Acute Oral Toxicity of Trichosetin in Mice (Mus Musculus L.)

Philippine Journal of Science Final Copy: 8 pages 143 (2): 99-106, December 2014 Date Printed: 05/11/14 ISSN 0031 - 7683 10:16 A.M. Date Received: ?? ???? 2014 Acute Oral Toxicity of Trichosetin in Mice (Mus musculus L.) Marilen M. Parungao-Balolong1, Marilet Lian B. Caluag1, Isabella Bianca D. Catibog1, Nelia P. Cortes-Maramba2, Isidro C. Sia2, Susie O. Sio2, Ernesto C. Balolong3, and Eufrocinio C. Marfori4* 1Department of Biology, College of Arts and Sciences, University of the Philippines Manila, Ermita, Manila 2Department of Pharmacology and Toxicology, College of Medicine, University of the Philippines Manila, Ermita, Manila 3Department of Biology, School of Sciences and Engineering, Ateneo de Manila University, Katipunan Avenue, Loyola Heights, Quezon City 4National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines Los Baños, College, Laguna This study characterizes the toxicity profile of trichosetin, a novel drug which showed pronounced activity against methicillin-resistant Staphylococcus aureus (MRSA). Different doses of this drug were orally administered to male and female ICR-strain Swiss Webster mice. Characterization of toxidromes was executed during a 14-d observation period. Gross examination of the liver, heart, spleen, kidneys, and lungs of mice was performed at the termination of the study. Trichosetin showed a dose-related increase in the magnitude of biological response observed in the toxidromes and body weights of mice. However, no dose-related changes were observed in the weight and gross morphological anatomy of the major internal organs of mice. The median effective dose -1 (ED50) for analgesia was 325.1 + 71.7 mg kg . The median toxic dose (TD50) for dyspnea was 417.6 + 67.4 mg kg-1. The No Observed Adverse Effect Level (NOAEL) of trichosetin was 160 mg kg-1 under the conditions of this study. The results of this study suggest that the possible target organ system of trichosetin toxicity is the central nervous system, which exhibited the most number of toxidromes. Key Words: Antibiotic, cleaning, dual culture, MRSA, toxicity, trichosetin INTRODUCTION ubiquitous microorganism colonizing the skin and mucous membranes of most of the human population. It is also an Trichosetin is a tetramic acid antibiotic (Fig. 1) produced opportunistic pathogen causing infection most commonly in the dual culture of Trichoderma harzianum H14 in tissues and sites with lowered host resistance such as in and Catharanthus roseus callus (Marfori et al. 2002a). diabetic individuals, old malnourished persons and other Belonging to a class that displays a wide spectrum of chronic cases (Rivera & Boucher 2011). Worldwide, many biological activity, this compound was found to have a strains of S. aureus have already been reported as resistant marked inhibitory activity against Gram-positive bacteria against standard antibiotics except vancomycin (Liu et such as Staphylococcus aureus. S. aureus causes a variety al. 2011). The increasing incidence of hospital-acquired of nosocomial and community-acquired infections infections caused by multi-drug resistant bacteria such as (Chambers & DeLeo 2009; Reddy et al. 2012). It is a methicillin-resistant Staphylococcus aureus (MRSA) has *Corresponding author: [email protected] been a serious clinical problem (Rehm 2008; Loomba et 99 Philippine Journal of Science Balolong et al.: Toxicity of trichosetin Vol. 143 No. 2, December 2014 OH Test Animals All mice were treated according to the guidelines of the O Philippine Association for Laboratory Animal Science Code of Practice for the Care and Use of Laboratory Animals in the Philippines (PALAS 1999). Six week old, NH specific pathogen-free (SPF), ICR-strain Swiss Webster HO mice, with weights ranging from 19-28 g, were obtained from the Department of Pharmacology and Toxicology, College of Medicine, University of the Philippines Manila. O All mice were housed individually in plastic observation cages and were kept at the Animal House, College of Arts and Sciences, University of the Philippines Manila. The mice were acclimatized for 2 week prior to the experiment proper. Rodent food and water were provided ad libitum throughout the acclimatization and observation periods. Administration of trichosetin Figure 1. Chemical structure of Trichosetin. The mice were randomly assigned to four treatment groups and one control group of three males and three females. The treatment groups were orally gavaged with trichosetin at 160, 250, 400, and 630 mg kg-1 body weight, while only al. 2010). The recent emergence of vancomycin-resistant distilled water was gavaged to the control group. enterococci and vancomycin-intermediate resistant S. aureus is now raising serious public health concerns (Thati Trichosetin was prepared as a 2% stock solution in distilled et al. 2011; Tarai et al. 2013). water. The volume of the stock solution administered to each mouse was calculated using the formula: Trichosetin was shown to have pronounced activity against MRSA (Diwa & Peji 2005). This has prompted us to work [dosage (g kg-1)] [weight (kg)] on the development of trichosetin as a therapeutic agent Volume of solution (mL) = concentration of the stock against MRSA. Previous studies on trichosetin included its solution (g mL-1) isolation and structural elucidation (Marfori et al. 2002a), biosynthesis (Marfori et al. 2002b), phytotoxicity (Marfori et al. 2003), efficacy against MRSA strains (Diwa & Peji In cases when volume of solution exceeded 1 mL, further 2005), and mutagenicity (Alad & Cocos 2008; Alad et al. administration of trichosetin was done 30 min after the 2009). However, basic knowledge of its toxicity in relevant first administration. All mice were starved of food and animal models is still lacking. The present paper reports water 3 h before the administration of trichosetin and 2 the acute oral toxicity of trichosetin in mice. Our findings h thereafter. will provide valuable information for clinicians to predict, diagnose, and prescribe treatment for acute poisoning to Characterization of lethal and non-lethal trichosetin. Moreover, information about the toxicity profile toxidromes in mice of trichosetin may aid in the assessment whether it will The mice were observed for clinical signs of toxidromes have a sufficiently low risk to be marketed as an antibiotic. (such as startle reaction, ataxia, loss of righting reflex, loss of screen grip, paralysis, dyspnea, pilomotor erection, catalepsy, analgesia, and others) immediately after administration, during the first 12 h, and twice daily MATERIALS AND METHODS thereafter for 14 d. In particular, the test for analgesia was done by pressing the toe of the hind foot of the mouse Preparation of Trichosetin using the fingernail. The test was negative for analgesia if Trichosetin was produced following the procedure as the mouse showed startle response with vocalization and/ described by Marfori et al. (2002a). The purified sample or attempts to bite or to escape, or there was an attempt to of trichosetin (purity >95 % by HPLC) was then sent to calmly withdraw the foot from the pressure. The test was Norberto R. Agcaoili Memorial Tissue Bank, Department positive for analgesia if the mouse showed no response. of Orthopedics, College of Medicine, University of the Philippines Manila-Philippine General Hospital for Mortality rates were monitored twice daily during the lyophilization. 14-d observation period. Also, the body weights were 100 Philippine Journal of Science Balolong et al.: Toxicity of trichosetin Vol. 143 No. 2, December 2014 determined prior to dosing, twice weekly, and at death or linear regression analysis, after which the dose-response termination. At the termination of the study, all mice that lines were drawn. Analyses were performed using the survived were sacrificed through cervical dislocation, BioStat 2008 Professional Software (AnalystSoft Inc., and were subjected to necropsies. Major internal organs Vancouver, Canada). of the body such as the heart, liver, left and right lungs, kidneys, and spleen of the treatment groups were excised and compared to the organs of the control group for any difference in the weight and gross morphological anatomy. RESULTS AND DISCUSSION Lethal and non-lethal toxidromes observed in mice after a Determination of the median effective dose (ED50), single administration of trichosetin at different dose levels median toxic dose (TD50), and no observed adverse are shown in Table 1. There was 0% mortality observed effect level (NOAEL) when mice were orally administered with 0- 400 mg kg-1 -1 ED50 and TD50 were defined as the dose that caused trichosetin. At the highest dose level of 630 mg kg , there analgesia and dyspnea (abdominal breathing and gasping), was one male mouse which died after five days accounting respectively, in 50% of the mice population. NOAEL was for only 17 % mortality. The non-lethal toxidromes, on the highest dose at which no adverse effect was detected. the other hand, were observed to vary with dose levels with % morbidity increasing in response to increasing Analysis of Data dose level. A dose-dependent increase in the severity Experiments were laid out in a completely randomized of adverse effects was also manifested by several organ design. The data on body weights were analyzed by systems with the central nervous system exhibiting the one-way ANOVA while the data on organ weights were most number of toxidromes such as loss of righting reflex, analyzed by two-way ANOVA to determine significant

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