We studied the effect of heavy metal cations: Fe 2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+ on the activity ofcathepsin D in human aorta homogenate and blood serum. The concentration of cations was 1 mmol/l. Hemoglobinwas the cathepsin D substrate. The activity of cathepsin D was determined at pH 3.5. Only Hg2+ cationsinhibit the activity of cathepsin D. Cations Hg2+ damage lysosomes and release cathepsin D from these organelles.We studied the effect of heavy metal cations: Fe 2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+ on the activity ofcathepsin D in human aorta homogenate and blood serum. The concentration of cations was 1 mmol/l. Hemoglobinwas the cathepsin D substrate. The activity of cathepsin D was determined at pH 3.5. Only Hg2+ cationsinhibit the activity of cathepsin D. Cations Hg2+ damage lysosomes and release cathepsin D from these organelles

Gacko, Marek

Grzegorczyk, Ewa

Karczewski, Jan K.

Karwowska, Alicja

Łapiński, Radosław

Żurawska, Joanna

Folia Histochemica et Cytobiologica

English

Alicja Karwowska

Radosław Łapiński

Marek Gacko

Ewa Grzegorczyk

Joanna Żurawska

Jan K. Karczewski

Crossref

Effect of heavy metal cations on the activity of cathepsin D (in vitro study)

Via Medica Journals

FOLIA HISTOCHEMICAET CYTOBIOLOGICAVol. 50, No. 3, 2012pp. 432–435©Polish Society for Histochemistry and CytochemistryFolia Histochem Cytobiol. 201210.5603/FHC.2012.0059www.fhc.viamedica.plORIGINAL STUDYCorrespondence address: A. Karwowska,Department of Hygiene and Epidemiology,Medical University of Bialystok,Mickiewicza Str. 2c, 15–089 Białystok, Poland;tel.: + 48 85 748 55 60;e-mail: alicja.karwowska@umb.edu.plEffect of heavy metal cations on the activity ofcathepsin D (in vitro study)Alicja Karwowska1, Radosław Łapiński2, Marek Gacko2, Ewa Grzegorczyk1,Joanna Żurawska2, Jan K. Karczewski11Department of Hygiene and Epidemiology, Medical University of Bialystok, Poland2Department of Vascular Surgery and Transplantation, Medical University of Bialystok, PolandAbstract: We studied the effect of heavy metal cations: Fe 2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+ on the activity ofcathepsin D in human aorta homogenate and blood serum. The concentration of cations was 1 mmol/l. Hemo-globin was the cathepsin D substrate. The activity of cathepsin D was determined at pH 3.5. Only Hg2+ cationsinhibit the activity of cathepsin D. Cations Hg2+ damage lysosomes and release cathepsin D from these or-ganelles. (Folia Histochemica et Cytobiologica 2012, Vol. 50, No. 3, 432–435)Key words: cathepsin D, lysosomes, heavy metalsIntroductionCations, Fe2+, Cu2+ and Zn2+ are natural componentsof the organism, while Cd2+, Hg2+ and Pb2+ can onlybe found in a state of intoxication. Whichever path-way heavy metals take to reach the body, they passthrough the blood and come into contact with plas-ma proteins, morphotic components and vascularendothelial cells [1–3]. In arterial disorders, changesin the content of these metal cations have been ob-served both in plasma and in vascular wall lesions[4–10]. Agarwal et al. found that high levels of serumcadmium are associated with cardiovascular and cere-brovascular disease [11]. Poręba et al. observed thatexposure to lead can be associated with increasedblood pressure and accelerated progression of ath-erosclerosis [12]. Moreover, they found that higherblood concentrations of lead and cadmium are inde-pendent risk factors for the incidence of arterial hy-pertension in subjects chronically exposed to heavymetals [13, 14]. Other researchers have claimed thatmercury can also be a risk factor for hypertension andatherosclerosis [15]. In hypertension, atherosclerosisand aneurysm, changes in the activities of lysosomalproteolytic enzymes, e.g. cathepsin D, occur in bloodplasma and arterial walls [16–20].The study objective was to determine the effect ofheavy metal cations of variable ionic radius (Fe 2+,Cu2+, Zn2+, Cd2+, Hg2+, Pb2+) on the activities ofcathepsin D in human aorta homogenate and bloodserum, and on their release from liver lysosomes.Material and methodsHemoglobin, (Difco Laboratories, USA); Folin-Ciocalteureagent, (Merck, Germany); Bradford reagent and trichlo-racetate acid, (Sigma, USA); biuret reagent and metal cat-ions (chlorides): Fe 2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+, (POCh,Gliwice, Poland).The whole abdominal aorta homogenate from ten or-gan donors was prepared according to the method describedpreviously [21]. Serum was obtained from ulnar vein bloodcollected from ten healthy subjects. The blood was incubat-ed for 1 h at 37°C and centrifuged (1,500 × g, 30 min., 2°C).Metal cations solutions (0.05 ml) at a concentration10.0 mmol/l (in control 0.15 mol/l KCl for homogenate and0.15 mol/l NaCl for serum) was added to 0.35 ml homoge-nate and 0.35 ml serum, pH 7.5. Following a 30-min preincu-433Effect of heavy metal cations©Polish Society for Histochemistry and CytochemistryFolia Histochem Cytobiol. 201210.5603/FHC.2012.0059www.fhc.viamedica.plbation at 37°C, the samples were adjusted to pH 3.5. ThepH 3.5 samples were supplied with 0.1ml of 6% hemoglo-bin (pH 3.5) and incubated at 37°C for 6 hours (both homo-genate and serum). The reaction was discontinued by add-ing 0.1 ml of 10% trichloracetic acid. Precipitated samplesat time 0 were treated as control. In the supernatant ob-tained through centrifugation, the amount of released ty-rosine was determined [22].Lysosomes-containing homogenate of six rabbit liverswas prepared following the method described previously[23]. A 0.15 ml 8.0 mmol/l cation solution (in control 0.15mol/l KCl), pH 7.5, was added to 1.05 ml of this homoge-nate (pH 7.5), and incubated for 30 min. at 37°C. When pH5.0 was obtained, the samples were centrifuged (1,500 × g,30 min., 2°C). The obtained cytosol was adjusted to pH 3.5,and the activities of cathepsin D were determined as de-scribed above. Their activities were also investigated inwhole liver homogenate [21].The activity of cathepsin D was expressed as the level ofreleased tyrosine (nmol) per one gram of tissue and perhour in homogenate and cytosol, and per milliliter and perhour in serum.The Bradford method was used to determine protein inhomogenate and cytosol [24]. Serum protein was determinedusing the biuretic method [25].ResultsOnly Hg2+ cations inhibited the activities of cathepsinD in the aorta homogenate (Table 1). The mean proteincontent in the aorta homogenate was 12.7 mg/g tissue.Hg2+ cations also inhibited the activity of cathep-sin D in blood serum (Table 2). The mean serum pro-tein was 72.4 mg/ml.The Hg2+ cation released the highest amounts ofcathepsin D from hepatocyte lysosomes (Table 3). Therelease of this cathepsin was accompanied by a slightrelease of protein.Table 3. Influence of heavy metals cations on the release of cathepsin D from lysosomes of liverCation [1 mmol/l] Cathepsin D Protein [mg/g], (%)Tyr [nmol/g/h] Release (%) [mg/g] Release (%)Fe2+ 86.6 ± 8.2 12.7 2.4 ± 0.2 6.3Cu2+ 89.5 ± 8.4 13.3 2.5 ± 0.2 6.5Zn2+ 84.2 ± 8.3 12.5 2.6 ± 0.3 6.9Cd2+ 87.6 ± 7.9 12.9 2.5 ± 0.3 6.7Hg2+ 98.8 ± 8.9* 14.9 2.2 ± 0.2 5.9Pb2+ 77.7 ± 8.0 11.6 2.7 ± 0.2 7.1KCl, 0.15 mol/l (control) 80.9 ± 7.6 12.0 2.7 ± 0.3 7.1Whole homogenate 674.2 ± 65.8 100.0 38.0 ± 4.1 100.0*p < 0.05 compared to the controlTable 1. Influence of heavy metals cations on activity ofaorta homogenate cathepsin DCation [1 mmol/l] Cathepsin DTyr [nmol/g/h] Inhibition (%)Fe2+ 709.2 ± 70.2 3.9Cu2+ 710.5 ± 71.8 3.8Zn2+ 724.3 ± 72.3 1.8Cd2+ 715.2 ± 72.3 2.9Hg2+ 629.8 ± 69.3* 14.6Pb2+ 728.6 ± 71.0 1.2KCl, 0.15 mol/l (control) 737.2 ± 73.0 0.0*p < 0.05 compared to the controlTable 2. Influence of heavy metals cations on activity ofserum cathepsin DCation [1 mmol/l] Cathepsin DTyr [nmol/g/h] Inhibition (%)Fe2+ 47.3 ± 4.8 8.9Cu2+ 48.5 ± 4.9 6.5Zn2+ 51.7 ± 5.4 0.4Cd2+ 50.2 ± 5.3 3.3Hg2+ 37.7 ± 3.9* 27.4Pb2+ 51.0 ± 5.6 1.7NaCl, 0.15 mol/l (control) 51.9 ± 5.2 0.0*p < 0.05 compared to the controlDiscussionDivalent metal cations bind to the carboxyl, amino,disulphide and thiol groups of proteins [26–29]. Withcarboxyl groups, they form salt-type bonds, with ami-434 A Karwowska et al.©Polish Society for Histochemistry and CytochemistryFolia Histochem Cytobiol. 201210.5603/FHC.2012.0059www.fhc.viamedica.plno and disulphide groups they form complex bonds,and with thiol groups they form sulphides.Binding of metal cations to carboxyl groups of theAsp33 and Asp231 residues of the catalytic site ofcathepsin D plays a major role in the inhibition of itsactivity [30]. This refers mainly to the mercuric cat-ion, which shows strong affinity for carboxyl groups.The affinity of metal cations for proteins dependson the dimension of their ionic radius and standardpotential [31]. The Hg2+ cation, with the ionic radiusof 102 pm and Hg2+/Hg standard potential of +0.851 V,exhibits the strongest inhibitory effect on cathepsin D[32]. Cations with a greater or smaller ionic radiusand higher and lower standard potential do not in-hibit cathepsin D activity.Increased permeability of the lysosomal mem-branes is caused by damage to the structure of mem-brane lipids and proteins directly by metal cations andreactive oxygen species formed through the action ofthese cations [3, 33–39]. This leads to cathepsin re-lease from lysosomes. However, the activity of cathe-psin D secreted to cytosole is reduced by the part in-activated by the respective cations.Specific binding proteins (metallothionein, trans-ferrin, ferratin, ceruloplasmin and others) prevent thetoxic action of heavy metal cations [40–42]. The met-al binding exogenous compounds include ethyleno-diaminotetraacetate acid, diethylenotriaminopenta-acetic acid, N-acetylocystein, penicillamine and def-eroxamine [43–45]. The concentrations of heavy metalcations used in the current experiment resembledthose in the blood and tissues of intoxicated subjects[46]. Their in vitro effects do not differ from the onesobserved in the acute intoxication of laboratory ani-mals, in which cathepsin D activity was reduced bothin blood plasma and in organs [34, 47, 48]. However,no such relationship was found in the wall of athero-sclerotic or aneurysmatic arteries [6, 17, 49, 50].AcknowledgementsThis study was supported by a grant from the Medi-cal University of Bialystok, no. 113-39900 LM.References1. Goyer RA, Clarkson T. W. 2001. Toxic effects of metals, in:Toxicology. The basic science of poisons, ed. C.D. Klaassen.McGraw-Hill, New York. 811.2. Nogaj E, Kwapuliński J, Nogaj P, Szczygiet Ł. Arterial vesselsas biomarkers of heavy metals exposure. Ann Acad Med Siles.2006;60:290–296.3. Poręba R, Gać P, Poręba M, Andrzejak R. Environmentaland occupational exposure to lead as a potential risk factorfor cardiovascular disease. Env Tox Pharm 2011;31:267–277.4. Iskra M, Patelski J, Majewski W. Relationship of calcium,magnesium, zinc and copper concentrations in the arterialwall and serum in atherosclerosis obliterans and aneurysm.J Trace Elem Med Biol. 1997;11:248–252.5. Gacko M, Głowiński S, Worowska A. The contents of zinc,magnesium, manganese, copper and iron in the wall of aorticaneurysm. Biul Magnezol. 1999;4:322–324.6. Antonowicz-Juchniewicz J. Środowiskowy i zawodowy wpływmetali ciężkich na rozwój zmian patologicznych w naczyniachkrwionośnych. Wyd. AM Wrocław, Wroclaw 2001.7. Hukałowicz K, Borawska M, Gacko M, Guzowski A,Czyżewska E. The contents of copper in serum, arterial walland parietal thrombus of patients with aortic abdominal an-eurysm. Metal Ions Biol Med. 2004;8:456–459.8. Nogaj E, Kwapuliński J, Nogaj P, Bogunia M, Ahnert B. Con-tent of heavy metals in femoral arteries changed by athero-sclerotic process in persons living in the industrial Silesianregion. Med Środ. 2005;8:117–126.9. Socha K, Borawska MH, Gacko M, Guzowski A, Markiewicz R.Diet and the content of cadmium in the blood, arterial walland parietal thrombus of the patients with aortic abdominalaneurysm. Bromat Chem Toksykol. 2005;37(Suppl.):51–55.10. Karamova LM, Larionova TK, Basharova GR. Criteria ofecologic safety for serum levels of heavy metals in humans.Med Tr Prom Ekol. 2010;6:21–23.11. Agarwal S, Zaman T, Tuzcu EM, Kapadia SR. Heavy metalsand cardiovascular disease: results from the National Healthand Nutrition Survey (NHANES) 1999–2006. Angiology.2011;62:422–429.12. Poręba R, Poręba M, Gać P, Andrzejak R. Ambulatory blondpressure monitoring and structural chan ges In carotid arte-rie In normotensive Wolkers occupationally expose to lead.Hum Exp Toxicol. 2011;30:1174–1180.13. Poręba R, Gać P, Poręba M, Andrzejak R. The relationshipbetween occupational exposure to lead and manifestation ofcardiovascular complications In persons with arteria hyper-tension. Toxicol Appl Pharmacol. 2010;249:41–46.14. Poręba R, Gać P, Poręba M et al. Relationship between chron-ic exposure to lead, cadmium and manganese, blond pres-sure values and incidence of arterial hypertension. Med Pr.2010;61:5–14.15. Skoczyńska A, Jędrejko M, Martynowicz H et al. The cardio-vascular risk In chemical faktory workers expose to mercutyvapor. Med Pr. 2010;61:381–391.16. Yamada E, Hazama F, Amano S, Sasahara M, Kataoka H.Elastase, collagenase and cathepsin D activities in the aortasof spontaneously hypertensive and renal hypertensive rats.Exp Molec Pathol. 1986;44:147–156.17. Gacko M, Chyczewski L. Activity and localization of cathep-sin B, D and G in aortic aneurysm. Int Surg. 1997;82:398–402.18. Gacko M, Ostapowicz R, Chrostek L, Worowska A, Kordec-ki K. Activity of enzymes with different subcellular localiza-tion in the blood plasma of patients with aortic aneurysm.Med Sci Monit. 2005;11:CR211–CR213.19. Jormsjo S, Wuttge DM, Sirsjo A et al. Differential expres-sion of cysteine and aspartic proteases during progression ofatherosclerosis in apolipoprotein E-deficient mice. AmJ Pathol. 2002;161:939–945.20. Petersen E, Wagberg F, Angquist KA. Proteolysis of the ab-dominal aortic aneurysm wall and the association with rup-ture. Eur J Vasc Endovasc Surg. 2002;23:153–157.21. Karwowska A, Gacko M, Worowska A, Krupkowska A. Tis-sue fragmentation for biochemical studies. Bromat ChemToksykol. 2006;39(Suppl.):199–202.22. Greczaniuk A, Roszkowska-Jakimiec W, Gacko M, Worow-ska A. Determination of cathepsin D activity in blood plas-435Effect of heavy metal cations©Polish Society for Histochemistry and CytochemistryFolia Histochem Cytobiol. 201210.5603/FHC.2012.0059www.fhc.viamedica.plma using hydrochloric acid denaturate haemoglobin. Diagn.Lab. 2000;36:97–101.23. Gacko M, Głowiński J, Skrzydlewska E, Worowska A,Głowiński S. Activity of enzymes of various subcellular local-ization in cytosol obtained after cell organelle precipitationat pH 5,0. Annal Acad Med Bialostocensis. 1996;41:341–346.24. Bradford MM. A rapid and sensitive method for the quanti-tation of microgram quantitation of protein utilizing the prin-ciple of protein-dye binding. Anal Biochem. 1976;72:248–254.25. Gornall AC, Bardawill CJ, David HM. Determination of se-rum proteins by means of the biuret reaction. J Biol Chem.1949;177:751–766.26. Vallee BL, ULMER DD. Biochemical effects of mercury,cadmium and lead. Ann Rev Biochem. 1972;41:91–128.27. Tukendorf A. Proteins and peptides binding heavy metals.Post Bioch. 1989;35:141–153.28. Haugaard N. Reflection on the role of thiol groups in biolo-gy. Ann NY Acad Sci. 2000;899:148–158.29. Manahan SE. Toxicological chemistry and biochemistry.PWN, Warszawa 2006; 267.30. Minarowska A, Gacko M, Karwowska A, Minarowski Ł. Hu-man cathepsin D. Folia Histochem Cytobiol. 2008;46,23–38.31. Worowski K. Mechanism of action of proteolytic enzymes.Post Hig Med Dośw. 1978;32:467–491.32. Bielański A. Basis of inorganic chemistry. PWN, Warszawa2002.33. Yonaha M, Ohbayashi Y, Ichinose T, Sagai M. Lipid peroxi-dation stimulated by mercuric chloride and its relation to thetoxicity. Chem Pharm Bull. 1982;30:1437–1442.34. Żak J, Chociłowska W. Effect of cadmium on lysosomes per-meability in the liver. Bromat Chem Toksykol. 1987;20:214–215.35. Stohs SJ, Bagchi D. Oxidative mechanisms in the toxicity ofmetal ions. Free Rad Biol Med. 1995;18:321–336.36. Stohs SJ, Bagchi D, Hassoun E, Bagchi M. Oxidative mecha-nisms in the toxicity of chromium and cadmium ions. J Envi-ron Pathol Toxicol Oncol. 2001;20:77–88.37. Havelaar AC, De Gast IL, Snijders S. Characterization ofa heavy metal ion transporter in the lysosomal membrane.FEBS Lett. 1998;436:223–227.38. Yu ZQ, Persson HL, Eaton JW, Brunk UT. Intralysosomaliron: a major determinant of oxidant-induced cell death. FreeRad Biol Med. 2003;34:1243–1252.39. Goch A, Goch JH. Lead-induced pathomechanism of hyper-tension. Pol Merk Lek. 2005;18:351–353.40. Fowler BA. Biological roles of high affinity metal-binding pro-teins in mediating injury. Comments Toxicol. 1989;3:27–46.41. Vasak M, Hasler DW. Metallothioneins: new functional andstructural insights. Chem Biol. 2000;4:177–183.42. Coyle I, Philcox JC, Carey LC, Rofe AM. Metallothionein:the multipurpose protein. Cell Med Life Sci. 2002;59:627–647.43. Chvapil M, Ryan JN, Brada Z. Effects of selected chelatingagents and metals on the stability of liver lysosomes. BiochemPharmacol. 1972;21:1097–1105.44. Kostyniak PJ, Clarkson TW. Role of chelating agents in met-al toxicity. Fund Appl Toxicol. 1981;1:376–380.45. Łukasiak J, Jamrogiewicz Z, Jackowska D, Ożarowski A,Krasicki A. Zastosowanie kwasu wersenawego i jego soli wterapii ostrych i przewlekłych zatruć metalami. Ord Lek.2006;6:26–30.46. Schulz M, Schmold A. Therapeutic and toxic blood concen-trations of more than 500 drugs. Pharmazie. 1997;52:895–910.47. Davidson SJ. Metal-ion effects on proteolysis and stability insecondary lysosomes of mouse kidney. Biochim Biophys Acta.1975;385:163–172.48. Skrzydlewska E, Worowski K, Moniuszko-Jakoniuk J,Gałażyn-Sidorczuk M. Ascertation of distribution and deter-mination of proteinases activity in hepatocytes in cadmiumintoxication. Acta Toxicol. 1994;2:83–88.49. Ohkawara S, Kaji T, Yamamoto C, Fujiwara Y, Sakamoto M,Kozuka H. Interaction between cadmium and zinc in the pro-duction and sulfation of glycosaminoglycans in cultured bo-vine vascular endothelial cells. J Toxicol Environ Health.1996;47:183–193.50. Skoczyńska A. Miażdżycowe działanie ołowiu i kadmu. Czyn-niki Ryzyka. 1999;4:20–25.Submitted: 9 December, 2011Accepted after reviews: 26 January, 2012

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Effect of heavy metal cations on the activity of cathepsin D (in vitro study)

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