Biochemical and ultrastructural changes in mouse hepatocytes after the administration of exogenous melatonin.

T. Król, M. Łysek-Gładysińska, H. A. Wieczorek, K. Staszczyk, W. Trybus, A. Król, E. Trybus, A. Kopacz-Bednarska.

Department of Cell Biology, Institute of Biology, Świętokrzyska Academy, Świętokrzyska 15, 25-406 Kielce, Poland.

Abstract

The experiment was performed on male mice of the Porton breed, aged 12 and 44 weeks. The animals were kept in standard conditions, with a constant access to water and feed (with a 16% protein content). In each age group, a control and an experimental group was chosen. Mice belonging to the experimental group were given, through a feeding probe, 0.5 mg/kg b.w of melatonin for 14 days. The control animals received a solution of 0.9% physiological salt through an analogical method.

Melatonin caused an increase in the activity of the analyzed lysosomal enzymes in 44-week old males and a decrease in the enzyme activity of 12-week old mice. These changes correlated with the morphological profile changes in the studied hepatocytes.

Exogenous melatonin administered for 14 days caused significant changes in the morphological profile of the cell and an increase in the activity of the studied lysosomal enzymes only in older specimens. It can therefore be concluded that melatonin stimulated the degradative processes only in 44-week old mice.

Key words: Melatonin, cathepsin D and L, acid phosphatase, β-glucuronidase, mouse, liver

Introduction

Much attention has lately been given to melatonin, because, as numerous papers suggest, it can positively influence the life-length and improve its “quality” [42,41]. It was discovered by Lerner in 1959 [12,31,59]. Melatonin (N-acetylo-5-metoksytryptamine) is formed in a four-step process from the amino acid precursor L-tryptophan . The key enzyme in the biosynthesis of melatonine is serotonine N-acetyltransferase [18,61]. Its biosynthesis takes place mainly in the pineal gland and can also happen in the retina and, to a point, in the Harder gland and the intestines [3].

Melatonin synthesized in the pineal gland is released into the bloodstream and the cerebrospinal fluid and through there reaches the tissues of the body, where it exerts its physiological effects [53,54].

Certain premises exist which suggest a connection between melatonin and the aging process. From the works of, among others, Hause [13,39], it seems that the rate of melatonin production isn’t constant and decreases with age (lowering slowly until the age of 40 to 50 and then much faster).

The available literature discusses the role of melatonin in delaying the aging process and the prevention of neoplasm therapy [40,51]. Melatonin is attributed, among its other effects, to remove damage arising as a result of the aging process [44,48].

However, many unexplained mechanisms of the action of melatonin still remain. Very little data can be found on the effect of melatonin on the lysosomal compartment, which is the first to react in situations of disturbed cell homeostasis [2,8,10,26,27,24,25,63,62,62]

Materials and Methods

The animals used in the experiment were 40 male mice of the Proton breed, aged 12 and 44 weeks. The animals were kept in a room with a naturally regulated light-to-dark ratio, LD 12:12, were feed with dry granulated Murigram feed with 16% protein content and had a constant access to water. In each age group the animals were divided into a control group and an experimental group. The experimental animals were given, through a feeding probe, daily for 14 days, at a set time (9⁰⁰), melatonin in the dosage of 0.5mg/kg b.w. The control animals were given a solution of 0.9% physiological salt in an analogical way.

After 14 days, the animals of all groups were decapitated, following which slices of liver were taken for biochemical studies. The liver tissue was homogenized in the temperature of +4 ^oC in a 0.25 M solution of sucrose (1 g tissue: 7 ml sucrose). The homogenate was then centrifuged differentially according to the method of Marzella and Glaumann [33].

In the obtained lysosomal fraction, the activity of select model lysosomal enzymes was marked: cathepsine D and L (Cath. D and L, EC 3.4.23.5., EC 3.4.22.15.) according to the method of LANGNER et al. [28]; acid phosphatase (AcP, EC 3.1.3.2.) according to the method of HOLLANDER [14]; β-D-glucuronidase (BGRD, EC 3.2.1.31.) by the method of BARRETT [4]. The total protein content was marked by a modified Lowry method [21]. The activity of the studied enzymes was expresses in μmol/mg of protein/hour. The results obtained were expressed as means and standard deviations.

Furthermore, fragments were taken for microscopic studies. Initial fixation was performed in 3% glutaraldehyde. For further fixation, 2% osmium tetroxide was used. Contrasting was done in 2% uranyl acetate. Dehydrated slices were sealed in Epon 812 (Serva, Germany). Additional contrasting of ultra-thin slices was done in uranyl acetate and lead citrate according to the method of Marzella and Glaumann [34]. The photographs were taken with the use of the TESLA BS 500 electron microscope.

The experiment was conducted according to the recommendations of the Ethical Committee for Animal Experimentation.

Results

The obtained results are shown in figures 1-2 and photographs 1-4. They present the changes in the activity of observed lysosomal enzymes in the liver of male 12- and 44-week old mice, caused by the administration of melatonin.

14-day action of melatonin in the dose of 0.5 mg/kg b.w. ( fig 1) caused, in 12 week old males, a statistically significant decrease in the activity of cathepsin D and L (to 71%, p<0,001) and acid phosphatase (to 57%, p<0,001). No statistically significant changes were observed in the case of β –D-glucuronidase (108%). The administered dose of melatonin did not reveal significant changes in the ultrastructure of the hepatocytes observed (phot. 2) in comparison to the control cell (phot. 1).

Fig. 1 Changes in the activity of lysosomal enzymes in mouse liver of 12-week old mice after 14 days of melatonin administration in the dose of 0,5 mg/kg b.w.

*** p< 0,001- statistically significant differences

Phot. 1. Electron micrographs of hepatocyte fragment of control group - 12 weeks old. (N) nucleus, (ER) endoplasmic reticulum, (M) mitochondria, (L) lysosomes, x 8.500

Phot. 2. Electron micrographs of hepatocyte fragment of mouse - 12 weeks old after administrating of melatonine in the dose of 0.5 mg/kg b.w. (N) nucleus, (ER) endoplasmic reticulum, (M) mitochondria, (L) lysosomes, (AG) Golgi apparatus, x 13.500

The consequence of administering melatonin to 44 week old males (fig. 2) was a highly statistically significant increase of the activity, of cathepsin D and L (to 199%, p<0,001), acid phosphatase (to 210%, p<0,001) and β-glucuronidase (to 262%, p<0,001).

The observation of the morphological profile of the cell revealed a well developed rough endoplasmatic reticulum and significantly enlarged mitochondria (phot. 4) in comparison to the control cell (phot. 3).

Fig. 2. Changes in the activity of lysosomal enzymes in mouse liver of 44-week old mice after 14 days of melatonin administration in the dose of 0,5 mg/kg b.w.

*** p< 0,001- statistically significant differences

Phot. 3. Electron micrographs of hepatocyte fragment of control group - 44 weeks old. (N) nucleus, (ER) endoplasmic reticulum, (M) mitochondria, (G) glycogen granule, x 13.000

Phot. 4. Electron micrographs of hepatocyte fragment of mouse - 44 weeks old after administrating of melatonine in the dose of 0.5 mg/kg b.w. (N) nucleus, (ER) endoplasmic reticulum, (M) mitochondria, (L) lysosomes, (G) glycogen granule, (VA) vacuole autophagic, (P) peroxisome, x 12.500

Discussion

Numerous papers show that melatonin can strengthen the immunological system, which gets weaker with age [54]. Increasing, among others, the production of lymphocytes T, which enable resistance to certain diseases, melatonin softens the side effects of chemotherapy and regulates the natural sleeping rhythm. The effectiveness of this hormone in treating sleeping disorders, resulting from, for example, a quick change of time zones or from old age, has been shown [5,9,13,20,29,30,46,47,55,58].

It has also been shown, that this hormone lowers the content of lipid peroxidation products, the increase of which accompanies certain diseases, such as the Parkinson disease or the Alzheimer disease [36,63]. The observations, that with age the concentration of endogenous melatonin lowers, in the result of which the activity of free radicals increases and so does the susceptibility to different diseases, are especially interesting [19,37,38,39,42,47, 45,52,56].

This hormone displays good dissolubility, which allows it to freely enter the interior of all cells, and its action is not limited to only cellular membranes [6,11]. The highest concentration of melatonin can be found in the nucleus, where it is specifically bound by nucleoproteins and takes part in protecting DNA from the chemical effect of carcinogens and acts in delaying the aging process [1].

The gradual decrease in melatonin secretion which accompanies aging may disturb membrane transport, as well as the cellular and organism metabolism, leading to a disturbance in the endogenous homeostasis and contributing to the arising of diseases associated with old age [32,35,50].

The analysis of the activity of the enzymes observed in 12-week old mice after 14 days of exogenous melatonin administration showed a statistically significant decrease in the activity of studied lysosomal hydrolases (fig.1). However, the morphological studies did not show any significant changes in the ultrastructure of the cell encumbered with melatonin, in comparison to the morphological profile of the control cell (phot.1,2). The above-mentioned results show a decrease in the degradative processes in the liver cells studied ( fig.1).

From the works of Parmar et al. [37] and Pawlikowski et al. [40] it can be concluded that taking prophylactic melatonin at an appropriate age can decrease the hormonal disturbances appearing as a result of a low level of this hormone in the body, especially during menopause or andropause.

Our data too show that melatonin caused, in the liver of aging (44-week old) male mice, biochemical changes expressing in a statistically significant increase in the activity of Cath D and L, AcP and BGRD (fig.2). Ultrastructural studies show an increase in the number of mitochondria of a slightly swelled structure (phot. 4). These changes are probably an expression of the cellular adaptation to the increasing energy need. The increased number of glycogen granules might also suggest an intensified operation of the cell. These changes correlate with the biochemical changes, which suggest a stimulation in degradative processes in 44-week old specimens.

When comparing the observed enzymatic reactivity of 44-week old males in comparison to 12-week old males, it was concluded that 14 days of melatonin administration caused a statistically significant increase in most of the studied hydrolases only in 44-week old males and not in the younger, 12-week old males.

The above-mentioned data suggest that exogenous melatonin stimulated degradative processes only in aging, 44-week old males. Numerous papers show [8,22,23,57] that the aging process is accompanied by changes in the lysosomal system, indicating, among others, an inhibition of the degradative processes within the cell. The results obtained (fig.2, phot. 3, 4) allow us to suspect that melatonin, by supporting the degradative process in aging (44-week old) specimens, prevents the intracellular gathering of damaged proteins, which are the source of many diseases associated with old age.

These results are a confirmation of numerous other works [7,17]. Other authors [16,49,60] suggest that melatonin, by, among other factors, its antioxidant properties, plays a significant part in slowing the aging process.

In conclusion, it can be said that exogenous melatonin caused specific, age-dependant biochemical and ultrastructural changes, which were an expression of a precisely defined cellular function.

Melatonin, by influencing the overall processes taking place in the cell, contributed to the restoration of degradative processes in aging mice. It can therefore be suggested, that the supplementation of the melatonin deficiencies, which increase with age, may be a chance for preventing diseases in aging specimens, as well as for delaying the aging processes.

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