The Journal of Basic & Applied Zoology (2012) 65, 299–308
The Egyptian German Society for Zoology The Journal of Basic & Applied Zoology
www.egsz.org www.sciencedirect.com
Influence of rifampicin and tetracycline administration on some biochemical and histological parameters in albino rats
M.B. Shabana, Hania M. Ibrahim *, Soheir E.M. Khadre, Marwa G. Elemam
Zoology Department, Faculty of Science, Alexandria University, Moharram Bey, Alexandria 1511, Egypt
Received 14 March 2012; accepted 8 July 2012 Available online 7 January 2013
KEYWORDS Abstract The present investigation was designed to evaluate the effect of two oral administered Rifampicin; antibiotics (Rifampicin and Tetracycline) on some physiological parameters in the serum of male Tetracycline; albino rats. Histopathological alterations in liver and kidney were also examined. Data showed that Albino rats; treatment of rats with rifampicin caused a significant increase in the total cholesterol, triglycerides Physiological parameters; and LDL-cholesterol levels, while HDL-cholesterol level showed a significant decrease. Moreover, Histopathological changes; significant increases in serum AST, ALT, bilirubin and urea were observed. Also, the levels of the Liver; total protein, albumin and alpha 1-globulin were significantly decreased. No significant changes Kidney were recorded in the rest of the globulin fractions (alpha 2-, beta- and gamma-globulin) and albu- min/globulin ratio (A/G) as well as creatinine level. In the serum of tetracycline-treated rats, the triglycerides, LDL-cholesterol, AST, ALT, bilirubin, urea, creatinine and gamma-globulin levels increased significantly. On the other hand, HDL- cholesterol, total protein, albumin levels and A/G ratio showed significant decreases. No significant changes in the total cholesterol, alpha 2- and beta-globulin levels were detected. Histological examination of the liver and kidney in the rifampicin-treated rats indicated that the liver pathology includes necrosis of hepatocytes, cytoplasmic vacuolation, and distended sinusoids with some lymphatic aggregations. In the kidney, the glomeruli increased in size, the mesangial matrix was expanded and the renal tubules were degenerated. Histological analysis of liver samples of tetracycline – treated rats revealed high vacuolation of the cytoplasm of hepatic cells, sinusoidal dilation, hepatocellular necrosis and disappearance of the cell membrane in some hepatocytes. The kidney sections of rats treated with tetracycline showed shrinkage of the glomeruli, widening of the Bowman’s space, in addition to necrosis and vacuolation of the renal tubules. ª 2012 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. All rights reserved.
* Corresponding author. E-mail address: [email protected] (H.M. Ibrahim). Introduction Peer review under responsibility of The Egyptian German Society for Zoology. Antibiotics are widely used nowadays to cure many types of diseases (Davey, 2000). Antibiotics have different courses of administration depending upon the seriousness of the infection Production and hosting by Elsevier and the potency of the antibiotic taken. The recovery time of 2090-9896 ª 2012 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jobaz.2012.10.009 300 M.B. Shabana et al. an individual may range from a week to a whole month kg of pyrazinamide) once daily for 12 weeks caused membrane (Wiegand et al., 2008). One of the foremost concerns in disintegration and loss of the polyhedral structure in the liver, modern medicine is antibiotic resistance. Over the last decade, in addition to the presence of other abnormalities like necrosis, almost every kind of bacteria has become stronger and less macro vesicular steatosis and inflammation. responsive to antibiotic treatment when it is really required Lee (1979) reported that rifampicin can cause hemolysis (Pearson, 2007). Although health benefits of antibiotics are and subsequently acute renal failure, it can also produce inter- often emphasized, the side effects of antibiotics are not stitial nephritis (direct toxic effect) which is really part of a commonly known (Koju et al., 2005). pan-nephropathy. Moreover, some renal lesions were ob- Rifampicin is a bactericidal antibiotic drug of the rifamycin served, these lesions may be due to the formation of immune group. It is a semisynthetic compound derived from strepto- complexes which were detected on capillary glomerular base- myces spp that is used as a first line drug for the treatment ment membranes by immunofluorescent and electron micro- of tuberculosis (TB) worldwide (Eminzade et al., 2008). Rifam- scopic techniques. The deterioration in renal function picin produces many metabolic and morphological aberrations typically appeared acutely, after the reintroduction of rifampi- in the liver due to the fact that the liver is the main detoxifying cin (Covic et al., 1998). site for these antitubercular drugs (Santhosh et al., 2007). The Tetracycline antibiotics are bacteriostatic agents with a effect of rifampicin on lipid peroxidation was a significant in- broad spectrum of antimicrobial activity. Besides their anti- crease after 4 weeks of treatment of mice with rifampicin microbial activity, it has been shown that tetracyclines may (20 mg/kg) intraperitoneally (Upadhyay et al., 2007). Also, be useful in the treatment of pathological conditions in which Tasduq et al. (2007) demonstrated that, there is a significant in- acute or chronic inflammations are involved, such as derma- crease in triglycerides and cholesterol levels of rats after tological, periodontal, rheumatic and neurodegenerative dis- administration with rifampicin (250 mg/kg/day) for 30 days. eases (Bastos et al., 2007). Although the tetracyclines retain Santhosh et al. (2006) detected significant increases in triglyc- important roles in both human and veterinary medicines, erides, cholesterol and free fatty acids in the serum of rats after the emergence of microbial resistance has limited their effec- receiving anti-TB drugs (200 mg rifampicin + 200 mg isonia- tiveness (Chopra and Roberts, 2001). Large doses of tetracy- zid) for 30 days. Slight declines in the level of HDL-cholesterol cline have been shown to induce hepatic dysfunction in rats with concomitant rise in LDL-cholesterol level were also noted (Yin et al., 2006) and humans (Wruble and Cummins, .Pal et al. (2008) and Rana et al., 2010 showed significant in- 1965). This dysfunction of the liver resulted in the disturbance creases in serum AST and ALT levels after treatment of rats of nitrogen metabolism, jaundice and other signs of hepato- with anti-TB drugs. Elevated levels of AST and ALT were re- cellular damage, e.g., increase of serum transaminases corded after treatment of patients (Balamurugan et al., 2009) (Bo¨ cker et al., 1982). It was found that the levels of the total and mice (Upadhyay et al., 2007) with rifampicin. The phar- cholesterol, triglycerides and LDL-cholesterol in the serum of macokinetics of rifampicin in healthy volunteers and tubercu- rats were significantly increased after treatment with 10 mg/ losis-patients was studied by Rafiq et al. (2010), they observed 100 g body weight/day of tetracycline for 7 days, while a a decrease in the level of albumin in the patients, as rifampicin significant decrease in HDL-cholesterol levels was noticed binds only to albumin. Santhosh et al. (2007) and Eminzade (Vijayalekshmi Amma and Leelamma, 1991). Also, Hunt et al. (2008) showed a significant decrease in total protein and Washington (1994) found a significant increase in AST and albumin levels in the serum of rats treated with anti-TB and ALT levels in the serum of female patients treated with drugs. Balamurugan et al. (2009) recorded a significant de- tetracyclines for 2 months. Balashev and Polosova (1981) crease in the total protein in the serum of rats after treatment studied the effect of tetracycline hydrochloride (25 mg/kg/ with rifampicin (10 mg/kg) for 21 days. Yanardag et al. (2005) day for 7 and 20 days) on the levels of the total protein recorded a significant increase in the serum bilirubin, urea and and separate protein fractions such as alpha 1-, alpha 2-, creatinine levels of patients that received antituberculous treat- beta- and gamma-globulins in the lymph and blood serum ments of isoniazid (300 mg/d), rifampicin (600 mg/d), pyrazin- of rabbits. They found a significant decrease in the lymph amide (2 g/d), and ethambutol (1 g/d) for 8 months. Santhosh and serum contents of both the total protein and the protein et al. (2007) observed a significant increase in the serum biliru- fractions. Moreover, Zallen (2009) recorded significant in- bin and a significant decrease in the serum urea levels of rats creases in the levels of serum bilirubin and urea in mice after treated with isoniazid and rifampicin (200 mg each/kg bw/ treatment with tetracycline. Also, Miller and McGarity (2009) day) for 30 days. Tasduq et al. (2007) recorded a significant in- demonstrated that patients treated with tetracycline (250 mg/ crease in serum bilirubin and urea levels, but there were no sig- kg) four times daily for 7 days had renal failure. In addition, nificant changes in the levels of creatinine in the serum of rats they found an increase in urea and creatinine levels. Machado treated with rifampicin (250 mg/kg) for 30 days. et al. (2003) observed a moderate amount of vacuolation in From a histological point of view, Scheuer et al. (1974) the convoluted tubules and Henle’s loop and foci of necrosis found a wide range of histological changes ranging from iso- in the tubular epithelium (that increased according to the lated steatosis (through varying degrees of portal inflammation dose) in the kidney of the offspring of the tetracycline-treated with neutrophil or mononuclear infiltrate) to confluent necro- rats for 10 days. sis in some patients who developed abnormal liver function With this background, the object of this study is to evaluate tests within 6 weeks of rifampicin therapy. Kalra et al. (2007) the effect of rifampicin and tetracycline on liver and kidney reported diffuse microvesicular fatty infiltration with mild por- function in male albino rats. This was achieved by determina- tal triaditis in rats treated with isoniazid (50 mg/kg/day) and tion of the lipid profile, total protein and its electrophoretic rifampicin (100 mg/kg/day) for 7 days. Also, Tasduq et al. pattern, albumin, creatinine, and urea in the blood of rats trea- (2005) pointed out that treatment of rats with anti-TB drugs ted with rifampicin and tetracycline, and the histopathological (250 mg/kg of rifampicin + 50 mg/kg of isoniazid + 100 mg/ study of the liver and the kidney of the treated animals. Influence of rifampicin and tetracycline administration on some biochemical and histological parameters in albino rats 301
Material and methods Data were tabulated and analyzed using SPSS-15 (Statisti- cal Package for Social Science version 15) according to Howell The present study was carried out on white male albino rats (1995). having body weight range of 120–160 g. After randomization into various groups, rats were acclimatized for 14 days in the Results new environment before carrying out the experiment. The drugs were administered orally by means of gastric tubes. Rats Effect of rifampicin administration on the biochemical were administered with rifampicin (200 mg/kg body weight/ parameters studied day, for 30 days) (Santhosh et al., 2007), or tetracycline (140 mg/kg body weight/day, for 7 days) (Vijayalekshmi Table 1 shows that administration of rifampicin to rats for Amma and Leelamma, 1991) before feeding. 30 days caused a significant increase in the total cholesterol, triglycerides and LDL-cholesterol levels (mg/dl); the mean val- Rats were divided into four groups ues increased by 6.0%, 27.3% and 21.0%, respectively, than that of the control, respectively. In contrast, the level of HDL-cholesterol in the serum of the treated animals was sig- Group 1: Control for rifampicin treated rats. Animals nificantly decreased by 18.8%, as compared to that of the con- received 2 ml saline solution (the solvent of rifampicin) trol group. orally for 30 days. As shown in Table 1, the levels of AST and ALT were sig- Group 2: Rats were treated with rifampicin orally in a dose nificantly increased in the serum of the treated rats. The mean of 200 mg/kg body weight/day (dissolved in 2 ml saline values increased by 218.7% and 92.7%, respectively, as com- solution) for 30 days. pared to that of the control groups. Group 3: Control for tetracycline treated rats. Animals Data also show that a significant increase was noticed in the received 2 ml distilled water (solvent of tetracycline) orally levels of bilirubin and urea in the serum of the treated animals for 7 days. as compared to that of the control groups. These increments Group 4: Rats in this group were treated with tetracycline were 45.3% in bilirubin and 62.9% in urea than that of the orally in a dose of 140 mg/kg body weight/day (dissolved control. On the other hand, the mean value of serum creatinine in 2 ml distilled water) for 7 days. did not show any significant difference in the treated group as compared to that of the control group. Blood collection was performed 24 h after the end of the Concerning the total protein and its electrophoretic pattern 7th and 30th days. Animals were anesthetized with ether, in the serum of the rifampicin-treated animals, it is clear from and the blood was collected from the abdominal aorta. The the data illustrated in Fig. 1 that the mean values of total pro- blood sample was left 30 min at 37 C, and then centrifuged tein, albumin and alpha1-globulin were significantly decreased at 5000g for ten minutes. The separated serum was used for as compared to that of the control group. The rest of the pro- the estimation of the following physiological parameters: tein fractions (alpha2-, beta-, gamma-globulin and globulin) and A/G ratio in the treated animals recorded non-significant 1. Total cholesterol level was determined according to changes due to administration of rifampicin. Deeg and Ziegemohrm (1982). 2. Triglyceride level was determined according to Stein Effect of tetracycline administration on the biochemical and Myers (1995). parameters studied 3. LDL and HDL-cholesterol levels were determined according to Valerie (2002). Table 2 shows that administration of tetracycline (140 mg/ 4. AST and ALT levels were determined according to kg body weight/day) to rats for 7 days caused a significant in- Schmidt and Schmidt (1963). crease in the levels of triglycerides and LDL-cholesterol by 5. Serum total protein level was estimated using the Biuret 186.1% and 81.3% respectively, as compared to that of the reaction (Young, 1990). control group. Meanwhile a significant decrease in the level 6. Albumin and creatinine levels were determined accord- of HDL-cholesterol was noticed in the serum of the treated ing to Newman and Price (1999). rats. The mean value decreased by 44.6% in comparison to 7. Electrophoretic separation of proteins was carried out that of the control group. according to Jeppsson et al. (1979). The levels of AST and ALT were significantly increased in 8. Urea level was determined according to Patton and the serum of the treated rats. The mean values increased by Crouch (1977). 322.5% and 333.5%, respectively as compared to that of the control groups. On the other hand, treatment with tetracycline Liver and kidney were removed and fixed in Bouin’s fluid for 7 days had no effect on the level of the total cholesterol in for 24 h. After fixation, the organs were then dehydrated the serum of the rats. through ascending grades of ethyl alcohol, and then they were Significant increases were also noticed in the levels of biliru- transferred to xylol. The organs were then placed in a mixture bin, urea and creatinine in the serum of tetracycline-treated of melted wax and xylol (1:1) for about 10 min and then animals as compared to that of the control group. The values embedded in paraffin wax (56 C). Sections were made, stained increased by 45.6%, 47.2% and 49.3% for bilirubin, urea and with Ehrlich’s hematoxylin, counter stained with eosin and creatinine, respectively when compared to that of the control examined by the light microscope (Culling, 1974). group (Table 2). 302 M.B. Shabana et al.
Table 1 Mean ± SD of total cholesterol, triglycerides, LDL-cholesterol, HDL-cholesterol, AST, ALT, bilirubin, urea, and creatinine levels in the serum of rats, 30 days after treatment with rifampicin. Parameter Control Treated Difference (%) Total cholesterol (mg/dl) 177.60 ± 21.03 188.33* ± 16.78 6.0 Triglycerides (mg/dl) 205.80 ± 21.32 262.00* ± 73.27 27.3 LDL-cholesterol (mg/dl) 63.20 ± 12.32 76.50* ± 12.66 21.0 HDL-cholesterol (mg/dl) 73.24 ± 8.73 59.43* ± 13.72 18.8 AST (U/L) 69.80 ± 8.17 222.50* ± 56.49 218.7 ALT (U/L) 68.30 ± 7.89 131.67* ± 39.27 92.7 Bilirubin (mg/dl) 0.75 ± 0.20 1.09* ± 0.32 45.3 Urea (mg/dl) 22.60 ± 4.16 36.83* ± 5.78 62.9 Creatinine (mg/dl) 0.37 ± 0.11 0.43 ± 0.13 16.2 Each tabulated value is the average of six individual samples. * Significant difference from the control at P < 0.05.
Control 8 Control 8 Treated Treated 7 7 6 6 5 5 4
4 3
3 2 concentrations (g/dl)
concentrations (g/dl) concentrations 2 1 0 1 Total Albumin Alpha 1- Alpha 2- Beta- Gamma- Globulin A/G protein globulin globulin globulin globulin 0 Total Albumin Alpha 1- Alpha 2 - Beta - Gamma- Globulin A/G protein globulin globulin globulin globulin Figure 2 Mean ± SD of the concentrations (g/dl) of total protein and its electrophoretic fractions in serum of the rats, Figure 1 Mean ± SD of the concentrations (g/dl) of total 7 days after treatment with tetracycline as compared to that of the protein and its electrophoretic fractions in the serum of rats, control group. Asterisk denotes a significant difference from the 30 days after treatment with rifampicin as compared to that of the control value at P < 0.05. control group. Asterisk denotes a significant difference from the control at P < 0.05.
respectively. On the other hand, treatment with tetracycline Regarding the effect of tetracycline administration on the caused a significant increase in the level of the gamma-globulin total protein and its electrophoretic pattern in the serum of fraction in the serum of the rats. The level increased from 0.18 the treated rats, data in Fig. 2 exhibited that the mean values to 0.77 g/dl. The rest of the protein fractions (alpha 1, alpha 2, of the total protein, albumin and A/G ratio were significantly beta-globulin and globulin) in the treated rats were not signif- decreased from 6.65 to 5.80, 4.04 to 2.66 and 1.53 to 0.87 g/dl, icantly changed (Fig. 2).
Table 2 Mean ± SD of total cholesterol, triglycerides, LDL-cholesterol and HDL-cholesterol, AST, ALT, bilirubin, urea, and creatinine levels in serum of the rats, 7 days after treatment with tetracycline as compared to that of the control group. Parameter Control Treated Difference% Total cholesterol (mg/dl) 113.33 ± 12.32 119.00 ± 13.31 4.76 Triglycerides (mg/dl) 39.67 ± 5.28 113.50* ± 48.46 186.11 LDL-cholesterol (mg/dl) 44.00 ± 7.62 63.50* ± 6.06 44.31 HDL-cholesterol (mg/dl) 81.57 ± 12.84 45.17* ± 16.80 À44.62 AST (U/L) 76.17 ± 18.12 321.83* ± 46.68 322.51 ALT (U/L) 68.17 ± 13.21 295.50* ± 78.57 333.47 Bilirubin (mg/dl) 0.57 ± 0.38 0.83* ± 0.31 45.61 Urea (mg/dl) 20.83 ± 4.96 30.67* ± 7.26 47.23 Creatinine (mg/dl) 0.69 ± 0.22 1.03* ± 0.28 49.27 Each tabulated value is the average of six individual samples. * Significant difference from the control at P < 0.05. Influence of rifampicin and tetracycline administration on some biochemical and histological parameters in albino rats 303
Histological findings
Liver Control for rifampicin-treated rats. Fig. 3 represents a vertical section of the liver of control for rifampicin treated rats (2 ml saline/day for 30 days). It exhibits a normal architecture of the hepatocytes presenting a homogenous cytoplasm and a large spherical nucleus (N) containing one or more nucleolus and a variable amount of dispersed and peripheral heterochro- matin. Hepatocytes were arranged in trabecules running radi- antly from the central vein and were separated by sinusoid (s) containing kupffer cells (K). The lumen of sinusoid contained mainly erythrocytes and white blood cells. Kupffer cells (K) were found on the luminal surface of the sinusoid’s Figure 4 Light micrograph of a vertical section in the liver of endothelium. albino rats after treatment with rifampicin for 30 days. Arrows point to hypertrophied hepatocytes, vacuolated cytoplasm hepa- Control for tetracycline-treated rats. The liver of control for tocytes (arrow heads), pycnotic nuclei (pyn), numerous kupffer tetracycline-treated rats (2 ml distilled water for 7 days) cells (K), and dilation of blood sinusoids (S). (HE) stain (X 400). revealed almost the same histological picture as control for rifampicin treated rats; it is composed of hexagonal or pentagonal lobules with central veins and peripheral hepatic tetrads embedded in connective tissue. Hepatocytes (arrows) are arranged and separated by sinusoids containing kupffer cells (K). They are regular and contain a large spheroid nucleus with a distinctly marked nucleolus and peripheral chromatin distribution.
Rifampicin-treated rats. The light microscopic study of the liver of albino rats treated with 200 mg/kg body weight, daily for 30 days, shows several changes. There are dilation and conges- tion of blood sinusoids (s) and numerous hypertrophied kupffer cells (k) in sinusoids (Fig. 4). The liver cells were degenerated, hypertrophy of hepatocytes which have pycnotic nuclei (pyn) and deformed nuclei (dN) is observed (Fig. 5). Furthermore, there is a disorganization of hepatocytes with ly- sis of cytoplasm and some cells are completely filled with large Figure 5 Light micrograph of a vertical section in the liver of vacuoles (head arrows-Fig. 4) presenting a balloon like appear- albino rats after treatment with rifampicin for 30 days. Arrows ance (Fig. 5). point to hypertrophied hepatocytes, degenerated hepatocytes (double arrows), hypertrophied kupffer cells (K), deformed nuclei Tetracycline-treated rats. The light microscopic study of the (dN) and pycnotic nuclei (pyn). (HE) stain (X1000). liver of albino rats treated with 140 mg/kg body weight daily for 7 days (Fig. 6) shows necrotic changes, a small pycnotic
Figure 6 Light micrograph of a vertical section in the liver of Figure 3 Light micrograph of a vertical section in the liver of albino rats 7 days after tetracycline administration demonstrating albino rats (the control group), showing a normal architecture hypertrophied hepatocytes (arrows), cytoplasmic vacuolation without pathological alterations. Hepatocyte (arrows), Spherical (arrows head), abundance of kupffer cells (K) and massive nucleus (N), Sinusoids (S), Blood vessel (BV), and Kupffer cells lymphatic aggregation (LA) inside the hepatic tissue and hepato- (K). (HE) stain (X400). cellular necrosis. (HE) stain (X400). 304 M.B. Shabana et al. nucleus with condensed chromatin, lack of nucleolus and a cytoplasm filled with vacuoles. The trabecular structure of the lobules is slightly or distinctly blurred. Some sinusoids are over filled with erythrocytes and the walls of most sinu- soids show numerous kupffer cells (K). Also, aggregations of lymphocytes (LA) inside the hepatic tissue are observed.
Kidney Control for rifampicin-treated rats. The kidney of the control rat has a number of nephrons. The nephron is made up of re- nal tubules (Fig. 7 arrows) and a renal corpuscle which consists of a cluster of capillaries (glomerulus: G) surrounded by the Bowman’s capsule (BC). The microscopic picture of the glo- merulus shows a capillary space covered by endothelial cells Figure 8 Light micrograph of a transverse section of the kidney on the inner side, which in their lumen contain nucleated blood of albino rats receiving 2 ml. distilled water/day for 7 days (control cells. On the inner side of the Bowman’s capsules, epithelial group for tetracycline treated rats). Arrows point to distal renal cells are found. The convoluted renal tubules [distal renal tu- tubules (Drt) and proximal renal tubules (Prt), glomerulus (G), bules (Drt) and proximal renal tubules (Prt)] are covered by glomerular capillaries (GC), erythrocytes (Er), Bowman’s capsule tall columnar cells with a weak eosinophilic cytoplasm, apical (BC), and Bowman’s space (BS). (HE) stain (X400). microvilli or brush border toward the lumen as shown in Fig. 7.
Control for tetracycline-treated rats. The kidney of control rat of this group (2 ml. distilled water for 7 days) is composed of normal renal glomeruli and renal tubules }distal renal tubules (Drt) and proximal renal tubules (Prt){. The renal tubules are lined with typical thick cubic epithelium. The tubules have a relatively regular distinct lumen. Lobular organization of the glomeruli and a flat epithelium lining the glomerular capsule can be seen (Fig. 8).
Rifampicin-treated rats. The light microscopic study of the kid- ney of albino rats treated with rifampicin (200 mg/kg body weight daily for 30 days) shows several changes (Fig. 9). The degeneration of renal tubules [distal renal tubules (Drt) and proximal renal tubules (Prt)] is observed in the kidney, includ- Figure 9 Light micrograph of a transverse section of the kidney ing lysis of cytoplasm and vacuolation (arrows). Also, the of albino rats after treatment with rifampicin for 30 days, it shows glomerulus shows frequent shrinkage. Moreover, atrophy of glomerular swelling (GS), tightly filling the Bowman’s capsule glomerular capillaries causing Bowman’s space dilation is (BC), degeneration of distal (Drt) and proximal (Prt) renal tubules noticed. In addition; there are casts in some renal tubules. Cel- (arrows), necrosis in the glomerulus and cellular proliferation lular proliferation appears in the mesengial area (M). appear in mesengial area (M). (HE) stain (X 400).
Figure 7 Light micrograph of a transverse section of the kidney Figure 10 Light micrograph of a transverse section of the kidney of albino rats receiving 2 ml saline/day for 30 days (control group of albino rats after treatment with tetracycline for 7 days. It shows for rifampicin treated rats). Arrows point to distal renal tubules the atrophy of glomerular capillaries (GC) with Bowman’s space (Drt) and proximal renal tubules (Prt), glomerulus (G), glomerular dilation (BS), degeneration of distal (Drt) and proximal (Prt) renal capillaries (GC), erythrocytes (Er), Bowman’s capsule (BC), and tubules (arrows), and some of the renal tubules contain casts (C). Bowman’s space (BS). (HE) stain (X400). (HE) stain (X400). Influence of rifampicin and tetracycline administration on some biochemical and histological parameters in albino rats 305
with both drugs. Increased lipolysis of triglyceride depot liber- ates free fatty acid from adipose tissue stores (Stenberg, 1976) and the free fatty acids liberated by the adipose tissue are also taken up by the liver tissue, leading to the hypertriglyceridemic condition (Santhosh et al., 2006). However, Machado et al. (2003) observed that the organelles which changed in the pres- ence of tetracycline were mainly mitochondria in which, the beta oxidation enzyme is inhibited, resulting in an accumula- tion of triglycerides inside the cytoplasm. They stated that hypertriglyceridemia may be due to increased release of lipo- proteins into the circulation. Vijayalekshmi Amma and Leelamma (1991) reported that the uptake of triglyceride rich lipoprotein from the circulation is also decreased which is evi- dent from the decreased activity of lipoprotein lipase of the Figure 11 Light micrograph of a transverse section of the kidney extrahepatic tissue. They stated that high density lipoproteins of albino rats after treatment with tetracycline for 7 days. Notice are believed to be involved in the transport of cholesterol from the degeneration of renal glomerulus (head of arrows), adhesion of the tissue to the liver for its catabolism. the glomerulus (G) to Bowman’s capsule (BC), arrows indicate Amino transferases are important classes of enzymes link- degeneration of distal (Drt) and proximal (Prt) renal tubules, and ing carbohydrate and amino acid metabolism. Alanine trans- space between some renal tubules (double arrow heads). (HE) aminase (ALT) and aspartate transaminase (AST) are well stain (X400). known diagnostic indicators of liver diseases. Farrell (1995) re- ported that in cases of liver damage with hepatocellular lesions Tetracycline-treated rats. The light microscopic study of the and parenchymal cell necrosis, these marker enzymes are re- kidney of albino rats treated with tetracycline (140 mg/kg body leased from the damaged tissues into the blood stream. The weight) daily for 7 days shows similar changes in the renal tu- significant elevation in the levels of ALT and AST in the serum bules and glomeruli as shown in rifampicin treated rats, but of of rifampicin or tetracycline-administrated rats in the present different intensity. Degeneration and necrosis of renal tubules study is in accordance with the results of Kikkawa et al. are evident, some tubules appear empty and have casts; C (2006) and Santhosh et al. (2006). Elevated levels of these en- (Fig. 10, arrows). The glomerulus shows frequent shrinkage zymes in the serum are presumptive markers of drug-induced and complete degeneration of renal glomeruli (Fig. 11, head necrotic lesions in the hepatocytes (Amr and Alaa, 2005). of arrows). Also, some tubules show separation of the epithe- The enhanced susceptibility of hepatocyte cell membrane to lial cells from their membranes causing a wide space between drug induced peroxidative damage might have resulted in an the renal tubules (Fig. 11, double arrow heads). increased release of these diagnostic marker enzymes into the systemic circulation. An increase in the AST and ALT levels Discussion indicates a reversible change of the cell membrane permeability (Bo¨ cker et al., 1982). Also, the activities of these enzymes were significantly lower in the liver tissue of the anti-TB drugs The major disorder encountered in antitubercular drug-in- administrated rats (Santhosh et al., 2007). duced hepatitis is fatty accumulation in the liver, which is The decrease in the total protein and albumin recorded in developed either due to excessive supply of lipids to the liver the present study agrees with the results reported by Santhosh or interference with lipid deposition indicating the antitubercu- et al., 2007; Eminzade et al. (2008), Balamurugan et al. (2009) lar drug-induced hypercholesterolemic condition (Santhosh and Rafiq et al. (2010). The reduction of total protein and et al., 2006). The increment of cholesterol levels in the serum albumin levels indicates that the administration of drugs has of rifampicin-administrated rats may be attributed to liver dys- caused an impairment of liver function, e.g. its capacity to syn- function that has been observed in the histological study as a thesize albumin from the hepatic parenchyma. Khan et al. result of degeneration and necrosis of the hepatocytes caused (2002) reported that there was a differential binding of tetracy- by the toxic action of rifampicin. Liver synthesizes many clines with serum albumin, while Shen et al. (2009) observed important substances needed by the body, such as cholesterol that albumin secretion of gel entrapped hepatocytes was re- which is packaged in the cell membrane of hepatocytes as an duced by tetracycline. The decrement of alpha 1-globulin in end product of metabolism, and then distributed to the body the serum of rifampicin-administrated rats could be due to li- to be used. So, the necrosis and damage in hepatocytes might ver dysfunction which affects the synthesis of alpha protein cause the release of the cholesterol from the hepatocytes into fractions in the liver. The increment of gamma-globulin level the blood stream (Santhosh et al., 2006). The present study in the serum of tetracycline-treated rats may be due to hyper- showed that the level of LDL-cholesterol was significantly in- plasia of the reticulo-plasmic tissue of the bone marrow in- creased, whereas the level of HDL-cholesterol was significantly duced by tetracycline administration (Mikaelian, 1975). decreased in the serum of rifampicin or tetracycline treated The elevation in the serum bilirubin which was observed in rats. These results are in agreement with those reported by rifampicin or tetracycline-treated rats is in agreement with the Santhosh et al. (2006). The abnormal cholesterol deposition data reported by Tasduq et al. (2005) and Santhosh et al. is favored by the dangerous tendency of cholesterol to passive (2007). Hepatotoxicity is a common adverse effect of these exchange between the plasma lipoproteins and cell membranes drugs which induce liver damage as judged by elevated serum (Brown and Goldstein, 1986). Also, the levels of triglycerides bilirubin (Eminzade et al., 2008; Pal et al., 2008 and Marzouk were significantly increased in the serum of animals treated et al., 2009). 306 M.B. Shabana et al.
Urea and creatinine are waste products eliminated from the The kidney provides the final common pathway for the blood through the kidney by blood filtration process. The sig- excretion of many drugs and their metabolites, and therefore nificant increase in the level of urea in the serum of rifampicin is frequently subjected to high concentrations of potentially and tetracycline-treated rats as well as the increase in creati- toxic substances. Drugs and their metabolites are taken up nine in the tetracycline-treated rats is in harmony with the re- selectively and concentrated by the renal tubular cells before sults of earlier studies (Yanardag et al., 2005; Tasduq et al., excretion into the urine. So, high intracellular concentrations 2007 and Miller and McGarity, 2009). The non-significant are attained, particularly in the renal medulla, which has rela- changes in the level of serum creatinine of rifampicin -treated tively little vasculature compared with the cortex (Aronson, rats could be interpreted by the fact that the level of serum cre- 2003). The present histological findings showed glomerular atinine does not rise until at least half of the kidney’s nephrons distortion or shrinkage. Some tubular cells showed hyaline- are destroyed or damaged (Bhattacharya et al., 2005). It has droplet formation and tubular dilation. Warner (1975) found been reported that the adverse effects of rifampicin occasion- similar results. Also, Boulton-Jones et al. (1974) showed diffuse ally include nephritis and hepatitis (Plumb, 1999). Several iso- mesengial proliferative glomerulonephritis in the renal biopsy lated cases of acute renal failure (ARF) following rifampicin of patients using rifampicin with subacute infective endocardi- therapy have been reported (Yanardag et al., 2005) Tetracy- tis, while Salih et al. (2008) observed interstitial nephritis and/ cline has been shown to induce various forms of nephrotoxi- or acute tubular necrosis after treatment with rifampicin. city (Bihorac et al., 1999 and Zallen, 2009). The increment in The mechanism of renal injury is thought to be due to an urea and creatinine levels may be attributed to the impaired re- allergic reaction to rifampicin or one of its metabolites causing nal function which has been shown in the present histological allergic interstitial nephritis. However, the renal biopsy does study as a result of the degeneration and necrosis in the glome- not always show heavy infiltration of mononuclear cells and ruli and renal tubules due to the toxic action of antibiotics on occasionally the picture is that of severe, diffuse or focal tubu- the kidney. This action may have prevented the filtration of the lar necrosis with mild interstitial changes (Grunfeld et al., waste products (urea and creatinine) from the blood stream 1993). The immune complexes get deposited in the blood ves- (Bihorac et al., 1999). sels or interstitium and cause glomerular endotheliosis leading The results of the histopathological examination reflect the to tubular injury thereby decreasing renal function (Muthuku- toxicity of rifampicin to the liver and show a remarkable dam- mar et al., 2002). This may be possibly, the cause of renal fail- age of the architecture of the liver cells. These results are in ure in our study. Likewise, tetracycline has been shown to agreement with those of Awodele et al. (2010) who showed that induce various forms of nephrotoxicity. The current work rifampicin induces hepatic damage. The present detected necro- showed degeneration of renal glomeruli, tubules and irregular sis of the hepatic cells of rats treated with rifampicin may be cytoplasmic vacuolization. The same results were observed by attributed to the formation of free radicals, which act as a stim- Mavromatis (1965). The kidney of rat’s offspring showed ulator of lipid peroxidation and a source for destruction and slight tubular vacuolation and necrosis, more prominent in damage of the cell membrane (Ravi et al., 2010). Also, the cur- the newborn, as well as signs of tubular regeneration on the rent study indicated portal triaditis and an increase in the num- tenth and twentieth days from treatment with tetracycline ber of kupffer cells. This is in accordance with the results of (Machado et al., 2003). The results of Chambers (2001) con- Ravi et al. (2010) who found necrosis with moderate to heavy cluded that the main mode of elimination of most tetracycline portal triaditis after treatment with anti-TB drugs (Rifampicin is via renal glomerular filtration, and it is also eliminated via and Isoniazid). Cytoplasmic vacuolation of hepatocytes was the biliary route. Approximately 60% of tetracycline adminis- observed in the present study. Tasduq et al. (2005) suggested tered is found in the urine in an unchanged form. that the vacuolation of hepatocytes accompanied by cytoplas- mic rarefaction results in the loss of polyhedral structure in Recommendations the liver of rats treated with rifampicin. The present data showed that hepatocytes in the liver of tetracycline treated rats exhibited intense diffuse vacuolization – Over consumption of antibiotics may nullify their benefits with many small and medium vacuoles. These data agree with and instead, lead to side effects. So, people do not tend to the findings of Machado et al. (2003). In the study of consume them carelessly and without medical advice. Hagel-Lewicka et al. (1980), animals treated for a long period – All patients receiving antibiotics should have their liver and with tetracycline presented a single large fatty vacuole in the kidney function tests periodically assessed. hepatocytes and more quantity of collagen fiber in the portal spaces, probably due to the continuity of the treatment. Amacher and Martin (1997) used single doses of tetracycline in their experiments and likewise noted the presence of cyto- References plasmic changes such as the formation of fatty vesicles in the Amacher, D.E., Martin, B.A., 1997. Tetracycline-induced steatosis in hepatocytes after a few hours of administration, suggesting primary canine hepatocyte cultures. Fundam. Appl. Toxicol. 40, metabolic changes with accumulation of triglycerides and lipid 256–263. inclusions after the use of tetracyclines. Kikkawa et al. (2006) Amr, A., Alaa, A.H., 2005. Oxidative stress mediates drug-induced also showed fatty changes and focal inflammatory cell hepatotoxicity in rats: a possible role of DNA fragmentation. infiltration in the liver after treatment with tetracycline. The Toxicology 208, 367–375. overall net effects on the expression of lipid metabolism genes Aronson, J.K., 2003. Drugs and renal insufficient. Medicine 1, 103– indicated an increase in cholesterol and triglyceride 109. biosynthesis and a decrease in b-oxidation of fatty acids (Yin Awodele, O., Akintonwa, A., Osunkalu, V.O., Coker, H.A.B., et al., 2006). 2010. Modulatory activity of antioxidants against the toxicity of Influence of rifampicin and tetracycline administration on some biochemical and histological parameters in albino rats 307
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