Volume 28, Issue 9 (12-2021)                   RJMS 2021, 28(9): 146-156 | Back to browse issues page

Research code: 93-01-45-7730
Ethics code: IR.SUMS.REC.1393.7730
Clinical trials code: 0

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Derakhshanfar A, Tavakkoli H, Moayedi J, Poostforoosh Fard A. Evaluation of the toxic effects of lysine and nano-lysine amino acids on weight, growth and histopathological lesions of the fetus using a chick embryo model. RJMS 2021; 28 (9) :146-156
URL: http://rjms.iums.ac.ir/article-1-6871-en.html
Shahid Bahonar University of Kerman , cbrc@sums.ac.ir
Abstract:   (1123 Views)
Background & Aims: Millions of people take dietary supplements for a range of health benefits, from weight loss to muscle building, but some supplements can be very harmful. Lysine is an essential amino acid for humans and has several physiological effects on body organs and the growth process. It is found in large amounts in the structure of most proteins like histones, but not produced enough in the human body and must be supplied through food intake. The use of food supplements containing lysine amino acids is increasing rapidly in the world; however, the toxicopathological effects of such amino acids have always been a major concern. Besides, the use of nanodrugs increases the permeability of compounds to body tissues; therefore, the use of various drugs, minerals, vitamins, and amino acids in nano forms have been considered in medical and nutritional sciences. Since the pathological and teratogenic effects of lysine and nano-lysine amino acids on human fetus have not been evaluated, the current study aimed to investigate the toxic effects of lysine amino acid and its nano-form in different concentrations using the chick embryo model. Due to ethical rules and regulations, no drug experiment on the human fetus is permitted; hence, the chick embryo model is used as an ideal opportunity to study the adverse effects of such amino acids.
Methods: In this experimental study, a total of 70 fertile chicken eggs (Ross 308) with the similar average egg-weight were purchased from the Mahan Breeder Company, Kerman, Iran. The eggs were incubated at 37.5 ºC and 60% relative humidity in an incubator. On the 4th day of the embryonic growth, the chicken eggs were randomly assigned into seven equal groups, 10 eggs each. The wider end of the eggs was disinfected by ethanol 70% and the eggshell was punctured. Embryos received treatment by direct injection into the yolk sac according to the standard techniques. In the control group, 0.5 cc of phosphate buffer saline and in experiment groups, 0.5 cc of lysine and nano-lysine amino acids at doses of 30 mg/kg, 50 mg/kg, and 100 mg/kg egg weight were inoculated. After treatment, the exposed hole was sealed with warmed paraffin and the eggs were placed back into the incubator under the mentioned condition. The viability of the embryos was checked throughout the incubation period by candling. Embryos were allowed to develop until day 18, after which they were humanely killed by placing on ice and the eggs were opened at the wider end. After washing in normal saline solution, embryos were observed under stereomicroscope to study any gross abnormalities on the external body surface. The membranes and yolk sac were also inspected. The average embryo-weight/egg-weight in grams and the average body length in millimeters of each group were also computed. Body weight was measured by a digital scale and body length was measured by a digital caliper (from the front border of the head to the base of the tail including the tip of the uropygial gland). The tissues including brain, heart, lung, liver, and kidney were sampled and fixed in 10% neutral buffered formalin. Following routine preparation of tissues, serial sections of paraffin embedded tissue samples of 5 μm thicknesses were cut using a microtome, and stained with hemotoxylin and eosin. Tissue slides were evaluated under light microscope by a blinded pathologiest. Statistical analyses were performed using SPSS version 20. The Fisher's exact test was used to determine the significant differences in lesion occurrence between experimental groups. One-way analysis of variance followed by Tukey's test was applied to assess the significance of differences in embryos weight and body length. A p-value of <0.05 was considered as statistically significant.
Results: All the embryos treated with doses of 30 mg/kg, 50 mg/kg, and 100 mg/kg egg weight of lysine and nano-lysine amino acids were normal; hence, there were no abnormality in color, feather, limb, and other external body features. Besides, there were no marked depression in the body length and the embryo-weight/egg-weight of lysine and nano-lysine amino acids treated groups; however, inoculation of nano-lysine amino acid at dose of 100 mg/kg was resulted in embryos weight loss and growth retard. Various pathological lesions were observed following the inoculation of doses of 30 mg/kg, 50 mg/kg, and 100 mg/kg egg weight of lysine and nano-lysine amino acids. In all experimental groups, congestion and edema were observed in the lung tissue but it was much more severe in those treated with doses of 50 mg/kg and 100 mg/kg egg weight of nano-lysine amino acid. Congestion and edema were also observed in the brain of all groups; however, microtrombosis was seen merely in those treated with doses of 50 mg/kg and 100 mg/kg egg weight of lysine amino acid. In liver tissues, congestion and fatty liver were obvious in those treated with doses of 30 mg/kg, 50 mg/kg, and 100 mg/kg egg weight of nano-lysine amino acid. However, the embryos treated with different doses of lysine amino acid showed severe dilation of central veins and sinusoids as well as hepatocellular degeneration. Although inoculation of lysine and nano-lysine amino acids at dose of 30 mg/kg lead to congestion in kidney tissue, the higher doses in both lysine and nano-lysine amino acids treated groups caused sever congestion, renal glomerular atrophy, dilation of vasculature, tubular dilatation, and tubular epithelial changes. The heart in those treated with 30 mg/kg egg weight of lysine amino acid was normal. Congestion and dilation of myocardial vasculature were observed in the heart tissues of other groups. Besides, rhabdomyolysis and replacement of cardiac muscle by myxomatous tissues were also seen in the heart of those treated with 100 mg/kg egg weight of nano-lysine amino acid.
Conclusion: Based on findings of this study, inoculation of doses of 30 mg/kg, 50 mg/kg, and 100 mg/kg egg weight of lysine and nano-lysine amino acids are toxic to the chicken embryo in a dose dependent manner. Embryogenesis in chick is similar to human; therefore, the use of lysine and nano-lysine during the pregnancy might have adverse effects on the internal organs of the human fetus. Therefore, it is recommended that the use of these compounds during pregnancy be taken with caution.
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Type of Study: Research | Subject: Pathology

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