Study of Necrosis in the Liver of Formaldehyde and Benzo(α)Pyrene Exposured-Mice

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1 THE JOURNAL OF TROPICAL LIFE SCIENCE OPEN ACCESS Freely available online VOL. 3, NO. 1, pp , January, 2013 Study of Nerosis in the Liver of Formaldehyde and Benzo(α)Pyrene Exposured-Mie Ahmad Soni, Sri Widyarti *, Aris Soewondo Biology Department, Faulty of MathematisandNatural Sienes, Brawijaya University, Malang, Indonesia ABSTRACT Formaldehyde and benzo(α)pyrene are ompounds that harmful for health. Misappliation of this ompound has an impat in the form of organ damage in the body. This study aims to determine the impat of the treatment of the ombined exposure of formaldehyde and benzo(α)pyrene to ell nerosis in the liver of mie (Mus musulus). Treatment of formaldehyde dose of 25 mg/kg BW to mie was given orally every day for 60 days. Treatment of benzo(α)pyrene via ip at a dose of 250 mg/kg BW were given after 30 days of inubation with four times injetion with one day interval. Liver organ histologial preparations were made through the HE staining and were observed by BX51 miroopy. The data obtained that is the perentage of ells nerosis and neroti foi. This researh used Completely Randomized Design (CRD) with 95% onfidene interval. Liver organ preparations observations indiate that the perentage of nerosis in the untreated ontrol, benzo(α)pyrene 250 mg/kg BW, formaldehyde 25 mg/kg BW, ombination of formaldehyde 25 mg/kg BW with benzo(α)pyrene in a row that is equal to 14.43% ± 0.91; 26.05% ± 3.75; 49.38% ± 2.66; ± The mean of neroti foi in liver organ formed in the untreatment ontrol, benzo(α)pyrene 250 mg/kg BW, Formaldehyde 25 mg/kg BW, and the ombination of formaldehyde 25 mg/kg BW with benzo(α)pyrene in a row, equal to 1.3 ± 0,07; 1.63 ± 0.61; 2 ± 0.51, and 3.4 ± This suggests that the ombined treatment had the highest level of toxiity ompared with other treatments. Keywords: benzo(α)pyrene, formaldehyde, liver, nerosis INTRODUCTION Formaldehyde are ompound that is naturally produed by the body through the metaboli proesses. However, the urrent misuse of formaldehyde had rossed the threshold, partiularly in developing ountries like Indonesia. Previous study, showed that treatment with formaldehyde may degrade the ative ell to divide, inhibition of spermatogenesis proess, and also the depletion of the basement membrane of the seminiferous tubules [1]. Exposure to formaldehyde also may inrease the risk of hanges in the gastri muosa of mie (Mus musulus). Changes in the gastri muosa of mie is shown by the widening of the parietal ells in gastri muosa and the destrution of parietal ells and hief ells [2]. Benzo(α)pyrene * Corresponding author: Sri Widyarti Biology Department, Faulty of Mathematis and Natural Sienes, Brawijaya University, Jl. Veteran, Malang, Indonesia swid@ub.a.id (BaP) are ompound whose the struture has a five-ring polyyli aromati with hydroarbons bonding. This substane is arinogeni and an lead to aner if these substanes enter the ell. Benzo[α]pyrene an initiate the ourrene of mutations in the genes if exposed ontinuously [3]. The indution of benzo(α)pyrene in liver an indue hanges in ell morphology, there is vakuolization, narrowing of the sinusoids and swollen nuleus. In the lungs there are hange in the form of proliferation of bronhial epithelial layer [4]. Besides, the indution of benzo(α)pyrene in the liver an auses piknosis and karyolysis on the first day of inubation [5]. In developing ountries like Indonesia, misappliation of formaldehyde as a food preservative and oupled with high air pollution due to smoke generated from motor vehiles and industrial smoke, an lead to inreased exposure to formaldehyde and benzo(α)pyrene on the population. Previous researh suggests that the effet of exposure to either formaldehyde or benzo(α)pyrene an inreased the expression of the gene PARP 1 [6], whih is the inreased JTLS J. Trop. Life. Siene 58 Volume 3 Number 1 January 2013

2 Soni A, et al., 2013 expression of these genes an initiate ell to undergoing nerosis that is onerned to have a greater likelihood for the ourrene of aner ompared to a tissue that has a normal PARP gene expression [6, 7]. Histologial observations of the organs of mie that will prove an inrease in the perentage of ells undergoing nerosis and neroti foi in the liver organs of mie (Mus musulus) after exposure to formaldehyde and benzo(α)pyrene. MATERIALS AND METHODS All proedures were performed has been aepted by the Researh Ethis Committee of the UB with the number 30-KE. Animals Treatment The animals that used in this study were twomonths old male mie weighing between grams. Animal treatment arried out for two months. The animals were divided into 4 groups i.e ontrol (no treatment), formaldehyde, BaP, formaldehyde and BaP.Formaldehyde 25 mg/kg BW was given orally for 8 weeks everyday. Total dose of BaP 250 mg/kg BW was givenafter 30 days of inubation with four times the injetion interval of one day. Histologial preparation The first step taken is the organ liver of mie (Mus musulus) that have been exposed to formaldehyde, benzo(α)pyrene and the ombination between formaldehyde and benzo(α)pyrene exposure were fixation first with 4% formaldehyde solution for 24 hours. Liver organ then soaked in 70% alohol for 3 times for 10 minutes respetively while shaken. The speimen was then dipped in 70% alohol for overnight. Followed by immersed in alohol 80%, 3 times for 10 minutes respetively while shaken. The speimens then soaked with alohol at a onentration of 90% and 96%. Followed by immersed in absolute alohol and shaken for 10 minutes. Next, the speimens were soaked with xylol for ± 30 minutes. Infiltration proess is arried out by omparison xylol : paraffin in a row equal to 3:1, 1:1, and 1:3 respetively for 30 min at 52 0 C. Continued with the provision of pure paraffin at a temperature of 56 C for 12 minutes. Next the speimens were bloking and setioning with a hunk size of ± 5 mirons. Tape the piees plaed on glass objets that have been given mayer albumin with a brush and put on the hot plate. Then, the speimens were stained by Haematoxylin-Eosin staining. Observation The first step is to perform observations and estimates of the perentage of nerosis in the field of view. Observations made using the Olympus BX51 binoular mirosope with a magnifiation of 200x and 400x for liver organ speimens. In the liver organ, the observation is determined by taking five random fields of view, then in eah field of view of the alulated number of total ells and subsequently in the average. The average results are then used as a referene the total number of ells in eah field of view. Then performed ounting the number of ells undergoing nerosis and the result is onverted to a perentage. Data Analysis The data that was obtained will be sorted first by eliminating the outliers of data using the upper and lower limits based on data obtained from observations. Furthermore, this study used Completely Randomized Design (CRD) with 95% onfidene interval. Analysis of signifiane by using ANOVA followed by Tukey-HSD test. The data used is the perentage of ells undergoing nerosis of the organ liver of mie exposed to benzo(α)pyrene and formaldehyde in toxi doses, and the average spotformed neroti foi in liver organ of mie. RESULTS AND DISCUSSION Results Nerosis and Neroti Foi in Liver Based on data analysis has been done, it is known that there is a differene between the ontrol without treatment, and treatment of benzo(α)pyrene (BaP) with formaldehyde treatment dose of 25 mg/kg BW and the ombined treatment of formaldehyde 25 mg/kg and benzo(α)pyrene in liver organ. Based on the analysis has been done, it is known that there are differenes in the perentage of ells undergoing nerosis of the liver organ in eah treatment (Figure 1). The perentage of nerosis in the untreated ontrol, benzo(a)pyrene 250 mg/kg BW, Formaldehyde 25 mg/kg BW, the ombined formaldehyde 25 mg/kg BW with benzo(α)pyrene in a row that is equal to 14.43% ± 0.91; 26, 05% ± 3.75.; 49.38% ± 2.66; ± It indiates that either the benzo(α)pyrene treatment in dose of 250 mg/kg BW, formalin dose of 25 mg/kg BW or the ombined treatment affets in the number of ells undergoing nerosis in the liver of mie. JTLS J. Trop. Life. Siene 59 Volume 3 Number 1 January 2013

3 Neroti ells (%) Average of NF Study of Nerosis in The Liver , b 3,5 3 2,5 ab b 20 a 2 1,5 a 10 0 K (-) BaP F25 F25B Treatments (a) (b) Figure 1. Data analysis of observations. (a) The perentage of ell nerosis in the liver organ. (b) Average of spots neroti foi on liver organ. K (-): Control; BaP : Benzo(a)pyrene; F25: Formaldehyde 25 mg/kg BW; F25B: Combination Treatment. 1 0,5 0 K (-) BaP F25 F25B Treatments Figure 2. Spot neroti foi in the liver organ. (a) ontrol without treatment, (b) treatment of BaP 250 mg/kg BW, () treatment of formaldehyde 25 mg/kg BW, (d) the ombined treatment of formaldehyde 25 mg/kg BW and BAP 250 mg/kg BW. Neroti foi showed by the blak arrows and white irle. (HE, Sale : 300 µm) JTLS J. Trop. Life. Siene 60 Volume 3 Number 1 January 2013

4 Soni A, et al., 2013 Thus, the more extreme treatment an ause the more ells undergoing nerosis in liver of mie. Observations of liver organ preparations indiate there are formation of neroti foi in eah treatment, but with a different distribution (Figure 1). The mean neroti foi are formed in the ontrol treatment with no treatment, benzo(a)pyrene 250 mg/kg BW, Formaldehyde 25 mg/kg BW, and the ombined formaldehyde 25 mg/kg BW with benzo(α)pyrene in a row, whih is 1.3 ± 0.07 ; 1.63 ± 0.61; 2 ± 0.51, and 3.4 ± Histologial ross setion of neroti foi in liver organ preparations is shown in the Figure 2. Disussion Nerosis is a proess of ell death that ours as a result of exposure to toxi substanes in high doses and our suddenly. Nerosis also known as the end result of a atastrophi bioenergeti aused by ATP depletion due to the level that does not allow ells to repair themselves or apoptosis. The ause of ATP depletion itself due to the toxi substanes that enter or the presene of physial damage. Nerosis an be haraterized morphologially, that is the presene vakuolation the ytoplasm, plasma membrane rupture and indue inflammation in ells undergoing nerosis by removing debris of ells undergoing nerosis and release several proinflamatory moleules. Furthermore, a olletion of debris ell and infiltration of lymphoid ells will form the neroti foi [8]. Observations that have been done, show that the neroti foi are formed mostly loated around blood vessels, that is in the hepatoytes where loated around the entral vein. Neroti foi formed by the ells undergoing lysis, so that the lysosomes of ells that have undergone ell lysis will be digestingaround its. This proess will then result in ell debris, so an initiating infiltration of phagoyti ells [8]. Observations on liver organ showed that in eah treatment reveal any ells undergoing nerosis, but in different perentages. This is due to nerosis is also a phenomenon that ours in any organism if they get an infetion, but in very small quantities [8], as well as in the ontrol treatment. Some types of nerosis that an be found in the liver is the organ preparations piknosis, kariorrhexis, and karyolysis with different distributions in eah treatment. Cells undergoing nerosis and neroti foi were also found in liver organ without treatment. This is due to nerosis is a phenomenon that ours in every living being, but the perentage is very small, so it doesn t interfere with the metabolism in the body itself. It is highly related to the metabolism that our in eah organ. As is known, the liver is a detoxifiation organ where toxi substanes that enter the body, so it is possible in these organs were also found to have nerosis of hepatoytes ells, although not treated with the treatment of toxi substanes. The previous researh showed that there was inreased expression of PARP-1 protein with inreasing doses of benzo(α)pyrene and formaldehyde treatment given to the liver of mie. Based on the PARP protein fragment, there are indiations that the event ours ell nerosis in liver of mie [9]. DNA damage in the initial level will not lead to apoptosis or nerosis due to the presene of PARP-1 protein responsible for DNA repair proess [10]. DNA damage levels are going to initiate ell apoptosis beause the ells are not able to repair DNA damage that ours, but ells in this ase still has the ATP in suffiient quantities to perform apoptosis. However, DNA damage at higher levels will initiate the ells to undergo nerosis due to the over expression of PARP-1 resulting in ATP depletion. This resulted in ATP in the ell does not have suffiient numbers to perform apoptosis, so ells will lead to nerosis [7,11,12,13]. The ells undergoing DNA damage will take the proess of DNA repair by ativating protein PARP-1. Ativation of this protein will lead to the ourrene of poly (ADP-ribosyl)ation there was a key proess in DNA repair by using NAD+ as substrate. Then, NAD+ will be onverted into niotinamide and ADP-ribose. At low level of DNA damage, the ell will ativate the protetive homeostati proess that an repair the damage. If a strand of DNA damage has been repaired, the ell will remain alive and ellular NAD+ level will be inreased again by onverting niotinamide. The onversion proess requires two ATP moleules per one moleule of niotinamide is onverted to NAD+. If the repair proess an not run properly, then the ell will make the proess of apoptosis through aspase-dependent mehanism, for example is aspase 3 [14]. However, if the DNA damage that ours is too severe, then the ell will not be able to repair suh damage and this ase an lead hyper ativation of PARP-1 protein. This hyper ativation proess resulting in depletion of ellular NAD+ and ATP that diret ell death through the mehanism of nerosis [13]. In this study, ombined treatment has the highest level of toxiity ompared with other JTLS J. Trop. Life. Siene 61 Volume 3 Number 1 January 2013

5 Study of Nerosis in The Liver treatments. This is evident from the distribution of neroti foi formed in the liver organ of mie. In this study suggests that high exposure to formaldehyde an ause infiltration of lymphoid ells in liver organ around the spaes between ells, and also the ourrene of dilatation of the artery. Aording to previous researh, showed that the benzo(α)pyrene may inrease the aumulation of p53 protein. Inreased aumulation of p53 protein is assoiated with an inreased number of bonds between the BPDE- DNA, where this bond an lead to the dissolution of single hains of DNA. The breakdown of single hains of DNA an ativate PARP-1 for DNA repair [15]. Furthermore, high doses of formaldehyde and benzo(α)pyrene treatment on this study an improve the over expression of the PARP protein itself, so as to diret the ells to undergo nerosis. This is what led to the ombined treatment of formaldehyde 25 mg/kg BW with benzo(α)pyrene 250 mg/kg BW had a more severe effets than other treatments. CONCLUSION This researh performed at eah treatment reveal any organ like liver undergoing nerosis, but with different perentages. In general, the perentage of ells undergoing nerosis inreased with inreasing treatment extremities. The highest perentage of nerosis seen in the ombined treatment of formalin 25 mg/kg BW with benzo(α)pyrene 250 mg/kg BW, while the highest rates of neroti foi are formed in the ombined treatment of formalin 25 mg/kg BW with a benzo(α)pyrene dose of 250 mg/kg BW. REFERENCES 1. Golalipour MJ, Azarhoush R,Ghafari S,Gharravi AM, Fazeli SA, Davarian A (2007) Formaldehyde exposure indues histopathologial and morphometri hanges in the rat testis. Folia Morphol. 66 (3) : Priambowo A (2010) Histopathology studies of gastri muosal mie (Mus musulus) after exposure to subroni formaldehyde. Skripsi Not Published. Biology Department. Brawijaya University. Malang. 3. Aygün SF, Kabadayi F(2005) Determination of benzo(α)pyrene in haroal grilled meat samples by HPLC with fluoresene detetion. International Journal of Food Sienes and Nutrition. 56(8) : Yunus M(1996) Indution benzapiren influene on liver histology and lung of white rat (Rattus norvegius). Skripsi not published. Biology Department. Brawijaya University. Malang. 5. Pinuji HM (1999) Liver histopathology and lien of young Rattus norvegius after indution benzapiren. Skripsi not published. Biology Department. Brawijaya University. Malang. 6. Putri PF(2010) Gene expression of PARP-1 mrna liver and lung of mie (Mus musulus) exposed to formalin and benzo(α)pyrene. Skripsi not published. Biology Department. Brawijaya University. Malang. 7. Filipovi DM, Meng X, Reeves WB (1999) Inhibition of PARP prevents oxidant-indued nerosis but not apoptosis in LLC-PK1 Cells.Am J Physiol Renal Physiol. 277 : Edinger AL, Thompson CB(2004)death by Design: apoptosis, nerosis and autophagy. Current Opinion in Cell Biology. 16: Maghfironi A(2010) Study of PARP-1 protein expression in liver and lung of mie exposed to formalin and benzopiren. Skripsi not published. Department of Biology. Brawijaya University. Malang. 10. Wang ZQ, Stingl L, Morrison C, Jantsh M, Los M, Shulze-Osthoff K, Wagner EF(1997) PARP is Important for genomi stability but dispensable in apoptosis. Genes and Development 11 : Bey EA, Bentle MS, Reinike KE, Dong Y, Chin- Yang R, Girard L, Minna JD, Bornmann WG, Gao J, Boothman DA(2007) An NQO1- and PARP-1-mediated ell death pathway indued in non-small-ell lung aner ells by β-lapahone. National Aademy of Sienes of USA. 28(104) : Erdelyi K, Bakondi E, Gergely P, Szabo C, Virag L(2005) Poly-ADP-ribosylation in health and disease pathophysiologi role of oxidative stressindued poly (ADP-ribose) polymerase-1 ativation: Fous on ell death and transriptional regulation. Cellular and Moleular Life Sienes. 62: Moubarak RS, Yuste VJ, Artus CD, Bouharrour AD, Greer PA, Muria JM,Susin SA (2007) Sequential ativation of poly (ADP-Ribose) polymerase 1. alpains, and bax is essential in apoptosis-induing fator-mediated programmed nerosis. Journal Moleular and Cellular Biology. 13(27): Chaitanya GV, Alexander JS, Babu PP(2010) PARP-1 leavage fragments: Signatures of elldeath proteases in neurodegeneration. Biomed Central. 8(31): Serpi R (2003) Mehanism of benzo(α)pyreneindued aumulation of p53 tumour suppressor protein in mouse. Department of Pharmaology and Toxiology, University of Oulu. Oulu, Finland. JTLS J. Trop. Life. Siene 62 Volume 3 Number 1 January 2013