[ Ana Sayfa | Editörler | Danışma Kurulu | Dergi Hakkında | İçindekiler | Arşiv | Yayın Arama | Yazarlara Bilgi | E-Posta ]
Fırat Tıp Dergisi
2008, Cilt 13, Sayı 2, Sayfa(lar) 092-097
[ Özet ] [ PDF ] [ Benzer Makaleler ] [ Yazara E-Posta ] [ Editöre E-Posta ]
The Effect of Melatonin Hormone on Formaldehyde-Induced Liver Injury: A Light Microscopic and Biochemical Study
Hıdır PEKMEZ1, Neriman ÇOLAKOĞLU CAMCI2, İsmail ZARARSIZ3, İlter KUS4, Murat ÖGETÜRK4, Hacı Ramazan YILMAZ5, Mustafa SARSILMAZ4
1Fırat University, Elazig School of Health Sciences, ELAZIĞ
2Fırat University, Faculty of Medicine, Department of Histology and Embryology, ELAZIĞ
3Mustafa Kemal University, Tayfur Ata Sökmen Medical School, Department of Anatomy, HATAY
4Fırat University, Faculty of Medicine, Department of Anatomy, ELAZIĞ
5Süleyman Demirel University, Faculty of Medicine, Department of Medical Biology and Genetics, ISPARTA
Keywords: Liver, Formaldehyde, Melatonin, Oxidative Stress, Light Microscope, Karaciğer, Formaldehit, Melatonin, Oksidatif Stres, Işık Mikroskop
Summary
Objectives: This study aimed to investigate histological and biochemical changes in the livers of formaldehyde exposed rats and possible effects of melatonin hormone on these changes.

Materials and Methods: A total of 21 male Wistar-albino rats were divided into three equal groups. Control rats in Group I received 0.9% NaCl alone intraperitoneally (ip), rats in Group II were injected with 10% formaldehyde (10 mg/kg, ip) every other day and rats in Group III received melatonin (25 mg/kg, ip) plus formaldehyde. At the end of 14-day experimental period, all animals were decapitated. The liver tissue samples were processed histologically and analyzed under light microscope. Additionally, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), xanthine oxidase (XO), and malondialdehyde (MDA) levels in the liver tissue samples were determined.

Results: SOD, GSH-Px, CAT, XO and MDA levels of formaldehyde exposed rats were significantly higher than those of the control group rats. However, these biochemical values of rats treated with melatonin plus formaldehyde were lower than the non-melatonin group. Light microscopic evaluation of liver tissue samples of formaldehyde-exposed rats revealed enlarged sinusoids filled with blood and mononuclear cell infiltration in the portal areas and around the central veins. In addition, some of the hepatocytes showed vacuolar degeneration, and some had a hyperchromatic nucleus. In PAS staining, the hepatocytes around the portal areas were PAS negative. The rats treated with melatonin plus formaldehyde had somewhat fewer histological changes induced by formaldehyde exposure.

Conclusion: The liver damage caused by formaldehyde may be partially prevented by melatonin administration. ©2008, Firat University, Medical Faculty

  • Top
  • Summary
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Introduction
    Formaldehyde (HCHO) is a highly water soluble, colorless agent with sharp odor and exists in the natural structure of the organism1. HCHO taken in an organism is metabolized into formic acid in the liver and erythrocytes and excreted by urine, feces, or expiration2. It is used in paint, plastic, textile and leather industries, sanitary and cosmetic products, construction materials, paper, ink, confectionery productions and is used as a preserving additive in drugs3,4. In medicine, it is also used for cadaver embalming, organ and tissue fixation, and disinfection and sterilization procedures1,2,5,6. Various studies have reported the structural and functional disorders of the respiratory, gastrointestinal, reproductive and nervous systems associated with toxic effects of HCHO 7-10. Allergic effects of HCHO have also been reported1.

    HCHO has adverse effects on the histological structure and functions of the liver11. When received through respiration, it has been found to decrease the liver weight and triglyceride level12,13. Furthermore, the toxic effects of HCHO have been reported to cause structural changes in the epithelial biliary cells and damage intrahepatic and extrahepatic biliary ducts14. HCHO exposure has led to disorders of oxidant and oxidant-antioxidant systems of the liver tissue and inflicted oxidative damage15.

    Melatonin is released from the pineal gland in the dark16,17. In addition to the pineal gland, melatonin has been shown in the retina, intestines, erythrocytes, leucocytes, and many other tissues. The organs and tissues exposed to oxygen radical formation such as the liver, lungs, brain, and skin produce intracellular melatonin at lower levels18-21. Melatonin hormone (N-acetyl-5-methoxytryptamine) acts in the regulation of many physiological functions such as endocrine rhythm, antigonatropic effects, protecting the nervous system, stimulation of the immune system, and the protection of free radicals17,22-24. Recent studies have shown melatonin to be an antioxidant substance25. Melatonin, which is both water and oil soluble, is available to each organelle of the cell26.

    This study aimed to investigate histological and biochemical changes in the liver of formaldehyde exposed rats and the effects of melatonin hormone on these changes.

  • Top
  • Summary
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Methods
    Animals and Treatments
    The subjects of the study were 21 male Wistar-albino rats (weighing 310-320 g). The animals were divided into three equal groups. All animals received humane care in compliance with guidelines of Firat University Research Council’s criteria. Control rats in Group I were injected intraperitoneal (ip) injection of 0.9% NaCl alone every other day. The rats in Group II received ip 10 mg/kg HCHO diluted 1/10 in 0.9% NaCl every other day. In addition to receiving HCHO, the rats in Group III received ip 25 mg/kg melatonin (Sigma Chemical Co.) diluted 1/10 in 0.9% NaCl every other day. To regulate endogenous melatonin secretion, all rats were kept in 12-hour light and 12-hour dark conditions throughout the experimental procedures. At the end of the 14-day experimental period, all animals were sacrificed.

    Microscopic examination of liver tissue specimens
    The liver tissue specimens were fixed in formaldehyde solution (10%). Tissue specimens were embedded in paraffin wax and sectioned (thickness, 5 µm). Paraffin sections were stained with hematoxylin-eosin (H&E), Masson’s trichrome, and PAS and examined with an Olympus BH2 photomicroscope.

    Biochemical analysis of liver tissues
    For biochemical evaluations, rapidly harvested liver biopsy samples were washed in 0.15 M cold (+4ºC) potassium chloride (KCI) and dried with blotting paper. The tissues were homogenized in KCI (0.15 M) at 16000 rpm for three minutes. The homogenate was centrifuged at 5000xg for one hour at +4ºC and the supernatant was obtained and stored at –40ºC for one week until analysis. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), xanthine oxidase (XO) and malondialdehyde (MDA) levels were measured in the supernatant by spectrophotometric methods.

    Determination of superoxide dismutase activity
    It was measured by the method of Sun et al.27, in which SOD enzyme values are based on the reduction of nitroblue tetrazolium (NBT) by superoxide produced by the xanthine-xanthineoxidase system.

    Determination of glutathione peroxidase activity
    Glutathione peroxidase (GSH-Px, EC 1.6.4.2) activity was measured by the method of Paglia and Valentine28. In a medium containing hydrogen peroxide, GSH-Px catalyzes the amplification of glutathione reductase (GSH) to oxidized form of glutathione. The oxidized glutathione is reduced to GSH by glutathione reductase and NADPH. GSH-Px activity was detected by decreased absorbance at 340nm during the conversion of NADPH to NADP+.

    Determination of catalase activity
    Catalase activity was measured according to the Aebi method 29, through the observation of hydrogen peroxide (H2O2) destroyed by the enzyme at 240 nm wavelength in a spectrophotometer.

    Determination of xanthine oxidase activity
    Tissue xanthine oxidase activity was studied according to the method of Prajda and Weber 30, by the observation of uric acid absorbance formed by xanthine at the level of 293 nm wavelength on spectrophotometer.

    Determination of malondialdehyde level
    MDA was measured by the Esterbauer and Cheeseman method 31. Malondialdehyde and thiobarbituric acid react at 90-100ºC and form a pink-colored compound. The absorbances of the samples obtained were read at 532 nm.

    Statistical analyses
    “SPSS 11.0 for Windows” statistical program was used. The distribution of the groups was evaluated through a onesample Kolmogorov-Smirnov Test, a nonparametric test. The group comparisons were performed by the use of parametric tests: one-way ANOVA test and LSD, a Post Hoc test. P<0.05 was considered statistically significant.

  • Top
  • Summary
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Results
    Light microscopic findings In light microscopic evaluations, all three zones of hepatic acinus in the liver sections of the control group were normal. With PAS staining, the hepatocytes in all three zones were intensely glycogen stained (Figure 1).


    Click Here to Zoom
    Figure 1: Group I. The glycogen content (arrow) in the hepatocytes around the portal area. PAS X240.

    Upon evaluation of the liver tissue preparations of the rats exposed to excessive HCHO for 14 days, enlarged sinusoids were blood filled, and there was mononuclear cell infiltration in the portal area and around the vena centralis (Figures 2 and 3).


    Click Here to Zoom
    Figure 2: Group II. Dilatation and congestion (*) in the sinusoids. H&E X240.


    Click Here to Zoom
    Figure 3: Group II. Mononuclear cell infiltration (arrow) around the portal area (p). H&E X240.

    Furthermore, some hepatocytes had vacuolated cytoplasm (Figure 4) and some had a hyperchromatic nucleus (Figure 5). With PAS staining, the hepatocytes around the portal area were PAS negative; thus, there was no glycogen content (Figure 6).


    Click Here to Zoom
    Figure 4: Group II. Glycogen content of hepatocytes (arrow) and vacuolization (double arrow) are visible. PAS X480.


    Click Here to Zoom
    Figure 5: Group II. Hepatocytes with hyperchromatic nucleus (arrow) and vascular congestion (*) were evident. Masson’s trichrome X240.


    Click Here to Zoom
    Figure 6: Group II. The hepatocytes around the portal area were PAS negative (*) and the glycogen containing hepatocytes around vena centralis (vc) were PAS positive (arrow). PAS X240.

    The evaluation of the liver tissue preparations of the rats exposed to both HCHO and melatonin showed that enlarged sinusoids were filled with blood, and there were hepatocytes with hyperchromatic nuclei at various locations (Figure 7). PAS staining indicated glycogen presence in hepatocytes of all three zones as seen in the control group (Figure 8). In this group also, sinusoidal enlargements were not as common as those in the HCHO group. Moreover, there was no cellular infiltration.


    Click Here to Zoom
    Figure 7: Group III. Enlargement and congestion of the sinusoids (*) are visible. Masson’s trichrome X240.


    Click Here to Zoom
    Figure 8: Group III. The hepatocytes with glycogen content around the portal area (arrow) are noticeable. PAS X240.

    Biochemical findings
    In HCHO-exposed rats, oxidative antioxidant enzymes SOD, GSH-Px, CAT values were significantly higher than those of the control group (p<0.05). MDA and XO levels of the same group were also statistically higher than the control group (p<0.05). In the HCHO and melatonin-exposed group, SOD, GSH-Px, CAT, XO and MDA levels were lower than the HCHO alone injected group (p<0.05). The biochemical findings are summarized in Table 1.


    Click Here to Zoom
    Table 1: MDA, XO, GSH-Px, CAT and SOD values of the study groups presented in mean ± SD (for each group n=7).

  • Top
  • Summary
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Discussion
    Previous studies have shown the toxic effects of formaldehyde (HCHO) on the skin and eyes, and on the respiratory, gastrointestinal, nervous, and reproductive systems7-10. In addition to its deleterious effects on histological structure and functions of the liver, HCHO has been reported to cause a decrease in liver weight12,13 and damage to the biliary ducts14.

    In our study, the light microscopic evaluation of the liver tissue sections revealed enlarged sinusoids filled with blood and cellular infiltration in the portal area and around vena centralis. Furthermore, some hepatocytes had cytoplasmic vacuolizations, while some had hyperchromatic nuclei. Strubelt et al.15 found that HCHO exposure leads to mitochondrial destruction and damages rough endoplasmic reticulum. Beall and Ulsamer32 reported centrilobular vacuolization and local cellular necrosis in the liver associated with HCHO exposure. In their study on rats, Dumont et al.14 detected structural changes in the biliary epithelial cells after HCHO administration.

    Free oxygen radicals are a result of metabolic intracellular process forming under natural conditions. They inflict oxidative damage on the cells by affecting membrane lipids, proteins, and nucleic acids. These potentially harmful effects are regulated by antioxidant defense mechanism. The antioxidant enzymes such as SOD and GSH-Px are needed for the maintenance of cellular balance and scavenging the free radicals away33. Malondialdehyde (MDA) is one of the products of lipid peroxidation and is commonly used parameter to indicate oxidative stress34.

    HCHO disturbs the oxidant-antioxidant balance in various tissues and cause oxidative stress in parallel with tissue damage. In previous studies, increased MDA levels in the lung, liver, and testicular tissues of the rats exposed to HCHO were reported34-36. In accordance with our findings, Strubelt et al.15 have reported increased MDA levels in the liver tissues of HCHO-exposed animals. Similarly, Teng et al.37 in their experimental study on isolated rat hepatocytes showed that HCHO at low concentrations leads to oxidative stress.

    Skrzydlewska and Farbiszewski38-40 noted methanol metabolized into HCHO and formic acid for the increased levels of SOD and GSH-Px levels in rat liver tissues. However, Datta and Namasivayam41 found that methanol decreases SOD levels and increases CAT and MDA levels in rat hepatocytes. In our study, SOD, GSH-Px, and CAT values increased in the HCHO-exposed rats. Increased SOD activity may be a response of increased oxidative stress in the liver tissue. CAT increase, however, may be indicative of high degree oxidative stress due to elevated endogenous H2O2. It may also be an adaptive response to oxidative stress induced by HCHO. GSH-Px is an important antioxidant enzyme acting in H2O2 elimination and lipid peroxidation. Increased GSH-Px activity suggests increased H2O2 products.

    Melatonin is known to be involved in a variety of physiological processes including the regulation of endocrine rhythm, antigonadotropic effects, neuroprotective effects, stimulation of the immune system, and free radical scavenging action17,22-24. In addition to these properties of melatonin, it is a potent antioxidant agent and exerts a protective effect against oxidative stress25,42,43. In our study, melatonin was found to partially prevent the liver damage against HCHO intoxication. The exact mechanism of melatonin-provided prevention of hepatic damage induced by formaldehyde is not completely clear. Considering the distinctive properties of melatonin and the results of the present study, it is plausible that both its radical-scavenging and antioxidant actions are involved in preventing tissue damage.

    We concluded that chronic exposure of formaldehyde causes structural degeneration and oxidative damage in the liver of rats. We also concluded that melatonin exerts a beneficial effect against formaldehyde toxicity in the liver appeared to be due to its antioxidant and free radical scavenger activity.

  • Top
  • Summary
  • Introduction
  • Methods
  • Results
  • Discussion
  • References
  • References

    1) Smith AE. Formaldehyde. Occup Med 1992: 42: 83-88.

    2) Usanmaz SE, Akarsu ES, Vural N. Neurotoxic effects of acute and subacute formaldehyde exposures in mice. Envir Toxicol Pharmacol 2002: 11: 93-100.

    3) Feron VJ, Till HP, de Vriger F, et al. Aldehydes: Occurrence carcinogenic potential, mechanism of action and risk assessment. Mutat Res 1991: 259: 363-385.

    4) Heck HD, Casanova M, Starr TB. Formaldehyde toxicity: New understanding. Crit Rev Toxicol 1990: 20: 397-426.

    5) Sarnak MJ, Long J, King A. Intravesicular formaldehyde instillation and renal complications. Clin Nephrol 1999: 51: 122-125.

    6) Cohen BI, Pagnillo MK, Musikant BL, Deutsch AS. Formaldehyde evaluation from endodontic materials. Oral Health 1998: 88: 37-39.

    7) Khamgaonkar MB, Fulare MB. Pulmonary effects of formaldehyde exposure: an environmental-epidemiological study. Indian J Chest Dis Allied Sci 1991: 33: 9-13.

    8) Restani P, Galli CL. Oral toxicity of formaldehyde and its derivates. Crit Rev Toxicol 1991: 21: 315-328.

    9) Taskinen H, Kyyrönen P, Hemminki K, et al. Laboratory work and pregnancy outcome. J Occup Med 1994: 36: 311-319.

    10) Kilburn KH. Neurobehavioral impairment and seizures from formaldehyde. Arch of Environ Health 1994: 49: 37- 44.

    11) Shizumi K, Sugita M, Yokote R, et al. Intestinal edema caused by ingested formalin. Chudoku Kenkyu 2003: 16: 447-451.

    12) Kamata E, Nakadate M, Uchida O, et al. Results of a 28-month chronic inhalation toxicity study of formaldehyde in male Fisher- 344 rats. J Toxicol Sci 1997: 22: 239-254.

    13) Rusch GM, Clary JJ, Rinehart WE, Bolte HF. A 26-week inhalation toxicity study with formaldehyde in the monkey, rat and hamster. Toxicol Appl Pharmacol 1983: 68: 329-343.

    14) Dumont M, D’Hont C, Moreau A, et al. Retrograde injections of formaldehyde into the biliary tree induce alterations of biliary epithelial function in rats. Hepatology 1996: 24: 1217-1223.

    15) Strubelt O, Younes M, Pentz R, Kuhnel W. Mechanistic study on formaldehyde-induced hepatotoxicity. J Toxicol Environ Health 1989: 27: 351-366.

    16) Susko I, Mornjakovic Z, Alicelebic S, Cosovic E, Beganovic A. Retinal and pineal melatonin-from a circadian signal to therapeutic use. Med Arh 2004: 58: 61-64.

    17) Vijayalaxmi, Reiter RJ, Tan DX, Herman TS, Thomas CR. Melatonin as a radioprotective agent: a review. Int J Radiat Oncol Biol Phys 2004: 59: 639- 653.

    18) Hardeland R, Reiter RJ, Poeggeler B, Tan DX. The significance of the metabolism of the neurohormone melatonin: antioxidative protection and formation of bioactive substances. Neurosci Biobehav Rev 1993: 17: 347-357.

    19) Lee PP, Pang SF. Melatonin and its receptors in the gastrointestinal tract. Biol Signals 1993: 2: 181-193.

    20) Pang SF, Allen AE. Extra-pineal melatonin in the retina: Its regulation and physiological function. In: Reiter RJ (Editor). Pineal Research Review. New York, NY: Alan R. Liss., 1986: 55-95.

    21) Stefulj J, Hortner M, Ghosh M, et al. Gene expression of the key enzymes of melatonin synthesis in extrapineal tissues of the rat. J Pineal Res 2001: 30: 243-247.

    22) Csernus V, Mess B. Biorhythms and pineal gland. Neuro Endocrinol Lett 2003: 24: 404-411.

    23) Kus I, Sarsilmaz M, Ogeturk M, et al. Ultrastructural interrelationship between the pineal gland and the testis in the male rat. Arch Androl 2000: 45: 119-124.

    24) Guerrero JM, Reiter RJ. A brief survey of pineal gland-immune system interrelationships. Endocr Res 1992: 18: 91-113.

    25) Longoni B, Salgo MG, Pryor WA, Marchiafava PL. Effects of melatonin on lipid peroxidation induced by oxygen radicals. Life Sci 1998: 62: 853-859.

    26) Arendt J. Melatonin. Clin Endocrinol 1988: 29: 205-229.

    27) Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988: 34: 497-500.

    28) Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967: 70: 158-169.

    29) Aebi H. Catalase. In: Bergmeyer U (Editor). Methods of Enzymatic Analysis. New York and London: Academic Press, 1974: 673-677.

    30) Prajda N, Weber G. Malignant transformation-linked imbalance: decreased xanthine oxidase activity in hepatomas. FEBS Lett 1975: 59: 245-249.

    31) Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hidroxynonenal. Methods Enzymol 1990: 186: 407-421.

    32) Beall JR, Ulsamer AG. Formaldehyde and hepatotoxicity: a review. J Toxicol Environ Health 1984: 14: 1-21.

    33) Husain K, Scott BR, Reddy SK, Somani SM. Chronic ethanol and nicotine interaction on rat tissue antioxidant defense system. Alcohol 2001: 25: 89-97.

    34) Kamal AA, Gomaa A, el Khafif M, Hammad AS. Plasma lipid peroxides among workers exposed to silica or asbestos dusts. Environ Res 1990: 49: 173-180.

    35) Dobrzynska I, Skrzydlewska E, Kasacka I, Figaszewski Z. Protective effect of N-acetylcysteine on rat liver cell membrane during methanol intoxication. J Pharm Pharmacol 2000: 52: 547- 552.

    36) Tang M, Xie Y, Yi Y, Wang W. Effects of formaldehyde on germ cells of male mice. Wei Sheng Yan Jiu 2003: 32: 544-548.

    37) Teng S, Beard K, Pourahmad J, et al. The formaldehyde metabolic detoxification enzyme systems and molecular cytotoxic mechanism in isolated rat hepatocytes. Chem Biol Interact 2001: 130-132: 285-296.

    38) Skrzydlewska E, Farbiszewski R. Lipid peroxidation and antioxidant status in the liver, erythrocytes, and serum of rats after methanol intoxication. J Toxicol Environ Health A 1998: 53: 637-649.

    39) Skrzydlewska E, Farbiszewski R. Liver and serum antioxidant status after methanol intoxication in rats. Acta Biochim Pol 1997: 44: 139-145.

    40) Skrzydlewska E, Farbiszewski R. Decreased antioxidant defense mechanisms in rat liver after methanol intoxication. Free Radic Res 1997: 27: 369-375.

    41) Datta NJ, Namasivayam A. In vitro effect of methanol on folatedeficient rat hepatocytes. Drug Alcohol Depend 2003: 71: 87-91.

    42) Gupta YK, Gupta M, Kohli K. Neuroprotective role of melatonin in oxidative stress vulnerable brain. Indian J Physiol Pharmacol 2003: 47: 373-386.

    43) Reiter RJ, Tan DX, Manchester LC, et al. Melatonin: detoxification of oxygen and nitrogen-based toxic reactants. Adv Exp Med Biol 2003: 527: 539-548.

  • Top
  • Summary
  • Introduction
  • Methods
  • Results
  • Discussion
  • References
  • [ Başa Dön ] [ Özet ] [ PDF ] [ Benzer Makaleler ] [ Yazara E-Posta ] [ Editöre E-Posta ]
    [ Ana Sayfa | Editörler | Danışma Kurulu | Dergi Hakkında | İçindekiler | Arşiv | Yayın Arama | Yazarlara Bilgi | E-Posta ]