placed in the same position below the horn antenna, and the same numbers of rat positions were reshuffled. A similar experiment was performed with the MW sham but without energizing the system. A similar experimental setup was adopted previously by Paulraj and Behari31,32 and Kesari and Behari.33 A full description of the exposure setup has been discussed by Kesari et al.34 The SAR was calculated for the average size of the small animals by following the work of Durney et al.35 For a plane-wave exposure with a random polarization and a power density of 0.21 mW/cm2, the SAR is 0.014 W/kg. Materials The caspase 3 assay kit (colorimetric; cat. no. CASP-3-C) was purchased from Sigma, USA. The creatine assay kit (cat no. K635-100) was purchased from BioVision Research Products (Mountain View, CA, USA), the ELISA melatonin kit (cat. no. E90908Ra) was purchased from Uscn Life Science Inc. (Wuhan, China), and the testosterone EIA kit (cat. no. 582701) was purchased from the Cayman Chemical Company (Ann Arbor, MI, USA). The remaining chemicals were purchased from Merck Chemicals, India. Melatonin Assay The melatonin level in the serum was estimated using an ELISA kit developed by Uscn Life Science Inc. Briefly, 50 ml of dilutions of the standards, the blanks and each sample were added to wells that were pre-coated with a polyclonal antibody specific for rat melatonin followed by the addition of 50 ml of detection reagent A to each tube. After incubation for one hour at 37˚C, the wells were washed with wash solution three times. A total of 100 ml of detection reagent B was added to each well and incubated for 30 min at 37˚C. The plate was washed again, which was followed by addition of the substrate solution to each well and incubation for 15 min at 37˚C. The color development was stopped by adding stop solution, and the intensity of the yellow color was measured using a spectrophotometer (450 nm). The concentrations of the unknown samples were calculated by comparison with a standard curve. The