|Year : 2017 | Volume
| Issue : 1 | Page : 35-42
Evaluation of the protective effect of ginseng against gentamicine-induced nephrotoxicity in adult, albino rats: a histochemical and immunohistochemical study
Sayed A Raheem1, Abdel R Meselhy2, Sohier A Hafiez3, Nasser A Naby1
1 Department of Pathology, Faculty of Medicine, Al-Azhar University, Cairo, Egypt
2 Department of Anatomy, Faculty of Medicine, Al-Azhar University, Damietta, Egypt
3 Department of Pathology, Faculty of Medicine, Assiut University, Assiut, Egypt
|Date of Submission||03-Jan-2017|
|Date of Acceptance||15-Mar-2017|
|Date of Web Publication||23-Aug-2017|
Sayed A Raheem
Associate Professor of Pathology, Department of Pathology, Faculty of Medicine, Al-Azhar University, Cairo
Source of Support: None, Conflict of Interest: None
Caspase-3 plays an important role in apoptosis. In this study, we determined the protective effect of ginseng against gentamicine-induced nephrotoxicity in albino rats. The aim of this study was to investigate the protective effect of ginseng on the expression of caspase-3 in gentamicine-induced nephrotoxicity.
Materials and methods
A total of 40, adult, albino rats weighing 250±20 g were divided into four groups (10 rats each) and treated by intraperitoneal injection for 10 days with 1 ml of isotonic saline (group 1), gentamicine 100 mg/kg/day (group 2), gentamicine 100 mg/kg/day plus ginseng 100 mg/kg/day (group 3), and gentamicine 100 mg/kg/day for 10 days and then ginseng 100 mg/kg/day for another 10 days (group 4). After the last injection, blood urea and creatinine levels were calculated, and tissue samples were obtained for haematoxylin and eosin, Masson trichrome, periodic acid-Schiff (PAS), and caspase-3 staining.
In group 2, body weight of rats decreased, serum urea and creatinine levels increased, and glomerular and tubular histological changes were observed, compared with the control group. When ginseng and gentamicine were given together (group 3) or when ginseng was given after gentamicine (group 4), body weight, serum urea and creatinine, and histological features showed improvement. Significant reactivity of caspase-3 in the distal renal tubules was observed in group 2 as compared with weak reactivity in group 3 and group 4.
Gentamicine has the ability to induce nephrotoxicity, mainly tubular, and ginseng may improve this nephrotoxicity.
Keywords: caspase-3, gentamicine-induced nephrotoxicity, ginseng
|How to cite this article:|
Raheem SA, Meselhy AR, Hafiez SA, Naby NA. Evaluation of the protective effect of ginseng against gentamicine-induced nephrotoxicity in adult, albino rats: a histochemical and immunohistochemical study. Al-Azhar Assiut Med J 2017;15:35-42
|How to cite this URL:|
Raheem SA, Meselhy AR, Hafiez SA, Naby NA. Evaluation of the protective effect of ginseng against gentamicine-induced nephrotoxicity in adult, albino rats: a histochemical and immunohistochemical study. Al-Azhar Assiut Med J [serial online] 2017 [cited 2020 Feb 25];15:35-42. Available from: http://www.azmj.eg.net/text.asp?2017/15/1/35/213587
| Introduction|| |
Aminoglycosides are natural products or semisynthetic derivatives produced by a variety of soil actinomycetes . High doses of gentamicine (40 mg/kg or more) can rapidly induce extended cortical necrosis leading to renal dysfunction in animals . Gentamicine nephrotoxicity accounts 10–15% of all cases of acute renal failure. The cells of the proximal renal tubules have the ability to concentrate gentamicine several folds more than plasma levels . Gentamicine-induced nephrotoxicity is reversible, and renal function recovers if the use of the drug is stopped . Histopathological findings show that administration of aminoglycosides causes apoptosis, intracellular edema, basal membrane interruption, glomerular narrowing of the Bowman’s capsule, and acute tubule necrosis leading to diminished creatinine clearance and renal dysfunction .
Herbal products including ginseng have been reported to possess protective effects against drug-induced nephrotoxicity in experimental animals . The mechanism by which ginseng exerts its activity is possibly through the hypothalamus–pituitary–adrenal axis . Ginseng may reduce cell damage induced by toxic substances and act to stabilize the cell membrane  and protect tissues from damage by inhibiting lipid peroxidation. These effects are due to the antioxidant nature of ginseng .
Caspases are essential in cells for apoptosis (programmed cell death) . The CASP3 protein is a member of the cysteine–aspartic acid protease (caspase) family. Caspase-3 interacts with caspase-8 and caspase-9. It is encoded by the CASP3 gene identified in numerous mammals . Sequential activation of caspases plays a central role in the execution phase of cell apoptosis. Caspase-3 is fully active under normal and apoptotic cell conditions . Caspase-3 is activated in apoptotic cells both by extrinsic (death ligand) and intrinsic (mitochondrial) pathways ,. Caspase-3 has a typical role in apoptosis, where it is responsible for chromatin condensation and DNA fragmentation .
The aim of this study was to evaluate the protective effect of ginseng against gentamicine-induced nephrotoxicity in adult, albino rats relying on biochemical, histological, histochemical [Masson trichrome (MT) and periodic acid-Schiff (PAS)], and immunohistochemical (caspase-3) results.
| Materials and methods|| |
A total of 40, adult, albino rats of both sexes weighing 250±20 g and aged 70 days were used for the present study. Rats were obtained from the breeding colony maintained at the animal house of the Nile Company for pharmaceuticals, Cairo, Egypt. Animals were housed at the animal facility of the Faculty of Medicine, Al-Azhar University, under normal conditions in special clear-sided cages with controlled temperature (23±3°C), humidity (about 60%), and a 12:12 h light–dark cycle. Rats were fed a standard rat diet and water ad libitum, and were randomly divided into four equal groups (10 rats each) as follows:
- Control group (10 rats): injected with 1-ml isotonic saline solution/day intraperitoneally (IP) for 10 days.
- Gentamicine group (10 rats): injected with gentamicine (100 mg/kg/day) IP for 10 days.
- Gentamicine+ginseng from the start (10 rats): received ginseng (100 mg/kg/day) orally and were simultaneously injected with gentamicine (100 mg/kg/day) IP for 10 days.
- Gentamicine for 10 days and then ginseng for another 10 days (10 rats): injected with gentamicine (100 mg/kg/day) IP for 10 days, and then 24 h later ginseng was given orally (100 mg/kg/day) for another 10 days.
Body weight of each rat was recorded twice weekly. At the end of the specified duration for each group and 24 h from the last dose, rats were anesthetized with ether inhalation. Blood samples were collected from the tail vein of each animal using heparinized capillary tubes. Serum was separated by centrifugation and used immediately for kidney function (serum urea and creatinine) tests. Rats were killed and both kidneys were excised, washed with saline, and fixed in 10% neutral buffered formalin. Paraffin blocks were prepared, and 4 µm sections were stained with hematoxylin and eosin, MT, and PAS; this was followed by caspase-3 immunohistochemistry.
Chemicals and stains
- Ginseng: ginseng syrup 120 ml (9.33 mg ginseng extract/ml) was obtained from ‘Pharco Pharmaceuticals, Alexandria, Egypt’. A dose of 100 mg/kg/day was calculated according to the body weight of each rat ,.
- Gentamicine: Garamycin (1 ml) ampoule (40 mg gentamicine sulfate/ml) was obtained from Memphis Company for Pharmaceutical and Chemical Industries (Cairo, Egypt). A toxic dose of 100 mg/kg/day was calculated according to the body weight of each rat .
- MT staining kit (product code: AR173; staining interpretation − fibrin: pink, collagen: blue, nuclei: blue or black, erythrocytes: red; control tissue: liver; Dako Corporation, Denmark).
- PAS [product code: AR165; staining interpretation − PAS-positive structures: magenta, nuclei: blue background and pink; control tissue: kidney for basement membrane (BM); Dako Corporation]. According to Tagboto and Griffiths , PAS staining was used to demonstrate the integrity of the tubular brush border, cell boundaries, nuclear details, and adhesion of cells to the BM. Each slide was scored according to the above-mentioned criteria as 1 (poor), 2 (moderate), or 3 (excellent). This resulted in a final histological score of between 12 (excellent renal preservation) and 0 (poor renal preservation).
- Caspase-3: rabbit polyclonal AB-3 was purchased from Lab Vision Co., Fremont, California, USA. The positive control was tonsils. Reactivity was predominantly cytoplasmic with some nuclear staining.
Data were analyzed using IBM statistical package for the social sciences advanced statistics version 22 (SPSS; SPSS Inc., Chicago, Illinois, USA). All tests were two-tailed. A P value less than 0.05 was considered significant.
| Results|| |
Body weight decreased in group 2 and relatively improved in groups 3 and 4 as shown in [Table 1].
Serum urea and creatinine were elevated in group 2 and relatively improved in groups 3 and 4 as shown in [Table 2].
Group 1 (control group): haematoxylin and eosin staining showed no histopathological changes in the cortex and medulla, as shown in [Figure 1] and [Table 3].
Group 2 (gentamicine group): glomerular hypercellularity with narrow or obliterated Bowman’s spaces were present. Renal tubules showed edematous and necrotic epithelial lining and intraluminal debris with proximal tubules (PT) showing loss of brush borders. Cortical interstitial edema, hemorrhage, dilated congested blood vessels, and inflammatory cellular infiltrates were observed, as shown in [Figure 2] and [Table 3].
|Figure 2 Group 2: glomerular hypercellularity, narrow Bowman’s spaces and tubular epithelial edema, and necrosis (red arrows)|
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Group 3 (gentamicine+ginseng from the start for 10 days): most of the glomeruli showed less cellularity than those of group 2. Bowman’s spaces of many glomeruli were patent, and the majority of tubules were less edematous, but a few of them were still markedly edematous with scattered necrotic/apoptotic tubular epithelial lining, as shown in [Figure 3] and [Table 3].
|Figure 3 Group 3: decreased glomerular cellularity and patent Bowman’s space with apoptotic tubular epithelial lining (red arrow)|
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Group 4 (gentamicine for 10 days+ginseng for another 10 days): the majority of glomeruli were normal with patent Bowman’s spaces. We observed less tubular edema compared with groups 2 and 3, as shown in [Figure 4] and [Table 3].
|Figure 4 Group 4: decreased glomerular cellularity, patent Bowman’s spaces, less edematous tubules, and interstitium. Hematoxylin and eosin, ×360|
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Histochemical and immunohistochemical results
Group 1: MT staining showed no fibrosis as shown in [Figure 5]. PAS stained renal tubules showed preserved brush borders, cell borders, and nuclear details, and the cells rested on the BM as shown in [Figure 6] and [Table 4] and [Table 5]. Caspase-3 showed weak reactivity in glomeruli with no reactivity in tubules as shown in [Figure 7] and [Table 6] and [Table 7].
|Figure 6 Group 1: renal tubules showing preserved brush borders (red arrow)|
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|Figure 7 Group 1: weak reactivity in glomeruli with no reactivity in tubules|
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Group 2: MT staining showed periglomerular and peritubular fibrosis as shown in [Figure 8]. PAS staining showed loss of brush borders and indistinct (poor) cell borders as shown in [Figure 9] and [Table 4] and [Table 5]. Caspase-3 showed marked reactivity in tubules with no reactivity in glomeruli as shown in [Figure 10] and [Table 6] and [Table 7].
|Figure 10 Group 2: marked reactivity mostly in distal tubules (red arrows) with no reactivity in glomeruli|
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Group 3: MT staining showed peritubular fibrosis as shown in [Figure 11]. PAS staining showed relatively (moderately) preserved brush borders as shown in [Figure 12] and [Table 4] and [Table 5]. Caspase-3 showed moderate reactivity in tubules (especially in distal tubules) with no reactivity in glomeruli as shown in [Figure 13] and [Table 6] and [Table 7].
|Figure 13 Group 3: moderate reactivity in distal tubules (red arrows) with no reactivity in glomeruli|
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Group 4: MT staining showed less peritubular fibrosis as shown in [Figure 11]. PAS staining showed preserved brush borders as shown in [Figure 14] and [Table 4] and [Table 5]. Caspase-3 showed mild reactivity in both glomeruli and tubules as shown in [Figure 6],[Figure 15],[Figure 16] and [Table 6], [Table 7], [Table 8], and [Table 9].
|Figure 14 Group 4: preserved brush borders (red arrows). Periodic acid-Schiff, ×360|
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|Figure 16 Group 4: mild reactivity in both glomeruli and tubules (red arrow). Caspase-3 immunostain, ×360|
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|Table 9 Statistical analysis of caspase-3 reactivity in different groups|
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| Discussion|| |
In the present study, daily injection of 100 mg/kg of gentamicine IP for 10 days significantly elevated blood urea and serum creatinine, suggesting renal damage and nephrotoxicity. These findings are in agreement with those of Derakhshanfar et al. , who mentioned that 10 days of treatment with gentamicine (80 mg/kg of body weight) produced remarkable nephrotoxicity that was characterized by an increase in blood urea nitrogen when compared with the control rats.
Our results showed that, in group 2 (positive control), gentamicine caused loss of body weight when given alone. However, when gentamicine was given with the ginseng, there was an improvement in body weight in groups 3 and 4; these findings are in agreement with ,.
Our results showed that gentamicine increased serum urea and creatinine levels when given alone; these findings are in agreement with those of Babu and colleagues ,, who considered serum creatinine as one of the most reliable indicators of the efficiency of renal function, and with Fekete et al. , who considered serum urea as a significant marker of renal dysfunction. Our results also showed improvement in these parameters in the ginseng-treated groups (groups 3 and 4), and these findings are in agreement with Lipsky and colleagues ,, who reported that there is an improvement in these effects by ginseng treatment.
Our study revealed decreased levels of serum urea and creatinine in rats belonging to group 1 and groups 3 and 4 that received ginseng, and these results are in agreement with Soliman et al. , Babu et al. , and Chaware et al. .
Our histological findings included increased glomerular cellularity, loss of brush borders, indistinct cell borders, intratubular cellular debris, and basal membrane interruption, and these results are in agreement with Bennett and colleagues ,,, who reported that aminoglycoside-induced nephrotoxicity is characterized by tubular necrosis, basal membrane disruption, mesangial cell contraction, proliferation, and apoptosis.
Our study showed marked cytoplasmic reactivity of caspase-3, mostly in the distal tubules, with no reactivity in glomeruli in group 2 (gentamicine group) and moderate and mild reactivity in tubules in groups 3 and 4, respectively. These results are in agreement with Yang et al. , who reported the presence of caspase-3 in the cytoplasm, and most of the distal renal tubular cells were positive for caspase-3, whereas only occasional cells showed caspase-3 positivity in proximal tubular epithelial cells. Most of the proximal tubular epithelial and glomerular cells were negative for caspase-3, and these findings are in disagreement with Lopez-Novoa et al.  and Alarifi et al.  who reported that tubular damage was more prominent in proximal convoluted tubules than in distal tubules and collecting ducts.
Our results are in agreement with Rudel and colleagues ,, who demonstrated that caspsase-3 activity was the best predictor of apoptosis, inflammation, and fibrosis, and in agreement with Meguid El Nahas et al. , who demonstrated a significant increase in caspase-3 activity-related apoptosis in a nonimmune-mediated chronic renal fibrosis model.
| Conclusion|| |
Form this study, we conclude that gentamicine can induce renal tubular damage, caspase-3 is strongly expressed in renal tubular cells, indicating the role of apoptosis in this nephrotoxicity, and that ginseng may improve gentamicine-induced nephrotoxic effects.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Chambers HF, MacDougall C. Section VII: Chemotherapy of microbial diseases. Chapter 54: Aminoglycosides. In: Brunton LL, Chabner BA, Knollmann BC, editors. Goodman and Gilman’s: the pharmacological basis of therapeutics. 12th ed. USA: Mc Graw-Hill professional; 2011. pp. 1505–1520.
Parker RA, Bennett WH, Porter GA. Animal models in the study of aminoglycoside nephrotoxicity, in the aminoglycosides: microbiology, clinical use and toxicology. In: Wi-Ielton A, Neu HC. The aminoglycoside. New York: Marcel Dekker Incorporated; 1982. pp. 235–267.
Rincon J, Romero M, Viera N, Pedreanea A, Mosquera J. Increased oxidative stress and apoptosis in acute puromycin aminonucleoside nephrosis. Int J Exp Pathol 2004; 85:25–33.
Rang HP, Dale MM, Ritter JM, Flower RJ, Henderson G. Chapter 50: Antibacterial drugs. 7th ed. Section 5: Drugs used for the treatment of infections, cancer and immunological disorders. Rang HP, Dale MM, Ritter JM, Flower RJ, Henderson G, editors. Rang and Dale’s pharmacology. London: Elsevier Churchill Livingstone; 2011. pp. 630–631.
Souza VB, Oliveira RFL, Ferreira AAA, De Araujo Junior RF. Renal changes by aminoglycosides. Arq Med 2008; 22:131–135.
Kang KS, Kim HY, Yamabe N, Nagai R, Yokozawa T. Protective effect of sun ginseng against diabetic renal damage. Biol Pharm Bull 2006; 29:1678–1684.
Liu RJ, Wang S, Liu H, Yang L, Nan G. Stimulatory effect of saponin from Panax ginseng on immune function of lymphocytes in the elderly. Mech Ageing Dev 1995; 83:43–53.
Tran QL, Adnyana IL, Tezuka Y, Harimaya Y, Saiki I, Kurashige Y et al.
Hepatoprotective effect of ginsenoside R2 the major saponin from Vietnamese ginseng (Panax Vietnamenesis). Planta Med 2002; 68:402–406.
Lee HC, Hwang SG, Lee YG, Sohn HO, Lee DW, Hwang SY, Moon JY. In vivo effects of Panax ginseng extracts on the cytochrome P450 dependent monooxygenase system in the liver of 2,3,7,8 tetrachlorodibenzo-p-dioxin exposed guinea pig. Life Sci 2002; 71:759–769.
González D, Bejarano I, Barriga C, Rodríguez AB, Pariente JA. Oxidative stress-induced caspases are regulated in human myeloid HL-60 cells by calcium signal. Curr Signal Transduct Ther 2010; 5:181–186.
Alnemri ES, Livingston DJ, Nicholson DW, Salvesen G, Thornberry NA, Wong WW, Yuan J. Human ICE/CED-3 protease nomenclature. Cell 1996; 87:171.
Stennicke HR, Salvesen GS. Biochemical characteristics of caspases-3, −6, −7, and −8. J Biol Chem 1997; 272:25719–25723.
Ghavami S, Hashemi M, Ande SR, Yeganeh B, Xiao W, Eshraghi M et al.
Apoptosis and cancer: mutations within caspase genes. J Med Genet 2009; 46:497–510.
Salvesen GS. Caspases: opening the boxes and interpreting the arrows. Cell Death Differ 2002; 9:3–5.
Porter AG, Jänicke RU. Emerging roles of caspase-3 in apoptosis. Cell Death Differ 1999; 6:99–104.
Qadir MI, Tahir M, Lone KP, Munir B, Sami W. Protective role of ginseng against gentamicine induced changes in kidney of albino mice. J Ayub Med Coll 2011; 23:53–57.
Khan MR, Badar I, Siddiquah A. Prevention of hepatorenal toxicity with Sonchus asper in gentamicine treated rats. BMC Complement Altern Med 2011; 11:113.
Tagboto S, Griffiths AP. The evaluation of renal ischemic damage: the value of CD10 monoclonal antibody staining and of biochemical assessments of tissue viability. BMC Clin Pathol 2007; 7:5.
Derakhshanfar A, Bidadkosh A, Kazeminia S. Vitamin E protection against gentamicine-induced nephrotoxicity in rats: a biochemical and histopathologic study. Iran J Vet Res 2007; 8:231–238.
Ali BH, Abdel Gayoum AA, Bashir AA. Gentamicine nephrotoxicity in rat: some biochemical correlates. Pharmacol Toxicol 1992; 70:419–423.
Chen X, Gillis CN, Moalli R. Vascular effects of ginsenosides in vitro. Br J Pharmacol 1984; 82:485–491.
Babu SV, Urolagin DK, Veeresh B, Pattanshetty N. Anogeissus latifolia prevents gentamicine induced nephrotoxicity in rats. Int J Pharm Sci 2011; 3:1091–1095.
Kore KJ, Shete RV, Kale BN, Borade AS. Protective role of hydro alcoholic extract of Ficus carica
in gentamicine-induced nephrotoxicity in rats. Int J Pharm Life Sci 2011; 2:978–982.
Fekete A, Rosta K, Wagner L, Prokai A, Degrell P, Ruzicska E, Ver A. Na+
-ATPase is modulated by angiotensin II in diabetic rat kidney − another reason for diabetic nephropathy? J Physiol 2008; 586:5337–5348.
Lipsky JJ, Cheng L, Sacktor B, Leitman PS. Gentamicine uptake by renal brush border membrane vesicles. J Pharm Clin Ther 1980; 215:390–393.
Yokozawa T, Zhou JJ, Hattori M, Inaba S, Okada T, Oura H. Effects of ginseng in nephrectomized rats. Biol Pharm Bull 1994; 17:1485–1489.
Soliman KM, Abdul-Hamid M, Othman AI. Effect of carnosine on gentamicine-induced nephrotoxicity. Med Sci Monit 2007; 13:73–83.
Chaware VJ, Chaudhary BP, Vaishnav MK, Biyani KR. Protective effect of the aqueous extract of Momordica charantia
leaves on gentamicine-induced nephrotoxicity in rats. Int J Pharm Tech Res 2011; 3:553–555.
Bennett WM, Wood CA, Houghton DC, Gilbert DN. Modification of experimental aminoglycoside nephrotoxicity. Am J of Kidney Dis 1986; 8:292–296.
Martinez-Salgado C, Henández-López FJ, Novoa-López JM. Glomerular nephrotoxicity of aminoglycosides. Toxicol Appl Pharmacol 2007; 223:86–98.
De Sousa VB, Dutra IJP, Lucena HE, Alves MSCF. Amikacin induces renal morphohistological alterations in Wistar rats. Arq Med 2009; 23:205–871. 959–969.
Yang F, Liu GS, Lu XY, Kang JL. Expression of caspase-3 in rat kidney with renal tubular damage induced by lipopolysaccharide and hypoxia. J South Med Univ 2009; 29:2091–2093.
Lopez-Novoa JM, Quiros Y, Vicente L, Morales AI, Lopez-Hernandez FJ. New insights into the mechanism of aminoglycoside nephrotoxicity: an integrative point of view. Kidney Int 2011; 79:33–45.
Alarifi S, Al-Doaiss A, Alkahtani S, Al-Farraj SA, Al-Eissa MS, Al-Dahmash B, Mubarak M. Blood chemical changes and renal histological alterations induced by gentamicine in rats. Saudi J Biol Sci 2012; 19:103–110.
Rudel T. Caspase inhibitors in prevention of apoptosis. Herz 1999; 24:236–241.
Thornberry NA, Rano TA, Peterson EP, Rasper DM, Timkey T, Garcia-Calvo M et al.
A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J Biol Chem 1997; 272:17907–17911.
Meguid El Nahas YBA, Thomas GL, Haylor JL, Watson PF, Wagner B, Timothy SJ. Caspase-3 and apoptosis in experimental chronic renal scarring. Kidney Int 2001; 60:1765–1776.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]