Med. Weter. 2014, 70 (12) 766
Praca oryginalna Original paper
Amphotericin B (AmB), a macro-cyclic natural anti- biotic produced by Streptomyces nodosus, has been used for more than 50 years. Despite its toxicity, AmB has been commonly used to treat fungal infections, e.g. visceral Leishmaniasis (5, 6), and is applied especially in immunocompromised states (AIDS, cancer) (12, 35). What is important is that pathogens rarely show resistance to AmB (8). This antibiotic has a higher af-finity for cell membranes of protozoa and fungi which contain ergosterol than for animal cell membranes with cholesterol (9, 10). AmB molecules undergo auto-oxidation and cause lipid peroxidation of animal membranes (2). This process, however, has significant toxic effects on the animal organism (21). Therefore, in order to improve AmB treatment, different aspects of the harmful side effects should be studied, especially with the use of model animals.
A good model organism, used in medical, especially epigenetic, nutrigenetic and geriatric research, is the honeybee (Apis mellifera) (4, 13, 19, 20, 25, 30, 32). This is because A. mellifera is biologically/genetically well known (1, 13) and therefore meets many criteria
set by scientists. The obtaining of the biological mate-rial is easy and cheap. Therefore, providing a required number of individuals (18) poses no problems in the case of A. mellifera. What is crucial is to successfully apply the results obtained with model animals in solv-ing medical problems. Because of many unexpected biological resemblances of the honey bee to man (15, 18), its conservative and unique genome (18), social lifestyle, phenotypic plasticity (the same genomes but different form and function), the honey bee can undoubtedly be used as a good experimental model to resolve human health problems (17).
The proteolytic and antioxidative systems and bio-chemical compounds employ substances responsible for resistance to environmental pressure and longevity (1, 11, 31) in both honey bees and humans. The system elements are probably synthesized in the apian fat body and then migrate to haemolymph. The fat body is considered an analogue of the mammalian liver (14) and haemolymph is the equivalent of mammalian blood. Therefore humans and honey bees have similar biochemical compounds employed in similar metabolic
Effect of amphotericin B on the biochemical markers
in the haemolymph of honey bees
MILENA BAJDA, ALEKSANDRA ŁOŚ, MALWINA MERSKA*Department of Biological Basis of Animal Production, Faculty of Biology and Animal Breeding, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland
*Department of Biochemistry and Toxicology, Faculty of Biology and Animal Breeding, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland
Received 27.06.2014 Accepted 28.07.2014
Bajda M., Łoś A., Merska M.
Effect of amphotericin B on the biochemical markers in the haemolymph of honey bees Summary
We examined the effect of amphotericin B (AmB) on the following enzymatic markers: aspartate amino- transferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and on non-enzymatic markers: glucose, triglycerides, and proteins in the haemolymph of a model organism, Apis mellifera. AmB is an antifungal antibiotic. Despite its toxicity, it is used to treat disease conditions. The haemolymph of honey bees is considered as an analogue of human blood, and changes in marker values indicate pathological states, both in humans and honey bees. Three groups of caged bees were fed sugar syrup (the control group). The syrup was supplemented with AmB at concentrations of 0.25 mg/ml (AmB-25) and 0.50 mg/ml (AmB-50). The authors observed that the biochemical markers were age-related in the control group. Decreased values of the enzymatic markers in the AmB-treated groups confirm that AmB has a negative effect on the organism. The higher the dose of the antibiotic, the greater the increase in the concentration of the non-enzymatic markers. Our research shows that honey bees are an important model for studying the effects of AmB.
Med. Weter. 2014, 70 (12) 767 pathways, e.g. aspartate aminotransferase, alanine
ami-notransferase, alkaline phosphatase and non-enzymatic markers: glucose, triglycerides, and proteins. Changes in marker values indicate pathological conditions in both human and apian organisms.
The aim of this study was to analyse the effect of AmB treatment on the important biochemical markers in the haemolymph of A. mellifera treated as a model organism. The study was performed at different apian ages, i.e. with different durations of treatment.
Material and methods
To evaluate the effects of both AmB doses and treatment durations (experimental factors) the following protocol was used. Three groups were established with 10 wooden cages (12 × 12 × 4 cm) per group, 50 one-day-old worker bees in each cage. The cages had glass front screens, but no combs or wax foundations inside. The sugar syrup (1 : 1) was administered ad libitum via inner-cage feeders. In the first group (control), AmB was not added, whereas in the second group, the syrup was supplemented with AmB at a concentration of 0.25 mg/ml (AmB-25), and in the third group, at a concentration of 0.50 mg/ml (AmB-50). The cages were kept within an air-conditioned chamber (26°C and 65% RH). Every second day, the feeders were replen-ished with the syrup, and dead workers were removed from the cages (compare; 3).
From 12 to 15 live workers were sampled 3 to 7 times from the each cage/group at days 1, 7, 14 and 21 (at dif-ferent ages, i.e. at difdif-ferent treatment durations). A glass capillary was introduced between the 3rd and 4th tergite of
each sampled worker. Haemolymph, taken in this way, was collected into sterile Eppendorf tubes containing 200 µl of ice-cooled 0.6% NaCl, according to Strachecka et al.’s (28, 29) method. From 3 to 7 pooled samples (tubes), containing 100 µl of fresh hemolymph each, were created in each of the groups by this protocol and immediately refrigerated at –25°C for further biochemical analyses.
The following parameters (biochemical markers) were analysed in the haemolymph samples:
– Activities of aspartate aminotransferase (AST), ala-nine aminotransferase (ALT) and alkaline phosphatase (ALP) were measured by the kinetic method, using Cormay (Lublin, Poland) monotests according to the producer’s procedure.
– Concentrations of trigycerides, glucose and protein were measured by the colorimetric method, using Cormay (Lublin, Poland) monotests according to the producer’s procedure.
The significance of experimental effects (AmB dose and treatment duration) and the significance of differences be-tween parameter means were tested by two-way ANOVA, plus LSD procedures. SAS statistical software (SAS In-stitute Version 9.13., 2002-2003 license 86636) was used.
Results and discussion
The activities of AST, ALT, and ALP and the concen-trations of glucose, triglycerides, and proteins did not differ between the groups only in the 1-day-old honey
bees (Fig. 1 and Fig. 2), i.e. after one day of treatment. Thus, there were no differences between the groups in the activities/concentrations of biochemical charac-teristics at the beginning of treatment. In the control group, the enzymatic marker activities increased with age. This tendency was reversed by AmB treatment, because in the treated groups the enzymatic marker activities decreased (or did not change) with age. The higher the dose of AmB was, the lower activities of AST, ALP and ALT were identified. The more the AmB treatment was prolonged, the greater decrease in the enzymatic marker activities in contrast to the control group was observed.
Fig. 1. Activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in the haemolymph of honeybees in the control (no amphotericin B treatment), AmB-25 (the workers were treated with 0.25 mg/ ml amphotericin B) and AmB-50 (the workers were treated with 0.50 mg/ml amphotericin B) groups
Explanation: Various lowercase letters: the differences between the groups (control and AmB-25- and AmB-50-treated) are sta-tistically significant within each of the age groups at P ≤ 0.05.
200 180 160 140 120 100 80 60 40 20 0 Activity of AST [U/l]
1-day-old 7-day-old 14-day-old 21-day-old
Days a a a a b c a b c a b c control AmB-25 AmB-50 120 100 80 60 40 20 0 Activity of AL T [U/l]
1-day-old 7-day-old 14-day-old 21-day-old
Days a a a a a b a b c a b c 35 30 25 20 15 10 5 0 Activity of ALP [U/l]
1-day-old 7-day-old 14-day-old 21-day-old
Days a a a a b c a b c a b c
Med. Weter. 2014, 70 (12) 768
The concentration of glucose decreased, whereas those of triglycerides and proteins increased with age (treatment duration) in the three groups. Glucose con-centration decreased with a lower dose and increased with a higher dose of AmB, independently of treat-ment duration, except for 21-day-old bees. Generally, the AmB treatment, independently of its duration, increased the concentrations of triglycerides and proteins. The higher the dose of AmB was, the higher concentrations of these compounds were obtained.
The composition of haemolymph is changed dur-ing apian development by environmental factors and various pathogens (23). Our studies also revealed that
activities of the enzymatic markers (ALT, ASP, ALP) in the control group were age-related (compare 16). This relation, however, was disturbed by the AmB treatment (Fig. 1). AmB leads to a reduction of AST, ALT and ALP activities, which can be regarded as symptoms of a metabolic disorder. We suppose that AmB can inhibit the formation of AST, ALT, and ALP in cells or suppress the release of the enzymes into haemolymph (24). AST, ALT, and ALP have already been used for monitoring bee health in metabolic stress conditions (22). Such pathogens as Nosema spp. and Varroa destructor (26), bacteria, fungi, and viruses (23), as well as antibiotics and other therapeutic substances (e.g. oxalic acid, formic acid, amitraz; 30, 32), can be sources of metabolic stress. AST, ALT, and ALP are used as liver function markers in humans. An increase in their activity may indicate chronic diseases, patho-logical changes and hepatotoxicity. Unlike in mam-mals, these enzymatic markers have lower activities in diseased honey bees (28, 29). Nevertheless, they still act as metabolic stress markers. In this study, the authors observed decreased values of the markers in the AmB-treated groups, which may confirm that AmB has a negative effect on the key metabolic pathways in bees. This corresponds with the results of Strachecka et al. (25), who found that AmB shortened the lifespan, reduced global DNA methylation levels and increased body-surface protein concentrations in A. mellifera.
Glucose levels in the haemolymph of healthy honey bees are higher than in mammal blood, whereas tri-glyceride values in haemolymph and blood are similar (22). Our research shows that glucose concentration in apian haemolymph depends on the AmB dose and treat-ment duration, and is different from that in the control, untreated group. Increased blood triglyceride levels indicate metabolic disorders in humans (34). Similar tendencies were observed in apian haemolymph, as a result of the AMB treatment. Strachecka et al. (27-29) confirmed our observations that protein concentrations increased at a young age and in maturity, but decreased in older bees. We also observed that a higher AmB dose increased the concentrations of glucose, triglycerides and proteins. This tendency was particularly evident for proteins. Most probably, this results from a very strong inhibition of membrane pumps, e.g. ATPase, which impedes active transport and leads to the deposition of certain metabolites (7, 33).
Conclusions:
1. Amphotericin B has a negative influence on the activities/concentrations of biochemical compounds in Apis mellifera. We may suppose that this antibiotic has similar harmful effects on insects and humans.
2. The negative influence of amphotericin B increas-es with the duration of therapy, and particularly with the antibiotic dose. Therefore, it is crucial to determine the proper treatment duration and antibiotic dose.
Fig. 2. Concentrations of glucose, triglycerides and proteins in the haemolymph of honeybees in the control (no amphotericin B treatment), AmB-25 (the workers were treated with 0.25 mg/ ml amphotericin B) and AmB-50 (the workers were treated with 0.50 mg/ml amphotericin B) groups
Explanation: Various lowercase letters – the differences between the groups (control and AmB-25- and AmB-50-treated) are sta-tistically significant within each of the age groups at P ≤ 0.05.
6 5 4 3 2 1 0 Protein [g/dl]
1-day-old 7-day-old 14-day-old 21-day-old
Days a a a a b c a b c a b c 4 3,5 3 2,5 2 1,5 1 0,5 0 T riglycerides [mmol/l]
1-day-old 7-day-old 14-day-old 21-day-old
Days a a a a b c a b c a b c 45 40 35 30 25 20 15 10 5 0 Glucose [mmol/l]
1-day-old 7-day-old 14-day-old 21-day-old
Days a a a a b c a b c a b c control AmB-25 AmB-50
Med. Weter. 2014, 70 (12) 769 3. Our results confirm that A. mellifera is a valuable
model for studying the effects of amphotericin B on the key metabolic pathways.
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Corresponding author: mgr inż. Milena Bajda, ul. Akademicka 13, 20-950 Lublin; e-mail: milena.bajda@wp.pl
