Histomorphometric analysis of the lung of Swiss mice treated with a fibrinolytic protease

Abstract

Fibrinolytic enzymes are considered promising alternative in the treatment of cardiovascular diseases by preventing fibrin clots. A protease from Mucor subtilissimus UCP 1262 was obtained by solid state fermentation and purified by ion exchange chromatography. The purified extract was administered at an acute dose of 2000 mg/mL to evaluate its toxic effects to the lungs of mice. After 14 days of treatment, a histomorphometric study was performed by the type 1 and 2 pneumocyte count and the evaluation of the lung area. As result, the experimental group showed a significant decrease of type 2 pneumocyte and although a decrease in the alveolar area was observed in relation to the control group, no significant pulmonary toxicity, emphysema, and fibrosis characteristics were detected. The in vitro tests suggest possible clinical applications for the enzyme.

Key words Fibrinolytic protease; thrombosis; Mucor; pneumocyte; thrombolytic agent

INTRODUCTION

Fibrinolytic enzymes have been used in the treatment of cardiovascular diseases because their capacity to prevent blood clots. On the other hand, other fibrinolytic agents such as urokinase, streptokinase, plasminogen activators (t-PA) and anticoagulants have been reported for risk of bleeding (Choi et al. 2013, Zapata-Wainberg et al. 2016).

Filamentous fungi are excellent sources of fibrinolytic enzymes (Germano et al. 2003, Liu et al. 2017, Deng et al. 2018) and the fungus Mucor subtilissimus UCP 1262 has been extensile researched to produce proteases have reported a promising non-toxic protease (Nascimento et al. 2015, 2016, 2017, 2020, Da Silva et al. 2019) for clinical application with potential to cleave fibrin and fibrinogen, but not through plasminogen activators. Some drugs induce lung diseases such as changes in lung immune cells (Kullberg et al. 2020) causing inflammatory reactions and apoptosis pathways (Zhang et al. 2020), inducing pulmonary fibrosis (Budas et al. 2018). Lung diseases are caused by several routes of administration: intravenous, oral and even inhalation (Bevan et al. 2018).

This study aims to evaluate the acute in vivo toxicity of the fibrinolytic protease from Mucor subtilissimus UCP 1262 in the lungs of Swiss mice based on histomorphometric study of lung tissue.

MATERIALS AND METHODS

Obtaining fibrinolytic protease

The filamentous fungus Mucor subtilissimus UCP 1262 was cultivated in Czapek medium and maintained at 30ºC. The inoculum preparation, fibrinolytic enzyme production by solid state fermentation, and enzymatic extraction, precipitation and purification were performed according to Nascimento et al. (2015).

Animals

Swiss mice (Mus musculus) weighting between 38 and 50 g were used, they were obtained from the Keiso-Asami Immunopathology Laboratory (LIKA-UFPE, Recife, Brazil) bioterium and kept in the bioterium of the Federal University of Pernambuco (Academic Center of Vitoria, Brazil) in appropriate polypropylene boxes at 21 ± 1ºC under a photoperiod of 12L:12D with access to food (labine), water and relative humidity of 50 ± 5%. The Animal Ethics Committee of the Universidade Federal de Pernambuco has approved all experimental procedures (process 0058/2018).

Lung evaluation of acute exposure to fibrinolytic enzyme

The fibrinolytic enzyme of M. subtilissimus was evaluated following the OECD 423 guideline known as “Acute Toxic Class Method” (OECD 423). Three animals per group were used in duplicate. One group was subjected to the oral application of 2000 mg/kg of water-diluted fibrinolytic protease, and the other group received only water (control). The animals fasted for 3-4 hours before administration of the enzyme. The food was suspended for another 1-2 hours and they were observed for 14 consecutive days. After this period the lung of each animal was removed by means of a thoracotomy and submitted to histological procedures

Histomorphometric analysis

Each lung was cleaved and dipped in formalin solution (10% v/v) for 48h. The fragments were dehydrated into ethyl alcohol in increasing concentrations, diaphanized by xylol, impregnated and embedded in paraffin. The blocks were cut into a microtome adjusted to 4 µm. Thus, the sections were maintained at 60ºC for 24 hours and submitted to the Hematoxylin-Eosin (H.E.) staining technique. The histological images of these slides were captured by a digital camera (Moticam 3000) coupled to the optical microscope (Nikon E-200), under fixed focus and field clarity, obtaining 20 fields per slide with 400X magnification to analyze the amount of type 1 and 2 pneumocytes, and 20 fields per slide in the 100X objective to analyze the area and perimeter of the alveoli. The photomicrographs were evaluated using ImageJ software version 1.44 (Research Services Branch, U.S. National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

A normality test using the Smirnov Kolmogorov program was developed, and then it was verified a non-normal distribution. From this, a non-parametric test (U of Mann-Whitney) was the applied. For this the GraphPad Prism 5.0 program was used and the data were expressed as mean ± SD, p <0.05. compared to the control.

RESULTS AND DISCUSSION

Lung diseases induced by drugs consist of an increasing cause of morbidity (Costabel 2000, Damas et al. 2006). Pacients treated with Amiodarone, a class III antiarrhythmic, showed multiple nodular lesions in the pathological examination (González Gordaliza et al. 2006). At the same time, many medications cause severe acute lung toxicity by presenting pulmonary infiltrates, eosinophilia, and rash (Fernández Álvarez et al. 1994). Free radicals from oxygen and various cytokines, such as methotrexate (MTX), produce lung toxicity and develop various forms of arthritis and other rheumatic conditions (Kurt et al. 2015). Thalidomide induces interstitial and alveolar alterations and is indicative of partial respiratory failure (Carrión Valero & Bertomeu González 2002). The obtention of non-toxic drugs is a prerequisit for clinical applications and justify the potential of the purified fibrinolytic enzyme from Mucor subtilissimus. The present study aims to demonstrate the action of that protease on the lung tissue structure based on its absence of cytotoxicity for tumor cells, kidney, spleen, and liver (Da Silva et al. 2019).

The macroscopic examination of the lung revealed no changes in the color or integrity of the tissue in any of the groups studied (Figure 1a, b, c, d). The lung parenchyma showed no changes in alveolar, septal or bronchiolar architecture. The two pneumocytes types [alveolar epithelial cells I (CEA1) and II (CEA 2)] have been targeted for infections caused by SARS-CoV-2, when a single tape RNA virus has as receptor the angiotensin 2 converting enzyme (ACE2), on the cell surface of the host. The ACE2 is a type I membrane protein expressed in cells in kidneys, heart, TGI, blood vessels and CEA 2 cells which are particularly prone to viral infections (Pascoal et al. 2020, Zhao et al. 2020, Andersen et al. 2020). Figure 1 demonstrates the absence of morphological changes in alveolar epithelial cells.

Figure 1
Photomicrographs of lung tissue sections of mice in the different groups studied (H&E). a and c= Preserved lung parenchyma (circle); b and d= Preserved alveolar sac (star), Pneumocyte type 1 (double arrow), Pneumocyte type 2 (arrow). HE staining with magnification of 10X (a and c) and 40X (b and d). Scale bar = 1 μm.

In the histomorphometric analysis, the lungs of mice exposed to an acute dose of fibrinolytic protease were measured according to the amount of type 1 and 2 pneumocytes. Table I shows that there was a significant decrease in the numner of CEA 2 cells, what is unusual (Castelo Branco et al. 2004). In addition, the alveolar area also showed a significant decrease (Table I). Absence of pulmonary emphysema could be evidenced by the fact that no alveolar changes [dilatation of the air spaces or destruction of the alveolar wall (Monteiro et al. 2004)] were observed. Our results suggest application of the fibrinolytic enzyme in clinical tests against pulmonary diseases.

Table I
Number of type and type 2 pneumocytes in the lungs of mice treated with the fibrinolytic enzyme produced by Mucor subtilissimus UCP 1262 (2000mg/mL). The results are expressed as mean ± SD. Statistical differences were determined by the Mann-Whitney U-test. CN: negative control; ENZ: fibrinolytic enzyme produced by Mucor subtilissimus. *P<0.05 vs Control.

	Pneumocyte type 1 (n of cells/ µm2)	Pneumocyte type 2 (n of cells/ µm2)	Alveolar Area (n of cells/µm2)
CN	20,38 ± 6,79	29,04 ± 9,58	580,30 ± 230,41
ENZ	17,74 ± 8,44	23,25 ± 11,55*	492,40 ± 242,72 *

CONCLUSIONS

The fibrinolytic protease produced by Mucor susbtilissimus UCP 1262 showed potential to reduce pulmonary toxicity, not developing cell characteristics of emphysema or fibrosis, what is indicative of its applicability in clinical tests.

ACKNOWLEDGMENTS

We are grateful to the following bodies for the grants awarded: CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior): Senior Internship (edital nº16/2016, case number: 88881.119817/2016-01 and the doctoral scholarship; FACEPE (Fundação de Amparo à Ciência e Tecnologia de Pernambuco): Researcher’s scholarship grant: BFP-0079-5.05/20 CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) process: 427153/2016-6 and we also thank the reviewers for their valuable comments and suggestions as these helped us to improve the manuscript.

REFERENCES

• ANDERSEN KG, RAMBAUT A, LIPKIN WI, HOLMES EC & GARRY RF. 2020. The proximal origin of SARS-CoV-2. Nat Med 26: 450-452. https://doi.org/10.1038/s41591-020-0820-9.
  » https://doi.org/10.1038/s41591-020-0820-9
• BEVAN RJ, KREILING R, LEVY LS & WARHEIT DB. 2018. Toxicity testing of poorly soluble particles, lung overload and lung cancer. Regul Toxicol Pharmacol 100: 80-91. https://doi.org/10.1016/j.yrtph.2018.10.006.
  » https://doi.org/10.1016/j.yrtph.2018.10.006
• BUDAS GR ET AL. 2018. ASK1 inhibition halts disease progression in preclinical models of pulmonary arterial hypertension. Am J Respir Crit Care Med 197: 373-385. https://doi.org/10.1164/rccm.201703-0502OC.
  » https://doi.org/10.1164/rccm.201703-0502OC
• CARRIÓN VALERO F & BERTOMEU GONZÁLEZ V. 2002. [Lung toxicity due to thalidomide]. Arch Bronconeumol 38: 492-494. https://doi.org/10.1016/S0300-2896(02)75272-1.
  » https://doi.org/10.1016/S0300-2896(02)75272-1
• CASTELO BRANCO NAA, MONTEIRO E, SILVA ACE, DOS SANTOS JM, REIS FERREIRA JM & ALVES-PEREIRA M. 2004. The lung parenchyma in low frequency noise exposed wistar rats. Rev Port Pneumol 10: 77-85. https://doi.org/10.1016/S0873-2159(15)30558-4.
  » https://doi.org/10.1016/S0873-2159(15)30558-4
• CHOI JH, SAPKOTA K, PARK SE, KIM S & KIM SJ. 2013. Thrombolytic, anticoagulant and antiplatelet activities of codiase, a bi-functional fibrinolytic enzyme from Codium fragile. Biochimie 95: 1266-1277. https://doi.org/10.1016/j.biochi.2013.01.023.
  » https://doi.org/10.1016/j.biochi.2013.01.023
• COSTABEL U. 2000. Doença pulmonar induzida pelas radiações ou pelos fármacos citostáticos. Rev Port Pneumol 6: 141-144. https://doi.org/10.1016/s0873-2159(15)30878-3.
  » https://doi.org/10.1016/s0873-2159(15)30878-3
• DA SILVA MM ET AL. 2019. Effect of acute exposure in swiss mice (Mus musculus) to a fibrinolytic protease produced by Mucor subtilissimus UCP 1262: An histomorphometric, genotoxic and cytological approach. Regul Toxicol Pharmacol 103: 282-291. https://doi.org/10.1016/j.yrtph.2019.02.009.
  » https://doi.org/10.1016/j.yrtph.2019.02.009
• DAMAS C, OLIVEIRA A & MORAIS A. 2006. Lung toxicity induced by rapamycin. Rev Port Pneumol 12: 715-724. https://doi.org/10.1016/S0873-2159(15)30463-3.
  » https://doi.org/10.1016/S0873-2159(15)30463-3
• DENG Y, LIU X, KATROLIA P, KOPPARAPU NK & ZHENG X. 2018. A dual-function chymotrypsin-like serine protease with plasminogen activation and fibrinolytic activities from the GRAS fungus, Neurospora sitophila. Int J Biol Macromol 109: 1338-1343. https://doi.org/10.1016/j.ijbiomac.2017.11.142.
  » https://doi.org/10.1016/j.ijbiomac.2017.11.142
• FERNANDEZ ALVAREZ R, GULLON BLANCO JA, RIESGO ALONSO C, MOLINOS MARTIN L & MARTINEZ GONZALEZ-RIO J. 1994. Acute lung toxicity induced by carbamazepine: A case report. Arch Bronconeumol 30: 471-472. https://doi.org/10.1016/S0300-2896(15)31023-1.
  » https://doi.org/10.1016/S0300-2896(15)31023-1
• GERMANO S, PANDEY A, OSAKU CA, ROCHA SN & SOCCOL CR. 2003. Characterization and stability of proteases from Penicillium sp. produced by solid-state fermentation. Enzyme Microb Technol 32: 246-251. https://doi.org/10.1016/S0141-0229(02)00283-1.
  » https://doi.org/10.1016/S0141-0229(02)00283-1
• GONZÁLEZ GORDALIZA MC, VICENTE BÁRTULOS A, SÁNCHEZ CORRAL JÁ & BERNAL MORELL E. 2006. Patrón alveolar nodular como forma de presentación de la toxicidad pulmonar por amiodarona. Radiologia 48: 99-102. https://doi.org/10.1016/S0033-8338(06)73135-6.
  » https://doi.org/10.1016/S0033-8338(06)73135-6
• KULLBERG S, RIVERA NV, ABO AL HAYJA M, GRUNEWALD J & EKLUND A. 2020. Changes in lung immune cells related to clinical outcome during treatment with infliximab for sarcoidosis. Clin Exp Immunol 0-3. https://doi.org/10.1111/cei.13438
  » https://doi.org/10.1111/cei.13438
• KURT A, TUMKAYA L, TURUT H, CURE MC, CURE E, KALKAN Y, SEHITOGLU I & ACIPAYAM A. 2015. Efectos protectores de infliximab sobre el daño pulmonar inducido por metotrexato. Arch Bronconeumol 51: 551-557. https://doi.org/10.1016/j.arbres.2015.03.018.
  » https://doi.org/10.1016/j.arbres.2015.03.018
• LIU X, KOPPARAPU N, KUMAR LI Y, DENG Y & ZHENG X. 2017. Biochemical characterization of a novel fibrinolytic enzyme from Cordyceps militaris. Int J Biol Macromol 94: 793-801. https://doi.org/10.1016/j.ijbiomac.2016.09.048.
  » https://doi.org/10.1016/j.ijbiomac.2016.09.048
• MONTEIRO R, JATENE FB, PAZETTI R, CORREIA AT, MANOEL LA, BERNARDO WM, RIVERO DHRF & OLIVEIRA SA DE. 2004. Avaliação das alterações morfológicas cardíacas secundárias ao enfisema pulmonar: estudo experimental em ratos. Rev Bras Cir Cardiovasc 19: 341-347. https://doi.org/10.1590/s0102-76382004000400003.
  » https://doi.org/10.1590/s0102-76382004000400003
• NASCIMENTO TP, CONIFF AES, MOURA JAS, BATISTA JMS, COSTA RMPB, PORTO CS, CAMPOS-TAKAKI GM, PORTO TS & PORTO ALF. 2020. Protease from Mucor subtilissimus UCP 1262: Evaluation of several specific protease activities and purification of a fibrinolytic enzyme. An Acad Bras Cienc 92: 1-12.
• NASCIMENTO TP, SALES AE, PORTO CS, BRANDÃO RMP, CAMPOS-TAKAKI GM, TEIXEIRA JAC, PORTO TS, PORTO ALF & CONVERTI A. 2016. Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate). J Chromatogr B Biomed Appl 1025: 16-24.
• NASCIMENTO TP, SALES AE, PORTO TS, COSTA RMPB, BREYDO L, UVERSKY VN, PORTO ALF & CONVERTI A. 2017. Purification, biochemical, and structural characterization of a novel fibrinolytic enzyme from Mucor subtilissimus UCP 1262. Bioprocess Biosyst Eng 40: 1209-1219.
• NASCIMENTO TP, SALES AE, PORTO CS, MARCOS R, BRANDÃO P, MARIA G, TAKAKI C, ANTÔNIO J, TEIXEIRA C, PORTO TS & PORTO ALF. 2015. Production and Characterization of New Fibrinolytic Protease from Mucor subtillissimus UCP 1262 in Solid-State Fermentation. Adv Enzym Res 3: 81-91. https://doi.org/10.4236/aer.2015.33009.
  » https://doi.org/10.4236/aer.2015.33009
• OECD. 423. 2001. O.G. for the T. of Chemicals, Acute Oral Toxicity, Acute Toxic Class Method. OECD Guideline for Testing of Chemicals, p. 1-14. doi: 10.1787/9789264070943-en.
• PASCOAL DB, CARVALHO ACS, MATA LELF, LOPES TP, LOPES LP & CRUZ CM. 2019. Sífilis em privados de liberdade em uma unidade prisional no interior de Rondônia. Braz J Hea Rev 2(2): 2195-2205. https://doi.org/10.34119/bjhrv3n2-138.
  » https://doi.org/10.34119/bjhrv3n2-138
• ZAPATA-WAINBERG G ET AL. 2016. Prognostic factors and analysis of mortality due to brain haemorrhages associated with vitamin K antagonist oral anticoagulants. Results from the TAC registry. Neurologia 33(7): 419-426. https://doi.org/10.1016/j.nrl.2016.07.005.
  » https://doi.org/10.1016/j.nrl.2016.07.005
• ZHAO Y, ZHAO Z, WANG Y, ZHOU Y, MA Y & ZUO W. 2020 Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan 2019-nCov. AJRCCM 202(5): 756-759. https://doi.org/10.1101/2020.01.26.919985.
  » https://doi.org/10.1101/2020.01.26.919985
• ZHANG X, GU W, MA Z, LIU Y, RU H, ZHOU J, ZANG Y, XU ZP & QIAN G. 2020. Short-term exposure to ZnO/MCB persistent free radical particles causes mouse lung lesions via inflammatory reactions and apoptosis pathways. Environ Pollut 261: 114039. https://doi.org/10.1016/j.envpol.2020.114039.
  » https://doi.org/10.1016/j.envpol.2020.114039

Publication Dates

• Publication in this collection
  09 Sept 2022
• Date of issue
  2022

History

• Received
  19 Feb 2020
• Accepted
  09 Sept 2020