Dose Related Histological Changes in Mouse Kidney After Administration of Manuka Honey.
Abstract Honey has been used for both nutritional purposes as well as medicinal purposes. The present study describes dose related histological effects of Manuka honey on mouse kidney. Another important purpose is to evaluate the dose tolerance of this natural product for its future exploitation as a therapeutic agent. Forty eight BALB/c mice were divided into six groups each of eight mice. Manuka honey was given orally at a dose of 5 10 15 25 and 35 mL/kg body weight to five groups for two weeks whereas the sith was kept as the control group. All the animals were sacrificed on day 15 their kidneys were dissected out and processed for histological analysis. No animal mortality occurred at any of the honey doses. Focal tubular epithelial degeneration tubular epithelial coagulative necrosis interstitial inflammation of chronic type comprising lymphocytes and plasma cells and glomerulus mesangial proliferation was observed in a dose dependent manner.
Manuka honey given orally in different doses was well tolerated by the experimental mice and no abnormal clinical signs and symptoms were recorded. The higher doses however brought some significant histological changes in mice kidney.
Key words: Manuka honey dose tolerance BALB/c kidney histology. INTRODUCTION
Honey has been used for the treatment of many ailments since ancient times (Olaitan et al. 2007). It is produced from many floral sources and its therapeutic efficacy varies with its origin and processing (Irish et al. 2011). It has been found to be effective against bacteria fungi and parasites (Mullai and Memon 2007; Maeda et al. 2008). In addition its role in promoting the healing process decreasing inflammation and stimulating tissue regeneration (Medhi et al. 2008; Jalali et al. 2007; Schencke et al. 2011) has been documented many times (Yusofa et al. 2007; Sukur et al. 2011). Honey contains more than 100 components in it which are responsible for its multi-dimensional performance (Simon 2009; Hannan 2009). Although there are lot more studies that narrate the therapeutic and biological properties of this natural product information regarding the dose tolerance and safety of honey is not much reported in the literature.
The aim of the present study was to evaluate dose tolerance of Manuka honey as well as the histological changes induced by it in the mouse kidney.
MATERIALS AND METHODS
This experimental study was conducted in the Department of Microbiology and Experimental Research Laboratory University of Health Sciences Lahore. The study was approved by the ethical committee of the university.
Manuka honey was obtained from Comvita New Zealand and was stored in refrigerator at 3-4C in its dark coloured bottle.
BALB/c mice 12-14 weeks of age weighing 25-30 g were obtained from University of Health Sciences Lahore. They were divided into 6 groups each of eight mice and were given honey as 5 10 15 25 and 35mL/kg body weight to five groups. One group was kept as control and was not given honey. The animals were housed group wise in separate cages duly labelled and having clean compact husk made beds with the room temperature maintained at 20-25C They were fed with the standard animal feed and water ad libitum and a 12 h day and 12 h night cycle was adopted (Munira et al. 2010).
All the mice were kept deprived of water 3 h before honey dose. Diluted honey (70% v/v) was given orally with the help of a feeding syringe. Mice were given honey in divided doses (BID) for 14 consecutive days. Several observations like mouse alertness urine colour stool consistency etc. were also made during and after feeding. On day 15 the animals were sacrificed by deep anaesthesia using chloroform (Sigma-Aldrich USA) (AVMA 2001). Kidneys were dissected out carefully and were fixed in 10% formal saline. After overnight fixation appropriate tissue sections about 4-6 m were cut from the representative areas of the specimen. These sections were processed through different gradations of alcohol and embedded in wax (Bancroft 2008).
From each tissue block 4-6 g thick sections were cut and then stained in haematoxylin and eosin.
Data was entered and analyzed using SPSS version 17.0. Percentages are given for qualitative variables. P-value shows association in between honey dose groups and histological changes induced. A p-value less than 0.05 is considered to be significant when Pearson Chi-square test applied.
No animal mortality occurred at any honey dose. Focal tubular epithelial degeneration (Fig. 1B arrow 2) was seen in 1 (12%) 8 (100%) 4 (50%) and 8 (100%) animals of 10 15 25 and 35 mL/kg body weight groups respectively (Fig. 2A). Tubular epithelial coagulative necrosis was seen only in 1 (12%) (Fig. 2B) animal of group 25 mL/kg (p-value 0.403). The rest did not demonstrate any necrotic changes. Interstitial inflammation (Fig. 1B arrow 4 and Fig. 1C) of chronic type comprising lymphocytes and plasma cells was observed in all the experimental mice other than the control group. It was of mild type in 5 (62%) 2 (25%) 8 (100%) and in 4 (50%) animals of 5 10 15 and 25 mL/kg (Fig. 2C) respectively. Moderate degree of interstitial inflammation was observed in 4 (50%) and in 1 (12%) animal in group 25 and 35 mL/kg respectively while 7 (88%) animals in the latter group demonstrated severe interstitial inflammation.
Six (75%) of animals in group 10 mL/kg demonstrated minimal interstitial infiltrate (p-value 0.000). Glomerulus mesangial proliferation (Fig. 1B arrow 1) was observed focally in 5 animals of group 10 l/kg and in all (n=8) of animals of groups 20 25 and 35 mL/kg respectively (Fig. 2D) (p-value 0.000). Glomeruli capillaries were unremarkable in nearly all animals except in 7 (88%) (Fig. 2E) animals of 35 l/kg group where thickened vascular walls (Fig. 1B arrow 3) were appreciated (p-value 0.000).
No animal mortality was observed from any experimental group throughout the experiment. A honey dose of 35mL/kg body weight brought some significant histological changes yet no mortality was observed. More than half of mice from all groups presented with increase in weight. Two of eight mice each from group 25 mL/kg and control C maintained their weight. This number was 3/8 for group 25 l/kg and 1/8 for group 20 and 25 l/kg respectively. Again 2/8 mice were observed to have a decrease in weight each from the groups 25 and 35 l/kg and 1/8 from group 20 l/kg. A significant difference was observed between weight of mice having different honey dose (p-value =0.05) when
ANOVA test was applied.
DISCUSSION An important and conspicuous finding was that there was no animal (n=48) mortality observed with any of the Manuka concentration given. The animals survived till the end of the experiment.
Ponte et al. (2007) in Brazil reported 70% mortality of rats at 35mL/kg honey dose. In our study urine colour was normal in all mice except two from the 35 ml/kg body weight group which passed honey coloured urine. One possibility for this urine colour might be that at 35ml/kg body weight honey dose kidney tubular degeneration was observed in all (100%) animals of that group and 12% animals exhibited kidney tubular necrosis also. Another possibility for kidney damage might be presence of sugars in it that can elicit nephrotoxicity when given in high doses (Yudkin et al. 1980; Triplitt 2012).
Badawy et al. (2004) using different brands of honey administered subcutaneously to mice determined 1 ml as a safe dose whereas higher dose produced mortality in mice. In contrast we documented no mortality at any dose of Manuka honey given orally. The reason may be the different route of administration and/or different dose and dose schedule of honey given to the experimental animals. In kidney honey induced tubular epithelial degeneration in a dose dependent manner. Tubular necrosis was observed in only one mouse (12%) kidney and that too at the maximum dose i.e. 35mL/kg body weight. No tubular necrosis was observed in any other animal from any group. Chronic interstitial inflammation and mesangial proliferation were also observed in a dose related manner. Thickening of the glomerulus vessel wall was observed only at the maximum honey dose. It is clear that only higher doses of honey could elicit nephrotoxic changes.
Onyije et al. (2012) evaluated effects of different doses (0.1 .15 and .02 mL/kg body weight) of unprocessed honey on rat kidney and reported elevation of urea and creatinine at higher doses. The maximum dose in this study is far less than the minimal honey dose we used in our study. This study however supports our results that higher doses can be nephrotoxic. Honey has been observed to be protective in cadmium induced nephrotoxicity at 0.05ml (50L) daily dose (Wafaa and Hemmat 2007) whereas in our study the least dose was 5mL/kg body weight (150L). This least honey dose given in our study induced minimal/no changes in the kidney tissues. Halawa et al. (2009) also documented 1.5 mL/kg body weight protective effects of honey against lead induced oxidative stress in rats.
Although it has been proven that honey is protective against chemical induced toxicity in kidney at lower doses' (Abdel-Moneim and Ghafeer 2007) the present study evaluated the dose tolerance of honey so that its uses as antimicrobial agent can be exploited in animal studies where high doses of honey might turn lifesaving. Hence we also emphasize that Manuka honey can be further explored for its hidden potential even if it used in high doses.
We are grateful to Sameer and Usman Arshad both Laboratory Technologists in University of Health Sciences Lahore and Atika Masood Assistant Professor Akhtar Saeed Medical College Lahore for their technical help.
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|Publication:||Pakistan Journal of Zoology|
|Date:||Feb 28, 2015|
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