The subacute toxicity study of an antimicrobial compound, 4-hydroxy nitrobenzene isolated from a Streptomyces species was carried out on long Evans rats. The compound, 4-hydroxy nitrobenzene (300μg) was administered daily for 14 days and its effect on body weight, hematological and biochemical parameters of blood was investigated. There was no significant difference between weight gain in rats, receiving compound and control (121±5.10 vs 115±7.2g, respectively). There were no significant changes detected in hematology (red blood cells, 5.63±0.23 vs 4.97±0.17; white blood cells, 12575±170.78 vs 10950±238; platelet, 312500 ± 9574 vs 31000±21602 (cells ml-1)x106; hemoglobin, 13.25±0.3 vs 13.27 ±0.36%, for experimental and control rats, respectively) or blood biochemistry (serum glutamic oxaloacetic transaminase, 11.5±1.29 vs 11.75±1.25IU L-1, serum glutamate pyruvate transaminase, 8.25±1.26 vs 7.25±1.5IU L-1, serum alkaline phosphatase 41.25 ±1.89 vs 44.25 ± 3.3IU L-1. Uric acid, 7.05±0.129 vs 7.3± 0.22; urea, 42.25 ± 1.708 vs 41.5 ± 2.64; creatinine, 0.85±0.05 vs 1.07±0.188mg dL-1, for experimental and control rats, respectively). Therefore, the compound, 4-hydroxy nitrobenzene (300μg day-1) over 14 days, had no toxic effects on rats.
PDF Abstract XML References Citation
How to cite this article
The concept of antibiosis opened a new field of research for isolation of antibiotics from micro-organisms and so far more than 4000 such antibiotics are known. In recent years, owing to indiscriminate use of antibiotics and other unknown reasons, the pathogenic organisms are gaining resistance to existence antimicrobial and chemotherapeutic agents (Roche, 1950). Literature survey revealed that a large number of antibiotics have been isolated from the Streptomyces species and these species has proved to be the potential sources of antibiotics. Therefore, a Streptomyces species was collected and identified. An active metabolite, 4-hydroxy nitrobenzene was isolated and its antimicrobial activity was conducted (Sathi et al., 2001a).
Pharmacology is a toxicology at higher doses, so, every drug shows toxicities at higher doses. Even at therapeutic doses many drugs shows unavoidable toxic effects. Moreover, toxicological data helps to make decision whether a new drug should be adapted for clinical use, for these assessments of a drug, it is necessary to investigate the toxicity to animals like, rats, guinapigs, dogs, monkeys, etc and in connection to these objectives, this work was conducted to report the toxicological studies of the compound 4-hydroxy nitrobenzene in rats.
MATERIALS AND METHODS
Collection and identification of the antagonistic organism: A Streptomyces species ( Holt et al., 1994) was isolated from Rajshahi (near Fine and Arts Department of the Rajshahi University), Bangladesh, at the depth of 0.75 meter using crowded plate technique (Hammond and Lambert 1978).
Isolation, purification and characterization of the compound: The compound 4-hydroxy nitrobenzene was isolated from the culture filtrate of Streptomyces species in Yeast Extract Glucose broth medium at pH 7 after 15 days incubation at the temperature of 37.5±0.5 °C having 3% salt (NaCl) concentration. The crude compound was resolved by thin layer chromatography (TLC) and was isolated from the chloroform extract (CHCL3) by preparative thin layer chromatography (PTLC) technique using solvent system CHCl3: CH3OH (10:1) .The compound was characterized on the basis of their UV, IR, NMR, Mass, HMBC and HMQC data. (Sathi et al., 2001b). Purity of the compound was checked by TLC (Touchston and Dobbins, 1978) using different solvent system.
Collection, maintenance and grouping of the experimental rats: Long Evans male rats were collected from International Center for Diarrheal Disease Research, Bangladesh (ICDDR, B). The rats were kept in properly numbered iron cages individually in a clean animal house with an optimal room temperature (25-300C) and were given ideal food (Hawk et al., 1954). The rats were maintained in this way for 15 days before drug administration and continued up to the end of the work.
On the basis of weight, the rats were grouped. The rats of group B (average weight 115.5g) were used for experiment while those of group A (average weight 106.5g) were used as control.
Sample administration: The compound was dissolved in distilled water with the help of tween-20, so that 0.3ml contained 300μg of the compound. The rats in group A and B were injected intraperitoneally with vehicle (300μl) and compound, 300μg rat-1 day-1, respectively.
Experimental Procedure: For hematological studies, blood was drawn from the tail veins of all the rats and blood smears were made on glass slides and stained with Leishmen reagent to perform TC (Total count), DC (differential count) and platelet count (Ghai, 1990). With the use of capillary tubes blood was drawn from each of the rat to estimate the hemoglobin percentage by Van Kampen-Zijlstras method (Ghai, 1990).
For the study of biochemical parameters such as SGOT (Serum glutamate oxaloacetate transaminase), SGPT (Serum glutamate pyruvate transaminase), serum alkaline phosphatase and serum creatinine, uric acid and urea were determined by using the procedures and reagents described in Enlehringer Mannheim GmbH Diagnostica (king and Armstrong, 1934; Reitman and Frankel, 1957; Fawcett and Scott, 1960; Coulombe and Favreau, 1963).
For histopathological study, liver, kidney, heart, lungs and spleen of all of the rats tissues were sliced into pieces and immersed in 10% formalin for three days, processed, stained with Harris Hematoxylin and eosin reagent, mounted on glass slides with diphenyl xyline mounting fluid and observed under microscope at the Bangladesh Sericulture Research Institute, Rajshahi, Bangladesh.
Statistical Analysis: Results are presented as the mean ± S.D, Students t-test was used for comparison between the experimental and control groups. p<0.05 was considered to be statistically significant (Gujarati, 1988).
RESULTS AND DISCUSSION
The structure of the compound, used for toxicological study is shown below:
|Table 1:||Body weight after daily intraperitoneal administration of 4-hydroxy nitrobenzene (300μg) for 14 days in rats|
|M1 and M2 indicate sample mean values, SD1 and SD2 = Standard deviations, n = Number of rats, + = Increase, NS =Not Significant|
|Table 2:||Hematological profiles after daily intraperitoneal administration of 4-hydroxy nitrobenzene (300μg) for 14 days in rats|
|Values are mean ± SD, n =4|
|Table 3:||Biochemical parameters after daily intraperitoneal administration of 4-hydroxy nitrobenzene (300μg) for 14 days in rats|
|Values are mean ± SD|
|SGPT||:||Serum glutamate pyruvate transaminase|
|SALP||:||Serum alkaline phosphatase|
|SGOT||:||Serum glutamic oxaloacetic transaminase|
Body weight: Table 1 showed the individual and average body weights of all the rats before and after drug administration. The body weight of all the rats increased after the drug administration, which were found to be statistically insignificant.
Hematological profiles: Hematological profiles were done to check the hematological abnormalities after intraperitoneal administration of the test sample. The changes in values of total count of RBC and WBC, differential count of WBC, platelet count and hemoglobin percentage before and after sample administration were very slight (Table 2). However, the changes with in the normal range (Islam et al., 1997)
Biochemical Parameters of blood: Biochemical parameters of blood e.g. SGOT, SGPT, SALP (Serum Alkaline Phosphatase), urea, uric acid and serum creatinine of both experimental and control rats, were determined to check any change of the parameters due to administration of the compound with respect to the control rats (Table 3). It was found that most of the parameters were slightly increased with respect to the control but remained with in the normal range. The results also revealed that the changes were statistically insignificant. These results indicated that the compound has no adverse effect on liver and kidney functioning (Anisuzzaman et al., 2001)
Histopathological Studies: After 14 day of drug treatment, the animals of both control and experimental groups were sacrificed and the organs such as liver, kidney, lung, spleen and heart were isolated and histopathological examinations were done. No detectable abnormality was observed between the control and the drug treated rats, indicating that the compound, 4-hydroxy nitrobenzene has no adverse effect on cellular structure.
From the experiment, conclude that the compound, 4-hydroxy nitrobenzene isolated from Streptomyces species has no toxic effects in rats at 300μg rat-1 day-1 for 14 day treatment.
The authors like to thank Dr. Naoki Sugimoto, National institute of Health Sciences, Tokyo, Japan for spectral analysis and Dr. Anwar Habib, Lecturer, Department of Pharmacology, Rajshahi Medical College, Rajshahi, Bangladesh for toxicological studies.
- Hawk, P.B., B.L. Oser and W.H. Summerson, 1954. Practical Physiological Chemistry. 13th Edn., McGraw Hill Book Co. Inc., New York, USA., pp: 573-575.
- Holt, J.G., N.R. Kreig, P.H.A. Sneath, J.T. Staley and S.T. Williams, 1994. Bergey's Manual of Determinative Bacteriology. 9th Edn., Lippincott Williams and Wilkins, Baltimore, USA., ISBN-13: 9780683006032, Pages: 787.