Apoptosis in Mice Liver Cells Caused by Formalin–containing Food: Normalization of HSP70 Overexpression by Chlorophyllin

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ScienceDirect Procedia Chemistry 14 (2015) 27 – 35

2nd Humboldt Kolleg in conjunction with International Conference on Natural Sciences, HK-ICONS 2014

Apoptosis in Mice Liver Cells Caused by Formalin–Containing Food: Normalization of HSP70 Overexpression by Chlorophyllin Alfonds Andrew Maramisa*, Mohamad Aminb, Sumarnoc, Aloysius Duran Corebimab a

Department of Biology, State University of Manado (UNIMA), Campus of UNIMA at Tondano, Minahasa 95618, Indonesia. b Biology Education Doctoral Program, State University of Malang, Jl. Semarang 5, Malang 65145, Indonesia. c Postgraduate Program Magister of Biomedic, Brawijaya University, Jl. Veteran, Malang 65145, Indonesia.

Abstract Repeated exposure to formalin–containing foodstuff results in mice to overexpressing HSP70 (Heat Shock Protein, 70 kDa) and induction of apoptosis. This study tried to assess the efficacy of supplementation of chlorophyllin in inhibiting this induced apoptosis. In this study, a 4 x 4 factorial experiment was conducted with treatment type and duration of exposure as independent variables. Expression of HSP70 was determined by immunohistochemistry (avidin–biotin sandwich assay). The number of cells expressing HSP70 was statistically analyzed using two–way ANOVA, followed by Duncan's Multiple Range Test (DMRT, α = 0.01). From the results, it can be concluded that chlorophyllin supplementation is able to inhibit the number of cells undergoing induced apoptosis due to repeated exposure of formalin–containing fish by normalizing the overexpressing of HSP70. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license © 2015 A.A. Maramis, M. Amin, Sumarno, A.D Corebima. Published by Elsevier B.V. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-reviewunder under responsibility of the Scientific Committee of HK-ICONS Peer-review responsibility of the Scientifi c Committee of HK-ICONS 2014 2014. Keywords: Chlorophyllin; formalin; HSP70; induced apoptosis; mice liver.

* Corresponding author. Tel.: +62 8132 5001 220. E-mail address:[email protected]

1876-6196 © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014 doi:10.1016/j.proche.2015.03.006

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Nomenclature g mass (gram, 1 000 miligram, 0.001 kilogram) L volume (liter, 1 000 mililiter) mg · L–1 mass concentration (miligram per liter) mg · kg–1 mass concentration (miligram per kilogram) % (w/v) volume concentration (percent weight per volume, mass of solute (g) for every 100 mL of solution) h time (hour) d time (day) mo time (month) 1. Introduction The misuse of formalin as a preservative of food is still frequent, although it has been banned since 26 years ago through the Regulation of the Minister of Health of the Republic of Indonesia. From the observations made by Kartikaningsih1 in Malang and surrounding areas, fishermen deliberately add formalin to fish to be sold in the market or at auction sites, while there was no formalin addition to fish sold to fish processing plants and or consumed by their family. The use of formalin as preservative does not meet the safety criteria because it can react with molecules in the cell and ultimately change the function, thus causing damage to the cellular, tissue, organ, until the organism level1-4. The previous studies have shown that repeated exposure of mice to fish meat containing formalin activates caspase–9 (an enzyme initiating apoptosis or programmed cell death) 5 and leads to overexpression of HSP70 (protein which is a marker of cellular stress)6 in liver cells, induces apoptosis of hepatic cells7, causes high elevated SGOT (serum glutamic oxaloacetic transaminase) and SGPT (serum glutamic pyruvic transaminase) level 8 which are a marker of functional disorders of liver tissue, physiological9 and pathological10 disorder of digestive organs, behavioral disorder11, and even somatic death of mice10. Based on the fact that the consumption of food containing formalin is still difficult to avoid, and the aforementioned multifaceted damage from the molecular to the organismic, it is needed to seek some prevention. The efforts of detoxification of foodstuffs containing formalin by treatment with derivative of chlorophyll, chlorophyllin, had been examined. Preliminary studies have shown that chlorophyllin supplementation can normalize the concentration of SGOT and SGPT 12 which elevated due to repeated exposure to formalin–containing foodstuff. To strengthen the evidence that chlorophyllin supplementation can detoxify formalin–containing food, more research is required. This research aimed to determine the ability of chlorophyllin supplementation in inhibiting induced apoptosis by overexpressing HSP70 in liver cells of mice that increased due to repeated exposure to formalin–containing foodstuffs. 2. Material and methods 2.1. Design of the research This experimental research uses a randomized block design, 4 × 4 factorial, with type of treatment and duration of exposure as independent variables. Treatment factor consist of: negative control (NC); fish containing formalin (FF); fish containing formalin plus chlorophyllin (FFChln); and chlorophyllin (Chln). Duration of exposure factor consist of daily repeated exposure over 0 (= control); 2 d; 14 d; and 62 d. 2.2. Animal cultivation, preparation, and induction of test substances Fourty–eight male Balb/c mice 2.5 mo old with 15 g to 25 g in weight were used. They were maintained in Animal Physiology Laboratory of Biology Department, Mathematics and Natural Science Faculty, Brawijaya University, Malang. Mice were housed in polyethylene plastic containers, maintained at (27 ± 2) °C with 12 h photoperiod and provided with fodder (standard fodder, ACT PBS Pellet produced by PT. Charoen Pokphand

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Indonesia Tbk.) and water ad libitum. All animal procedures conformed to the institutional ethic regulations concerning the protection of animals13,14. Stock solutions of the four treatments were made as follows: x Negative control (NC) — The NC stock solution consisting only of demineralized water that serves as placebo administration toward gavage (force-feeding) procedure. The volume of water which force-feeding by gavage tube (with the range of 0.01 mL to 0.1 mL) were adjusted to the body weight of mice. x Fish containing formalin (FF) — The fish used as food models in this study was Oreochromis niloticus. The primary stock of FF was set to 100 mg · kg–1 by mixing 10 mL of 10 000 mg · L–1 formaldehyde solution (diluted from 37 % (w/v) formaldehyde PA grade, Merck, Germany) and 1 kg of refined meat of O. niloticus. This concentration was chosen according to the research of Kartikaningsih1 which found that the average of formaldehyde concentration in fish be sold in traditional market in Malang City, East of Java, was 100 mg · kg–1. The primary stock of FF was diluted 10 times in order to obtain the FF secondary stock solution, with a concentration of 10 mg · L–1. Based on the research of Kartikaningsih1 (the average of formaldehyde concentration in fish) and assuming that the average consumption of fish by an adult (50 kg of body weight) i.e. 100 g · d–1 per adult, then the weight of formaldehyde exposure in every 1 kg of adults body was 0.2 mg. This weight of formaldehyde was used as exposure reference of formalin-containing fish, which adjusted to the body weight of mice. The following are the calculations of [FA] (concentration of formaldehyde) that will be exposed in mice weighing 20 g: x 20 g (mice body weight) (1) [FA] = (0.2 mg) × ---------------------------------- = 8 × 10–5 mg 50 000 g (adult body weight) In accordance with the volume range of test materials which will be exposed (0.01 mL to 0.1 mL), then to mice weighing 20 g the volume of the test material which is used as a benchmark was 0.04 mL. Thus, the [FF] (concentration of fish stock solution of formalin) is as the following: 8 × 10–5 mg [FF] = ------------------ = 0.002 mg · mL–1 = 2 mg · L–1 0.04 mL

(2)

The FF final stock solution (2 mg · L–1) prepared by diluting (five times) the FF secondary stock solution. x Fish containing formalin plus chlorophyllin (FFChln) — The FFChln stock solution was made by mixing 100 mL of the primary stock of FF and 200 mL of commercial chlorophyllin from alfalfa plants (Medicago sativa, 3 000 mg · L–1, Synergy WorldWide, Utah, USA) in 1 L volumetric flask and then adding demineralized water to the gauge line. The concentration of chlorophyllin was chosen according to the assumption of molar ratio of formaldehyde and chlorophyllin, i.e. 1 : 2, refers to the reaction of crosslink formation by one molecule of formaldehyde15. The FFChln final stock solution prepared by diluting (five times) the FFChln stock solution. x Chlorophyllin (Chln) — The Chln stock solution was made by inserting 200 mL of commercial chlorophyllin (3 000 mg · L–1) in 1 L volumetric flask and then adding demineralized water to the gauge line. The Chln final stock solution prepared by diluting (five times) the Chln stock solution. These four test substances were repeated exposed to each group of mice (once a day) by force-feeding using a gavage tube. At each selected times of repeated exposed (0 d, 2 d, 14 d, and 62 d), the group of mice were euthanized by cervical dislocation and then dissected to taking the liver organ16,17. 2.3. Tissue fixation and cutting–of the paraffin block Liver specimens were washed with PBS (phosphate buffered saline), inserted into fixative solution (10 % (w/v) formaldehyde) for a day, and then gradually dehydrated in 85 % (w/v) alcohol for 1 h to 2 h, 96 % (w/v) alcohol for 1 h to 2 h, and absolute alcohol for 2 h to 3 h. The specimens were cleared with xylol : absolute alcohol = 1 : 3 for 1 h, 2 : 2 for 1 h, 3 : 1 for 1 h, first pure xylol for 1 h, and second pure xylol for 1 h. Infiltration was done in the oven

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with xylol : paraffin = 1 : 1 (45 °C to 50 °C) for 1 h, first paraffin (65 °C to 70 °C) for 1 h, and second paraffin (65 °C to 70 °C) for 1 h. The specimens were inserted in a paper box, covered with liquid paraffin, and then labeled. Finally, the paraffin block was cut with a rotary microtome (4 μm thickness), and placed on the polysineTM slide (Thermo Scientific, Germany)18. 2.4. Immunohistochemistry detection of HSP70 Immunohistochemical staining was performed on liver sections on slides that were deparaffinized using xylol and rehydrated in a series of alcohol to water. The endogenous peroxidase activity was blocked with 3 % (w/v) H2O2. For detection of HSP70, the slides were incubated overnight with monoclonal anti–heat shock protein 70 (HSP70) clone BRM–22 (Sigma–Aldrich, Germany). Biotinylated secondary antibody, an avidin–biotin complex, and diaminobenzidine chromogene (Universal Dako LSAB®+ Kit Peroxidase, Dako North America, Inc., USA) were applied for visualization of the immunoreaction19,20. Histological expression of HSP70 was assessed on Meyer’s hematoxylin stained sections, and counted with double–blind manner21,22. 2.5. Data Analysis The data of HSP70 expression in the liver cell of mice (dependent variable, in three replicates) were analyzed using two way analysis of variance, after fulfilling normality (Kolmogorov–Smirnov test) and homogeneity (Levene test) requirements. The obtained data could be transformed while it does not meet the normality and or homogeneity test. The Duncan Multiple Range Test (DMRT) was applied to the data that show significant difference. All statistical tests (from normality to post–hoc test, with α = 0.01) were done using SPSS version 1523–25. 3. Result and discussion 3.1. Immunohistochemistry of HSP70 The immunohistochemical staining by anti–HSP70 visualizes cells expressing HSP70 as brown ring–shaped spots with the inner section reflected in higher intensity. The brown ring–shaped spot reflects the HSP70 in the cytoplasm of liver cell, while the brown bold inside reflects the protein in the cell nucleus.

Fig. 1.Immunohistochemical staining of liver cells of mice using anti–HSP70.

Determination of HSP70 using immunohistochemical techniques is based on the concept of antigen–antibody specific binding. The cells expressing HSP70 as antigen will be recognized specifically by anti–HSP70 antibody. This specific bond is amplified by avidin–biotin complex sandwich assay26-28. The series of immunoreaction and staining techniques will differentiate cells expressing HSP70 from other cells.

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3.2. The expression of HSP70 based on treatment and time factor Data of the number of mice liver cells that express HSP70 based on treatment factors are presented in Table 1 and Fig.2A. The number of cells that express HSP70 increases by the treatment with fish flesh containing formalin, and is almost reduced to the control level by additional treatment with chlorophyllin. Table 1. The results of DMRT Test (1 %) on the data of square root transformation of the mean of HSP70–expressing cell based on treatment factor. Type of treatment

N

HSP70–expressing cell ± SE

HSP70–expressing cell ± SE (square root transformation)

Retransformation

Negative control (NC)

12

6.2 ± 0.57

2.447a ± 0.127

5.988a

Fish containing formalin (FF)

11

19.9 ± 3.44

4.228b ± 0.451

17.876b

a

Fish containing formalin plus chlorophyllin (FFChln)

12

8.0 ± 1.07

2.748 ± 0.201

7.551a

Chlorophyllin (Chln)

12

6.4 ± 0.61

2.496a ± 0.130

6.230a

Note: The obtained data meet the homogeneity but does not meet the normality test, thus square root transformation was applied to the data.

Fig. 2.

Bar graph of the data of square root transformation of the mean of HSP70–expressing cell based on: (A) treatment factor (NC for negative control, FF for fish containing formalin, FFChln for fish containing formalin plus chlorophyllin, and Chln for chlorophyllin); and (B) time factor. The mean of HSP70–expressing cell is the average number of liver cells of mice which expressing HSP70 that was counted with double–blind manner in five different fields of view per liver section.

Table 2. The results of DMRT Test (1 %) on the data of square root transformation of the mean of HSP70–expressing cell based on time factor. Duration of repeated exposure (d)

N

HSP70–expressing cell ± SE

HSP70–expressing cell ± SE (square root transformation)

Retransformation

0

12

3.917 ± 0.398

1.947a ± 0.106

3.791a

2

12

14.250 ± 2.588

3.624 ± 0.318

13.133c

14

12

12.250 ± 2.990

3.275bc ± 0.372

10.726bc

62

11

9.915 ± 1.306

c

b

2.968 ± 0.193

8.809b

Note: The obtained data meet the homogeneity but does not meet the normality test, thus square root transformation was applied to the data.

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Data of the number of mice liver cells expressing HSP70 as a function of the time of treatment are presented in Table 2 and Fig.2B. The difference of the number cells that express HSP70 between the control of time (0 d) which lower than repeated exposure groups (2 d, 14 d, 62 d) showed that repeated treatment of test substance significantly influence to enhance HSP70 expression. The expression of HSP70 looks peaked on 2 nd d, a slow decline on 14th d, and continuously decline to 62nd d. 3.3. Formaldehyde–induced apoptosis through mitochondrial and death receptor pathway There have been many reports about the damage caused by exposure of experimental animals and cell cultures to formaldehyde. Formaldehyde can induce DNA–protein crosslinks2,15,29–32. DNA–protein crosslink (DPC) is formed when a protein binds to DNA by covalent bonds. Chemical compounds that induce DPC are quite diverse and a number of different methods have been used for the analysis of DPC, as one form of DNA damage15. The mechanism of DPC induction by formaldehyde include the reaction of these compounds with amino and imino groups of proteins (such as lysine and arginine side chains) or from nucleic acids (such as cytosine) to form a Schiff base (alkaline condensation products of aldehydes and amines) which then reacts with another amino group15,32. The level of crosslinking between DNA and proteins has been used as biomarker of formaldehyde and other DPC inducer agents exposure in mammalian cells33. As mentioned above, DPC is one of the DNA damage induced by formaldehyde exposure. According to Shankar and Srivastava34, the protein p53 (tumor suppressor protein) will be activated in response to DNA damage. This protein directly or indirectly modulates the expression of proteins that control mitochondrial membrane permeability, resulting in the release of mitochondrial proteins such as cytochrome c. Cytochrome c together with Apaf–1 (apoptotic protease activating factor 1) and dATP (nucleotides precursor) then form the apoptosom that activates Caspase–9 (initiator Caspase). In the context of apoptosis (programmed cell death), the Caspase group has been known to participate as a trigger of the cell death program, as a regulatory element in the program, and finally as part of the effector elements of the program itself 35. Caspase activation is a key requirement in the reaction of cells to undergo apoptosis34. In the process of apoptosis much evidence has suggested that caspases are activated in a hierarchical flow (caspases cascade). This cascade consists of caspase initiator (Caspase–8, –9, and –10) and executor (Caspase–3, –6, and –7) together with their inhibitors and activators. Initiator caspase is activated through autoproteolysis induced by binding to specific activators. Each initiator Caspase is specifically activated in response to certain cytotoxic stimuli. For example, Caspase–9 is activated in response to cellular stress such as drugs that are cytotoxic, heat shock, and ionizing radiation. Activation of the initiator caspase multiplies the death signals directly through proteolytic activation of executor caspase. Executor caspase then arrange the dismantling of cellular structures, disrupt cellular metabolism, deactivate cell death inhibitor proteins and activate additional destructive enzymes36. Previous research on the effect of formaldehyde on the activation of caspase been done by Hester et al37. Based on their research it was reported that formaldehyde (40 mL, 400 mM) dropped into the nostrils of male F344 rats may activate Caspase–3, –6, and –7 (executor caspase) through the induction of FasL (Fas ligand) and TNFR (tumor necrosis factor receptor) in the nasal epithelium cells. FasL is a specific ligand for the Fas receptor (transmembrane proteins in the cell membrane), while TNFR a specific receptor of the TNF ligand. The death receptor systems both Fas ligand–receptor or TNF are important signaling pathway in the regulation of apoptosis34,38–41. 3.4. The expression of heat shock protein as stress response Every organism shows a response resembling homeostasis when faced with rapid changes in their environment. One concept which is associated with response to changes in the environment is known as heat shock or stress response. When confronted with an increase in temperature or physiologically relevant stimuli the cells of every living organism respond with a rapid increase in the synthesis of a group of proteins known as heat shock proteins (HSP). The induction of HSP is not only caused by temperature stress but also by other stimuli such as exposure of cells by a variety of metals, amino acid analogues, hypoxia, oxidative stress, nutritional deficiencies, ultraviolet radiation, viral infections, wounds caused by ischaemia–reperfusion, and agent or treatment which leads to reduced ATP levels42.

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HSP70 is a prominent group of stress response proteins. In normal conditions the concentration of HSP is around 10% of the total protein content and increased by about two or three times under stress conditions. In the context of induced apoptosis HSP70 as a form of defense mechanism is synthesized to inhibit the formation of Caspase–9 in mitochondrial pathway and several protein kinases such as ASK1 (apoptosis signal–regulating kinase 1), p38– MAPK (p38 mitogen–activated protein kinase), and JNK (c–Jun N–terminal kinase) in death receptor pathway43,44. The results of this study indicate that the exposure of fish containing formaldehyde can increase the expression of HSP70 up to three times when compared to the control treatment. This fact shows that the consumption of fish containing formaldehyde can induce cellular stress at a high level. Related to the duration of exposure, the results showed that the increase of cellular stress due to consumption of fish containing formalin experienced a peak on the 2nd d after repeated exposure was done, decreased gradually on 14th d, and lasts until the 62nd d. The decline that occurred indicate an adaptive response to the consumption of fish containing formalin, as a result of repeated exposure. The expression ofHSP70 which began to decline on the 2nd d until the end of treatment showed a decrease in the ability of the organism to cope with stress stimulus or no longer able to tolerate stress, in line with the aging process of the organism itself45. 3.5. Chlorophyllin as detoxifier Chlorophyllin is a semi–synthetic form of natural chlorophyll that soluble in water46,47. Many studies have shown that chlorophyllin can be used in detoxifying a wide variety toxins at the molecular level. Chlorophyllin have strong and broad chemoprotective activity. Chlorophyllin significantly inhibit the mutagenic activity of various xenobiotic through mechanisms such as direct antioxidant activity and the formation of complexes with mutagens/carcinogens. Judging from its role as an antioxidant chlorophyllin can reduce damage to mitochondrial membrane lipids and potential oxidative damage48,49. The other mechanisms, chlorophyllin form complexes with mutagens/carcinogens through the strong interaction, which continues to facilitate the excretion of these carcinogens 50–56. Associated with the concept of DPC, chlorophyllin can lock mutagens/carcinogens and prevent these agents to form adducts with DNA46,57–59. The results showed that chlorophyllin supplementation could decrease the high expression of formaldehydeinduced HSP70 equivalent to the control treatment. This fact indicates that chlorophyllin can normalize cellular stress caused by the consumption of fish containing formalin. This study reinforces the previous study12 related to the effect of chlorophyllin supplementation in normalizing the concentration of SGOT and SGPT elevation due to repeated exposure of fish containing formaldehyde. 4. Conclusion The overexpression of HSP70 is closely linked to the suppression process of induced apoptosis. The overexpression of HSP70 is also closely linked to the defense of cells against various stress stimuli (including oxidative stress). The expression of HSP70 that increased in early repeated exposure then slowly decreased suggesting an adaptive response to formalin–containing foodstuff as oxidative stressor. HSP70 expression correlates with the suppression of formaldehyde–induced apoptosis by chlorophyllin, indicating that this substance could be categorized as a detoxifier of formalin–containing foodstuff. Acknowledgements Author would like to thanks to Directorate General of Higher Education, Ministry of National Education, Republic of Indonesia, for supporting this research partially through Dissertation Research Grant with a contract number of 495/SP2H/PP/DP2M/VI/2010.

References 1. Kartikaningsih H. Pengaruh paparan berulang ikan berformalin terhadap kerusakan hati dan ginjal mencit (Mus musculus) sebagai media pembelajaran keamanan pangan [Repeat exposure effect of fish containing formalin against damage of liver and kidney of mice (Mus musculus) as an instructional media for food safety]. Malang: Postgraduate Program, State University of Malang; 2008 [Bahasa Indonesia].

33

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Alfonds Andrew Maramis et al. / Procedia Chemistry 14 (2015) 27 – 35

2. Shaham J, Bomstein Y, Gurvich R, Rashkovsky M, Kaufman Z. DNA–protein crosslinks and p53 protein expression in relation to occupational exposure to formaldehyde. Occupat Environ Med 2003;60:403–409. 3. Schmid O, Speit G. Genotoxic effects induced by formaldehyde in human blood and implications for the interpretation of biomonitoring studies. Mutagenesis 2006;22(1):69–74. 4. Mahdi C. Suplementasi yogurt pada tikus (Rattus norvegicus) yang terpapar formaldehid dalam makanan terhadap aktivitas antioksidan, kerusakan oksidatif, profil, dan karakter protein jaringan hepar [Yoghurt supplementation for rat (Rattus norvegicus) which exposed to foodstuff containing formaldehyde against antioxidant activity, oxydative damage, profile and character of protein of liver tissue]. Malang: University of Brawijaya; 2008 [Bahasa Indonesia]. 5. Corebima AD, Maramis AA. The activation of Caspase–9 in liver cell of mice increases due to repeated exposure of formalin–containing fish. Int J Biol Ecol Environ Scie 2012;1(1):15–20. 6. Maramis AA, Amin M, Sumarno, Corebima AD. The expression of HSP70 in liver cell of mice increases due to repeated exposure of foodstuff containing formalin. Int J Biol Ecol Environ Scie 2012;1(1):8–14. 7. Maramis AA, Amin M, Sumarno, Corebima AD. The effect of repeated exposure of formalin–containing fish against apoptosis of the liver cell of mice. In: Kusumawinahyu WM, Hartanto DP, Firdausi R, Atsomya MF, editors. Proceedings of 2ndBasic Science International Conference. Malang: Brawijaya University; 2012. p. B24–B29. 8. Maramis AA, Amin M, Sumarno, Corebima AD. Pengaruh paparan berulang ikan berformalin terhadap gangguan fungsional hepar mencit [The effect of repeated exposure of formalin–containing fish against liver functional disorder in mice]. In: Sajidan, editor. Proceeding of 7th National Seminar of Biology Education of FKIP. Surakarta: Sebelas Maret University; 2010.p. 447–456 [Bahasa Indonesia]. 9. Maramis AA, Amin M, Sumarno, Corebima AD. The physiology of organs and organism of Mus musculus induced repeatedly with formalin– contaminated fish and chlorophyllin. In: Dewanti–Hariyadi R, Nuraida L, Gitapratiwi D, Immaningsih N, Hariyadi P, editors. Proceedings of International Seminar ‘Current Issues and Challenges in Food Safety: Science–based Approach for Food Safety Management’. Bogor: SEAFAST Center IPB; 2010. p. 329–344. 10. Maramis AA. Fisiopatologi mencit yang terpapar secara berulang dengan ikan berformalin dan klorofilin [Physiopathology of mice that repeatedly exposed with formalin–containing fish and chlorophyllin]. Jurnal Sains 2010;38(2) [Bahasa Indonesia]. 11. Maramis AA. Perilaku mencit yang terpapar secara berulang dengan ikan berformalin dan klorofilin [Behavior of Mice that Repeatedly exposed with formalin–containing fish and chlorophyllin]. In: Nugroho DB, Wibowo NA, Andini S, editors. Proceedings of 9th National Seminar on Science and Science Education. Salatiga: Science and Mathematic Faculty Satya Wacana Christian University; 2013. p. 577–585 [Bahasa Indonesia]. 12. Maramis AA, Amin M, Sumarno, Corebima AD. Chemopreventive effect of chlorophyllin against liver functional disorder in mice repeatedly induced with foodstuff containing formalin. In: Limantara L, Heriyanto, Sadtono E, editors. Proceedings of Natural Pigments Conference for South–East Asia. Malang: Ma Chung Research Center for Photosynthetic Pigments; 2010. p. 60–66. 13. Sprengel K, Eshkind L, Hengstler J, Bockamp E. Improved models for animal research. In: Conn PM, editor. Sourcebook of Models for Biomedical Research. Totowa, NJ: Humana Press; 2008. p. 17–24. 14. Drummond G B. Reporting ethical matters in The Journal of Physiology: standards and advice. J Physiol 2009;587(4):713–719. 15. Barker S, Weinfeld M, Murray D. DNA–protein crosslinks: their induction, repair, and biological consequences. Mutation Research 2004;589:111–135. 16. Lee SH, Kim M, Yoon BW, et al. Targeted Hsp70.1 disruption increases infarction volume after focal cerebral ischemia in mice. Stroke 2001;32:2905–2912. 17. Dong Z, Wolfer DP, Lipp HP, Büeler H. Hsp70 gene transfer by adeno–associated virus inhibits MPTP–induced nigrostriatal degeneration in the mouse model of Parkinson disease. Mol Therapy 2005;11(1):80–88. 18. Riawan W, Kusuma TS, Tarmono. Up–regulasi TNF–R1 dan aktivasi HIF–1a sel epitel pada tubuli ginjal kelinci yang diberikan Pseudomonas sp secara intraureter [Up–regulation of TNF–R1 and HIF–1a activation of epithelial cells on rabbit kidney tubuli which treated with Pseudomonas sp intraureter]. Brawijaya Med J, 2008;24(1):9–14 [Bahasa Indonesia]. 19. Antaliková L, Koubková M, Rozinek J, Jílek, F. Immunocytochemistry of heat shock protein Hsp70 in pig liver after a parasitic invasion. Vet Med Czech 2000;45(1):5–11. 20. Malusecka E, Zborek A, Krzyzowska–Gruca S, Krawczyk Z. Immunohistochemical detection of the inducible heat shock protein Hsp70: A methodological study. J Histochem Cytochem 2006;54(2):183–190. 21. Soini Y, Pääkkö P, Lehto VP. Histopathological evaluation of apoptosis in cancer. Am J Pathol 1998;153(4):1041–1048. 22. Pizem J, Coer A. Detection of apoptosis cells in tumour paraffin section. Radiol Onco 2003;37(4):225–232. 23. Betensky RA, Nutt CL, Batchelor TT, Louis DN. Statistical considerations for immunohistochemistry panel development after gene expression profiling of human cancers. J Mol Diagn 2005;7(2):276–282. 24. SPSS Inc. SPSS base 15.0 user’s guide. Illinois: SPSS Inc; 2006. 25. Todgham AA, Hoaglund EA, Hofmann GE. Is cold the new hot? Elevated ubiquitin–conjugated protein levels in tissues of antarctic fish as evidence for cold–denaturation of proteins in vivo. J Comparat Physiol B: Biochem Syst and Environ Physiol 2007;177(8):857–866. 26. Haugland RP, You WW. Coupling of antibodies with biotin. In: McMahon RJ, editor. Avidin–biotin interactions: methods and applications. Totowa NJ: Humana Press; 2008. p. 13–24. 27. Zwart SR, Lewis BJ. Optimization of detection and quantification of proteins on membranes in very high and very low abundance using avidin and streptavidin. In: McMahon RJ, editor. Avidin–biotin interactions: methods and applications. Totowa NJ: Humana Press; 2008. p. 25–34. 28. Freitag R, Hilbrig F. Use of the avidin (imino) biotin system as a general approach to affinity precipitation. In: McMahon RJ, editor. Avidin– biotin interactions: methods and applications. Totowa NJ: Humana Press; 2008. p. 35–50. 29. Solomon MJ, Varshavsky A. Formaldehyde–mediated DNA–protein crosslinking: a probe for in vivo chromatin structures. Proc Natl Acad Sci 1985;82:6470–6474. 30. Shaham J, Bomstein Y, Meltzer A, Kaufman Z, Palma E, Ribak J. DNA–protein crosslinks, a biomarker of exposure to formaldehyde: in vitro and in vivo studies. Carcinogenesis 1996;17(1):121–125.

Alfonds Andrew Maramis et al. / Procedia Chemistry 14 (2015) 27 – 35 31. Quievryn G, Zhitkovich A. Loss of DNA–protein crosslink from formaldehyde–exposed cells occurs through spontaneous hydrolysis and an active repair process linked to proteosome function. Carcinogenesis 2000;21(8):1573–1580. 32. World Health Organization (WHO). Formaldehyde: concise international chemical assessment document. Geneva: International Programme on Chemical Safety, World Health Organization; 2002. 33. Speit G, Merk O. Evaluation of mutagenic effects of formaldehyde in vitro: detection of crosslinks and mutations in mouse lymphoma cells. Mutagenesis 2002;17(3):183–187. 34. Shankar S, Srivastava RK. Death receptors: mechanisms, biology, and therapeutic potential. In: Srivastava RK, editor. Apoptosis, cell signaling, and human diseases. Totowa NJ: Humana Press; 2007. p. 219–261. 35. Slee EA, Adrain C, Martin SJ. Serial killers: ordering caspase activation events in apoptosis. Cell Death and Differentiation 1999;6:1067– 1074. 36. Creagh EM, Adrain C, Martin SJ. Caspase detection and analysis. In: Hughes D, Mehmet H, editors. Cell proliferation &apoptosis. Oxford: BIOS Scientific Publishers Ltd; 2005. p. 242–259. 37. Hester SD, Barry WT, Zou F, Wolf DC. Transcriptomic analysis of F344 rat nasal epithelium suggests that the lack of carcinogenic response to glutaraldehyde is due to its greater toxicity compared to formaldehyde. Toxicologic Pathology 2005;33:415–424. 38. Karna P, Yang L. Apoptotic signaling pathway and resistance to apoptosis in breast cancer stem cells. In: Chen GG, Lai PBS, editors. Apoptosis in carcinogenesis and chemotherapy. Hong Kong: Springer Science+Business Media B.V.; 2009. p. 1–25. 39. Karam JA, Hsieh JT. Anti–cancer strategy of transitional cell carcinoma of bladder based on induction of different types of programmed cell deaths. In: Chen GG, Lai PBS, editors. Apoptosis in carcinogenesis and chemotherapy. Hong Kong: Springer Science+Business Media B.V.; 2009. p. 25–50. 40. Li Y, Martin RCG. Apoptosis in carcinogenesis and chemotherapy: esophageal cancer. In: Chen GG, Lai PBS, editors. Apoptosis in carcinogenesis and chemotherapy. Hong Kong: Springer Science+Business Media B.V.; 2009. p. 127–156. 41. Liu ZM, Chen GG. Carcinogenesis and therapeutic strategies for thyroid cancer. In: Chen GG, Lai PBS, editors. Apoptosis in carcinogenesis and chemotherapy. Hong Kong: Springer Science+Business Media B.V.; 2009. p. 347–374. 42. Vallespi MG, Garcia I. Heat–shock proteins in inflammation and cancer. Biotecnologia Aplicada 2008;25:208–215. 43. Pockley AG. Heat schock proteins as regulators of the immune response [Internet]. Accesed on September 17th, 2009 from http://image.thelancet.com/extrs/02art9148web.pdf. 2003. 44. Fiers W, Beyaert R, Declercq W, Vandenabeele P. More than one way to die: apoptosis, necrosis and reactive oxygen damage.Oncogene 1999;18:7719–7730. 45. Hall DM, Xu L, Drake VJ, et al. Aging reduces adaptives capacity and stress protein expression in the liver after heat stress. Journal of Applied Physiology 2000;89:749–759. 46. Life Extension Foundation (LEF). Chlorophyllin and cancer prevention [Internet]. Accesed on December 21st, 2008 from http://www.lef.org. 2003. 47. Limantara L. Daya penyembuhan klorofil [Chlorophyll’s healing power]. Malang: Ma Chung Press; 2009 [Bahasa Indonesia]. 48. Kamat JP, Boloor KK, Devasagayam TP. Chlorophyllin as an effective antioxidant against membrane damage in vitro and ex vivo. Biochim Biophys Acta 2000;1487(2–3):113–127. 49. Boloor KK, Kamat JP, Devasagayam TP. Chlorophyllin as a protector of mitochondrial membranes against gamma–radiation and photosensitization. Toxicology 2000;155(1–3):63–71. 50. Ong T, Whong WZ, Stewart JD, Brockman HE. Chlorophyllyn: a potent antimutagen against environmental and dietary complex mixtures. Mutat Res 1986;173(2):111–115. 51. Ong T, Whong WZ, Stewart JD, Brockman HE. Comparative antimutagenicity of 5 compound against 5 mutagenic complex mixtures in Salmonella typhimurium strain TA98. Mutat Res 1989;222(1):19–25. 52. Tachino N, Guo D, Dashwood WM, Yamane S, Larsen R, Dashwood R. Mechanisms of the in vitro antimutagenic action of chlorophyllin against benzo[a]pyrene: studies of enzyme inhibition, molecular complex formation and degradation of the ultimate carcinogen. Mutat Res 1994;308(2):191–203. 53. Dashwood RH, Yamane S, Larsen R. Study of the forces of stabilizing complexes between chlorophylls and heterocyclic amine mutagens. Environ Mol Mutagen 1996;27(3):211–218. 54. Dashwood RH, Negishi T, Hayatsu H, Breinholt V, Hendricks J, Bailey G. Chemopreventive properties of chlorophylls towards aflatoxin B1: a review of the antimutagenicity and anticarcinogenicity data in rainbow trout. Mutat Res 1998;399(2):245–253. 55. Egner PA, Stansbury KH, Snyder EP, Rogers ME, Hintz PA, Kensler TW. Identification and characterization of chlorine(4)ethyl ester in sera of individuals participating in the chlorophyllin chemoprevention trial. Chem Res Toxicol 2000;13(9):900–906. 56. Fahey JW, Stephenson KK, Dinkova–Kostova AT, Egner PA, Kensler TW, Talalay P. Chlorophyll, chlorophyllin and related tetrapyrroles are significant inducers of mammalian phase 2 cytoprotective genes. Carcinogenesis 2005;26(7):1247–1255. 57. Guo D, Schut HA, Davis CD, Snyderwine EG, Bailey GS, Dashwood RH. Protection by chlorophyllin and indole–3–carbinol against 2– amino–1–methyl–6–phenylimidazo[4,5–b]pyridine (PhlP)–induced DNA adducts and colonic abberant crypts in the F344 rat. Carcinogenesis 1995;16(12):2931–2937. 58. Harttig U, Bailey GS. Chemoprotection by natural chlorophylls in vivo: inhibition of dibenzo[a,l]pyrene–DNA adducts in rainbow trout liver. Carcinogenesis 1998;19(7):1323–1326. 59. Egner PA, Jin–Bing W, Yuan–Rong Z, et al. Chlorophyllin intervention reduces aflatoxin–DNA adducts in individuals at high risk for liver cancer. PNAS 2001;98(25):14601–14606.

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