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 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

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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), 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

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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 analgesia, fasciculations and tremors. Other prominent differences. Treatment means were compared using toxic syndromes displayed by mice were paralysis of the hind legs, dyspnea, catalepsy, ataxia, loss of screen grip, Fisher’s LSD test at 5% level of significance. The ED50 and TD values were estimated by probit analysis via and pilomotor erection. No difference was observed in the 50 toxidromes exhibited by male and female mice.

Table 1. Lethal and non-lethal toxidromes of Trichosetin observed in ICR-strain Swiss Webster mice during the 14-day observation period. DOSE Lethal Toxidrome Non-lethal Toxidrome -1 mg kg Mortality (%) Time to death Morbidity (%) Observation 0 0 Not applicable 0 (+) Startle reaction 160 0 Not applicable 0 (+) Startle reaction 250 0 Not applicable 33 (+) Startle reaction Ataxia Catalepsy Recovery before day 14 400 0 Not applicable 67 No startle reaction Ataxia Pilomotor erection Analgesia Dyspnea Hind legs lay flat and stretched out when walking Confusion No recovery until day 14 630 17 5 days 100 No startle reaction Analgesia Dyspnea Pilomotor erection Ataxia Catalepsy Loss of righting reflex Paralysis of forelegs, hind legs and head Loss of screen grip No recovery until day 14

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Analysis of body weights showed a decrease in both the relapse. During necropsy of the mice, no superficial organ treatment and control groups during the first three days of damage in gross morphological anatomy was observed. the study. It was only after the third day that body weights The organ weights of the treatment and control groups increased. As shown in Figure 2, body weights of both in both male and female mice were also not significantly male and female mice from the control and treatment different (Fig. 3). groups were not significantly different, except that of the treatment group administered with the highest dose Among the non-lethal responses exhibited by the mice level of 630 mg kg-1 likely due to reduced food and water upon administration of trichosetin, we regarded analgesia intake resulting from prolonged periods of narcosis and as another possible therapeutic effect of trichosetin (aside from it as being an antibiotic). The dose-response

A. Male mice

B. Female mice

Figure 2. Mean body weights of male (A) and female (B) ICR-strain Swiss Webster mice administered with different dose levels of Trichosetin over a 14-day observation period.

102 Philippine Journal of Science Balolong et al.: Toxicity of trichosetin Vol. 143 No. 2, December 2014 curve for analgesia is shown in Figure 4 illustrating a toxidrome that affected the general well-being of 42% shallow, positive, linear slope. A positive, linear slope of the total mice population in treatment groups over the suggests that as the dose of trichosetin is increased, a 14-d observation period. As shown in Figure 5, the TD50 corresponding increase in the observed analgesia can be of trichosetin in male and female mice was 417.6 + 67.4 noted. The shallow slope indicates that a large increase mg kg-1. Similar with analgesia, the dose-response line in the dose of trichosetin will only merit a corresponding of trichosetin for dyspnea illustrated a shallow, positive, low change in its analgesic effect. The ED50 value of linear slope. trichosetin based on the detectable analgesia over the -1 14-d observation period was 325.1 + 71.7 mg kg-1, i.e. The mice at the dose levels of 400 and 630 mg kg exhibited analgesia, dyspnea, catalepsy and ataxia. Only at this dose, 50% of the mice will have the inability to -1 feel pain. catalepsy and ataxia were recorded for 250 mg kg , and no adverse effects were observed for mice administered In preclinical safety and toxicity testing, it is important with 160 mg kg-1. The similar body and organ weights and to know that all drugs are toxic at some dose (Abrams gross organ morphology compared with that of the control et al. 2003; Berkowitz 2004). If the toxic effect is the group supported our observation that there was no adverse -1 death of the animal, the (LD50) may effect caused by trichosetin at 160 mg kg . Hence, the dose be experimentally defined. Since there was only a single level of 160 mg kg-1 was identified to be the NOAEL for death observed at the highest dose level of 630 mg kg-1, trichosetin under the conditions of the study. the LD50 cannot be derived; hence, only the median toxic dose (TD50) was computed. Dyspnea was the basis of the

TD50 value of trichosetin because it was evidently the

A. Male mice B. Female mice

Figure 3. Mean weights of the major internal organs of male (A) and female (B) ICR-strain Swiss Webster mice 14 days after administering different dose levels of Trichosetin. Legend: (♦)- liver; (▲)- right lung; (∆)- left lung; (■)- kidney 1; (□)- kidney 2; (●)- heart; (○)- spleen.

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Figure 4. Dose-response line based on analgesia showing 325.1 mg kg-1 body weight (pointed by the arrow) as the median effective dose

(ED50) of Trichosetin.

Figure 5. Dose-response line based on dyspnea showing 417.6 mg kg-1 body weight (pointed by the arrow) as the median toxic dose

(TD50) of Trichosetin.

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CONCLUSION AND ALAD P, COCOS A, BALOLONG EC, PARUNGAO RECOMMENDATIONS MM, MARFORI EC. 2009. Mutagenicity potential of the novel drug trichosetin using the rec assay and Based on the results of this study, trichosetin showed micronucleus test. Philipp J Sci 138(2): 119-124. a dose-related increase in the magnitude of biological response observed in the toxidromes and body weights of BERKOWITZ BA. 2004. Development and regulation of mice. However, no dose-related changes were observed drugs. In: Basic and Clinical Pharmacology, 10th ed. in the weight and gross morphology of the major internal Katzung BG, ed. New York: McGraw-Hill Companies, organs of mice. Inc. pp. 64-74. CHAMBERS HF, DELEO FR. 2009. Waves of resistance: For future toxicity studies, it is recommended to evaluate Staphylococcus aureus in the antibiotic era. Nat Rev trichosetin using at least two rodent species and one non- Microbiol 7:629–641. rodent species as experimental animals. These studies should include drug exposure data, together with a more DIWA LT, PEJI RC. 2005. Antimicrobial activity detailed histology and clinical pathology data. This is of trichosetin against local clinical methicillin- important since results may suggest differences in the resistant Staphylococcus aureus (MRSA) isolates. absorption, distribution, and biotransformation of the [Undergraduate Thesis]. Manila, Philippines: drug. Chronic, teratogenic, and oncogenic toxicity tests in University of the Philippines Manila. 37p. (Available relevant animal models are also recommended to establish at the UP Manila Library) its long-term effects. This is to detect the unexpected LIU C, BAYER A, COSGROVE SE, DAUM RS, toxic effects that might not be observed after a single FRIDKIN SK, GORWITZ RJ, KAPLAN SL, dose administration only, but might become evident KARCHMER AW, LEVINE DP, MURRAY BE, J after its prolonged use. Furthermore, the mechanism of RYBAK M, TALAN DA, CHAMBERS HF. 2011. action of its toxicity should be studied as this will yield Clinical practice guidelines by the Infectious Diseases very substantial information required for drug labeling. Society of America for the treatment of methicillin- Since trichosetin displayed potential analgesic properties, resistant Staphylococcus aureus infections in adults the comprehensive evaluation of its analgesic capacity and children. Clin Infect Dis 52(3): 285-292. through efficacy studies is also highly recommended. LOOMBA PS, TANJEA J, MISHRA B. 2010. Methicillin- and vancomycin-resistant S. aureus in hospitalized patients. J Glob Infect Dis 2: 275-283. ACKNOWLEDGMENTS MARFORI EC, KAJIYAMA S, FUKUSAKI E, This study was funded by Department of Science and KOBAYASHI A. 2002a. Trichosetin, a novel tetramic Technology-Philippine Council for Advanced Science acid antibiotic produced in the dual cuture of and Technology Research and Development (DOST- Trichoderma harzianum H14 and Cathranthus roseus PCASTRD) and the University of the Philippines, Los callus. Z Naturforsch 57c: 465-470. Baños (UPLB) Basic Research. MARFORI EC, BAMBA T, KAJIYAMA S, FUKUSAKI E, KOBAYASHI A. 2002b. Biosynthetic studies of the tetramic acid antibiotic trichosetin. Tetrahedron 58: 6655-6658. REFERENCES MARFORI EC, KAJIYAMA S, FUKUSAKI E, ABRAMS WB, BAGDON RE, ZBINDEN G. 2003. KOBAYASHI A. 2003. Phytotoxicity of the tetramic Techniques of animal and clinical toxicology. In: acid metabolite trichosetin. Phytochemistry 62:715- Animal and Clinical Pharmacologic Techniques in 721. Drug Evaluation. Nodine JH, Siegler PE, eds. Chicago: Year Book Medical Pub Inc. pp. 45-57. [PALAS] Philippine Association for Laboratory Animal Science Code of Practice for the Care and Use of ALAD P, COCOS A. 2008. Mutagenicity potential Laboratory Animals Guidelines.1999. Administrative of trichosetin, a novel drug from the dual culture Order no. 40 Series of 1999. of Catharanthus roseus (L.) G. Don callus and Trichoderma harzianum H14, using Rec Assay and REHM SJ. 2008. Staphylococcus aureus: the new micronucleus test. [Undergraduate Thesis]. Manila, adventures of a legendary pathogen. Cleveland Clinic Philippines: University of the Philippines Manila. 95p. J Med 75(3): 177-192. (Available at the UP Manila Library) RIVERA AM, BOUCHER HW. 2011. Current concepts

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in antimicrobial therapy against select gram-positive organisms: Methicillin-resistant Staphylococcus aureus, penicillin-resistant Pneumococci, and vancomycin-resistant Enterococci. Mayo Clin Proc 86: 1230-1242. TARAI B, DAS P, KUMAR D. 2013. Recurrent challenges for clinicians: emergence of methicillin-resistant Staphylococcus aureus, vancomycin resistance, and current treatment options. J Lab Physicians 5:71-78. THATI V, SHIVANNAVAR CT, GADDAD SM. 2011. Vancomycin resistance among methicillin resistant Staphylococcus aureus isolates from intensive care units of tertiary care hospitals in Hyderabad. Indian J Med Res 134: 704-708.

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