* Department of Transplantation and Surgery, Semmelweis University, Budapest, Hungary, 1082;
Department of Cardiovascular Surgery, Semmelweis University, Budapest, Hungary, 1122;
Department of Forensic Pathology, Semmelweis University, Budapest, Hungary, 1091; and
National Institute of Forensic Toxicology, Budapest, Hungary, 1146
Received September 12, 2003; accepted October 29, 2003
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ABSTRACT |
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Key Words: Tinuvin 770; cardiotoxicity; catecholamine; L-type Ca2+ channels, light-stabilizers; hemodialysis.
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INTRODUCTION |
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Our previous study has indicated the time-dependent nature of Tinuvin 770 cytotoxicity on isolated rat cardiomyocytes caused by altered Ca2+ metabolism. The cardiomyocytes culture, suspended in 25 nM Tinuvin 770 solution for 60 min, showed hypercontraction in 53% of the cells, along with marked decrease in ATP and creatine-phosphate (CP) concentration. After 120 min, 60% of the suspended cardiomyocytes showed irreversible damage with further drastic fall in ATP and CP level (Sótonyi et al., 2001).
The significance of Tinuvin 770 content of medical industry products is underlined by fact that the molecule can come to direct contact with the circulation. During hemodialysis 250300 ml/min blood flows trough a 11.8 m2 plastic dialysis membrane for 34 h. This treatment is usually repeated three times a week in patients with chronic renal failure. Our previous analytical study showed that Tinuvin 770 is extractable from four different types of hemodialysis membranes (Sótonyi et al., in press), underlining the significance of experimental toxicological analysis of this chemical.
The literature is extensive on the adverse effects of hemodialysis, caused by the method itself, and directly or indirectly by the plastic membranes. Some of the most important adverse effects include anaphylactic reaction to ethylene oxide (Bommer and Ritz, 1987) and formaldehyde (Kessler et al., 1988
) used for sterilization, the reuse of the hemodialysis membranes (Sótonyi et al., 1996
), intoxication of chloramine (Tipple et al., 1991
) and fluorine (Arnow et al., 1994
), pyrogenic reactions (Roth and Jarvis, 2000
) caused by bacterial contamination, shock-syndrome (Tielemans et al., 2000
) due to interaction of ACE inhibitors and AN-69 membrane, and other unexpected cardiopulmonary events (Tielemans et al., 1996
) using different types of membranes and dialyzing solutions.
In 2001, several countries (Spain, Croatia, United States, Italy, Germany, Taiwan, and Columbia) reported separately cases of unexpected chest pain, dyspnea, and circulatory collapse followed by deaths, during or soon after hemodialysis. Subsequent toxicological studies showed the presence of perfluorohydrocarbon (PF5070) in pulmonary capillaries (Sass et al., 1976). PF5070 is a material used in premarket testing of membrane permeability. The investigation revealed remnants of this material, which was not removed completely during manufacturing and formed an emulsion with blood at a normal body temperature and low oxygen pressure. It is apparent that the interaction between plastics and the human organism can cause a wide range of unexpected reactions (Canaud, 2002
).
Hypotension is the most common complication of hemodialysis, occurring in about 1520% of patients (Sato et al., 2001). Clinical symptoms can develop subsequent to a drop in systolic blood pressure of a minimum 3050 mm Hg. Most commonly observed symptoms are nausea, vomiting, dizziness, and sometimes loss of consciousness. Decreased blood volume, peripheral vasoconstriction, and cardiac factors can be held responsible for the development of hypotension (Daugirdas, 1991
). Because Tinuvin 770 displays the L-type Ca2+ channel blocking effect on both the peripheral vessels and the myocardium, this molecule might potentially contribute to hypotension.
Based on our formerly published observations on myocardial cell cultures, we decided to study Tinuvin 770-induced morphologic changes of myocardium of rats.
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MATERIALS AND METHODS |
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Animal exposure and tissue analysis.
Animals were randomly selected into five groups (IV), with 10 animals in each (five males and five females). Group I served as control, receiving a mixture of equal amount of physiologic saline solution (Salsol solution, Human, Gödöll, Hungary) and 96% ethanol (Riedel de Haen, Seelze, Germany) intraperitoneally (ip) during the full length of the experiment. Groups IIV, were injected ip 15 times during a 5-week period (three times a week: Mondays, Wednesdays, and Fridays) with varying doses of Tinuvin 770 [bis(2,2,6,6-teramethyl-4-piperidinyl) sebacate, Ciba Geigy Co., Basel, Switzerland], in a 1-ml solution containing equal amount of physiologic saline and 96% ethyl alcohol. The applied Tinuvin 770 doses were calculated on the basis of parallel execution of an acute hemodynamic dog model, where the acute effective dose range (leading to hypotension and diminished ventricular contractility) was from 1 to 100 mg on dogs with 25 kg body weight (EC50
0.137 mg/kg body weight, LD50
4 mg/kg body weight). The Tinuvin 770 doses were: 1 µg for group II, 10 µg for group III, 100 µg for group IV, and 1 mg for group V. Animals were decapitated following deep ether narcosis. Myocardial samples for morphologic studies were taken from the left ventricular myocardium, the interventricular septum, and the papillary muscles. Myocardial tissues were fixed in phosphate buffered 4% formaldehyde (pH 7.2). After routine histological processing, hematoxylin-eosin (H&E), azan, phosphotungstic acid-hematoxylin (PTAH), Van Gieson, and PAS stains were used. For transmission electron microscopic studies, the myocardial samples were fixed in 3% glutaraldehyde, buffered with 0.1 M Na-cacodylate, postfixed in 1% osmium tetroxide and embedded in araldite. Toluidine blue stained semithin sections were used to select areas for ultrastructural investigation. Following araldite removal, further semithin sections were stained with PTAH and Movat methods. Ultrathin sections were contrasted with uranyl acetate and lead citrate, coated with carbon, and examined with JEOL 100B electron microscope at 60 kV accelerating voltage.
For cytochemical detection of Ca2+ lead-acetate method was used (Carasso and Favard, 1966). Alterations in adrenergic innervation were followed by the glyoxylic acid technique (Shwalev and Zhuchkova, 1979
; Zhuchkova et al., 1984
). Evaluation was performed by using Jenalumar fluorescent microscope (VEB Carl Zeiss, Jena, Germany) with exciting filter VG1/3.5+BG52/2 and closing filter GG9+OG4.
Urinalysis.
Last urine samples were collected by direct urinary bladder puncture and analyzed for noradrenalin using high performance liquid chromatography (HPLC). Extraction was performed using Discovery C18 column; the mobile phase was a mixture of 3% acetonitrile, 96% potassium dihydrogen phosphate (pH 3, 25 mM), EDTA, octane-sulphophosphoric acid (0.05 mM). Noradrenalin was detected by dual electrochemical method (Coulochem 2 coulometer, 550 mV).
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RESULTS |
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In group III, small disseminated hemorrhages were presented on the upper third of the interventricular septum at gross examination. On histology, slight, small, focal hypercontraction, interstitial edema, and myocytolysis were found (Fig. 2A). In groups II and III, catecholamine release did not increase at the adrenergic nerve terminals, and there were no differences in Ca2+ content compared to the control group.
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Histological examination of other organs (lungs, spleen, brain, kidneys, blood vessels) did not result any significant changes, except the livers in groups IV and V, where mild unspecific intracellular vacuolization was observed in hepatocytes.
Noradrenalin urine level was approximately 10 times higher in the treated groups (although without substantial differences between them) compared to the control one. Results are demonstrated in Table 3.
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DISCUSSION |
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No significant structural myocardial changes were seen in group II, where low dose (1 µg), of Tinuvin 770 was given. At the dose of 10 µg (group III), only slight alterations were observed. In groups IV and V, where higher doses of Tinuvin 770 (100 µg, 1 mg) were applied, an intense effect occurred. The L-type Ca2+ channel blockage may be counterbalanced and suspended by reflex sympathetic adrenergic activation. In support of this finding, our results demonstrated increased catecholamine activity by fluorescence microscopy and marked intracellular Ca2+ accumulation in the myocardium, showing myocytolysis and hypercontraction necrosis. Our experiments elegantly demonstrate the dose-dependent nature of this process.
In view of the above, our observation is seemingly contradictory, becauseas opposed to Ca2+channel blockersTinuvin 770 causes a dose-dependent myocardial damage (myocytolysis, hypercontraction necrosis). Tinuvin 770 can induce hypotension followed by reflex-induced increased sympathetic tone (data from our unpublished dog experiment), similar to effects caused by dihydropyridines. The cascade of events can lead to diminished oxygen supply, myocardial ischemia, and damaged myocardial contraction.
Myocytolysis and hypercontraction necrosis can be a final common pathway for many different events, including hypoxia, direct toxic effects, and left ventricular pressure overload. The described morphology is characteristic for catecholamine-induced myocardial damage. Elevated urine noradrenalin levels could be a marker for stress condition of the treated animals. Sudden increase in catecholamine blood level can induce hypermetabolism and forced myocardial contractility with increased work capacity and oxygen demand (Burniston et al., 2003). Catecholamines, in physiologic concentrations, can increase the contractility function of the myocardium by intensifying the consumption of high-energy phosphates. Using catecholamines in a higher than physiologic concentration, causes a dramatic increase in Ca2+influx, leading to the collapse of cellular energy and ionic homeostasis, and subsequent functional and structural cellular damage (Bristow, 1995
; Lehr, 1981
; Shinay et al., 2000
; Zimmer, 1997
). Catecholamine-induced myocardial damage is primarily a metabolic event. The aerobic oxidation will gradually shift toward anaerobic and fatty acid oxidation. ATP production in anaerobic glycolysis will be insufficient, because myocytes will be unable to maintain the ionic gradients, and the decreased amount of ATP is inadequate to maintain intracellular Ca2+ balance (Dhalla, 2001
). In the case of decreased intracellular ATP level, relaxation of myocardium is suffered (Van der Velden, 1998
). As a consequence, a rise in intracellular Ca2+ accumulation and decreased diastolic outflow develop. An increased intracellular Ca2+ accumulation will also appear in the mitochondria, affecting their structure and function.
The observed phenomenon is fully supported by the earlier in vitro studies on cardiomyocyte cultures (Sótonyi et al., 2001), that the cell membrane of myocytes lost their integrity, leading to irreversible damage of the isolated cells.
Summarizing the results of our morphological study, we can conclude that the toxic effects of Tinuvin 770 on the myocardium emerge when 100 µg or higher doses are administered long-term.
The above-described phenomenon should attract special attention, because Tinuvin 770 is a widely used industrial plastic additive, due to its excellent attribute as a UV light stabilizer, and due to its cost effectiveness. Long-term hemodialysis treatment could serve as a basis for contamination with Tinuvin 770, because of the long-lasting, direct interaction between blood and plastic surface. The likelihood emerges that Tinuvin 770 may play a role in the development of certain adverse events in chronic hemodialysis treatment. Our results aim to attract attention to the fact that any plastic device containing Tinuvin 770 can harbor the chance of contamination when it comes into contact with the human body.
We would like to call attention to the emerging latent problem of using Tinuvin 770 and initiate further interdisciplinary investigations. Development of systematized and extensive regular toxicological monitoring should also be obligatory for the safety of medical plastics.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Bommer, J., and Ritz, E. (1987). Ethylen oxide (ETO) as a major cause of anaphylactoid reactions in dialysis (a review). Artif. Organs 11, 111117.[ISI][Medline]
Bristow, M. R. (1995). Spontaneous reversibility of catecholamine-induced cardiotoxicity in rats. Am. J. Cardiovasc. Pathol. 5, 7988.[Medline]
Burniston, T. I., Clark, C. W., and Yeelan, N. G. (2003). Characterisation of adenoceptor involvement in skeletal and cardiac myotoxicity induced by sympathomimetic agents: Toward a new bioassay for beta-blockers. J. Cardiovasc. Pharmacol. 41, 518525.[CrossRef][ISI][Medline]
Canaud, B. (2002). Performance liquid test as a cause for sudden deaths of dialysis patients: Perfluorohydrocarbon, a previously unrecognized hazard for dialysis patients. Nephrol. Dial. Transplant. 17, 545548.
Carasso, N., and Favard, P. (1966). Mise en évidence du calcium dans les myonénes pédunoclaries de ciliés péritriches. J. Microsc. 5, 759770.[ISI]
Daugirdas, J. T. (1991). Dialysis hypotension: A hemodynamic analysis. Kidney Int. 16, 233246.
Dhalla, N. S. (2001). Role of oxidative stress in catecholamine-induced cardiac sarcolemmal Ca2+ transport. Arch. Biochem. Biophys. 387, 8592.[CrossRef][ISI][Medline]
Epstein, M., (Ed). (1998). Calcium Antagonist in Clinical Medicine. Hanley & Belfus Inc. Medical Publisher, Philadelphia, PA.
Flores, C. M., Rogers, S. W., Pabreza, L. A., Wolfe, B. B., and Kellar, K. (1992). A subtype of nicotinic cholinergic receptor in rat brain is composed of 4 and ß2 subunits and is up-regulated by chronic nicotine treatment. Mol. Pharmacol. 41, 3137.[Abstract]
Gächter, R, Müller, H., and Klemchuk, P. P. (1990). Plastic Additives Handbook, 3rd ed., pp. 129270. Hanser Publisher, Munich.
Glossmann, H., Hering, S., Savchenko, A., Berger, W., Friedrich, K., Garcia, M. L., Goetz, M. A., Liesch, J. M., Zink, D. L., and Kaczorowski, G. J. (1993). A light stabilizer (Tinuvin 770) that elutes from polypropylene plastic tubes is a potent L-type Ca2+ -channel blocker. Proc. Natl. Acad. Sci. U.S.A. 90, 95239527.[Abstract]
Gong, L., Zhang, W., Zhu, Y., Zhu, J., Kong, D., Page, V., Ghadirian, P., LeLorier, J., and Hamet, P. (1996). Shanghai trial of nifedipine in elderly (STONE). J. Hypertens. 14, 12371245.[ISI][Medline]
Kessler, M., Cao Huu, T., Mariot, A., and Chanliau, J. (1988). Oxide and formaldehyde. Contrib. Nephrol. 62, 1323.[Medline]
Lehr, D. (1981). Studies on the cardiotoxicity of alpha and beta adrenergic amines. In Cardiac Toxicology (T. Balazs, Ed.), Vol. II, pp. 75108. CRC Press, Inc. Boca Raton, FL.
Marque, D., Feigenbaum, A., and Riquet, A. M. (1996). Repercussion of plastic packing on the compatibility with packaged foodstuff. J. Chem. Phys. et Physico-Chem. Biol. 93, 165168.
McDonald, T. F., Pelzer, S., Trautwein, W., and Pelzer, D. (1994). Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells. Physiol. Rev. 74, 365507.
Papke, R. L., Grey Craig, A., and Heinemann, S. F. (1994). Inhibition of nicotinic acetylcholine receptors by bis(2,2,6,6-tetramethyl-4-piperidinyl) sebacate (Tinuvin 770), an additive to medical plastics. J. Pharmacol. Exp. Ther. 268, 718726.[Abstract]
Pelzer, D., Pelzer, S., and McDonald, T. F. (1992). Calcium channels in heart. In The Heart and Cardiovascular System. (H. A. Fozzard, Ed.), pp. 10491089. Raven Press Ltd., New York.
Pitt, B. (1997). Diversity of calcium antagonists. Clin. Ther. 19 (Suppl. A), 37.[CrossRef][ISI][Medline]
Roth, V. R., and Jarvis, W. R. (2000). Outbreaks of infection and/or pyrogenic reactions in dialysis patients. Semin. Dial. 13, 9296.[CrossRef][ISI][Medline]
Sass, D. J., Van Dyke, R. A., Wood, E. H., Johnson, S. A., and Didisheim, P. (1976). Gas embolism due to intravenous FC 80 liquid fluorocarbon. J. Appl. Physiol. 40, 745751.
Sato, M., Horigome, I., Chiba, S., Furuta, T., Miyazaki, M., Hotta, O., Suzuki, K., Noshiro, H., and Taguma, Y. (2001). Autonomic insufficiency as a factor contributing to dialysis-induced hypotension. Nephrol. Dial. Transplant. 16, 16571662.
Shinay, I., Shinichi, M., Mitsuhiro, Y., Shoei, K., and Sakan, M. (2000). Isoproterenol-induced myocardial injury resulting in alerted S100A4 and S100A11 protein expression in the rat. Pathol. Int. 50, 480485.[CrossRef][ISI][Medline]
Shwalev, W. N., and Zhuchkova, N. I. (1979). A simple method for revealing adrenergic nerve structure in human and animal tissues using glyoxylic acid solution. Arch. Anat. Histol. Embryol. 6, 114116.
Sótonyi, P., Járay, J., Pádár, Z., Woller, J., Füredi, S., and Gál, T. (1996). Comparative study on reused haemodialysis membranes. Int. J. Artif. Org. 19, 387392.[ISI][Medline]
Sótonyi, P., Keller, É., Járay, J., Nemes, B., Benk, T., Kovács, A., Tolokán, A., and Rajs, I. (2001). A light stabilizer Tinuvin 770-induced toxic injury of adult rat cardiac myocytes. Forensic Sci. Int. 119, 322327.[CrossRef][ISI][Medline]
Sótonyi, P., Kovács, A., Volk, G., Járay, J., and Benk, A. (2004). Detection of Tinuvin 770 a light stabilizer of plastic materials from dialysis membranes by high performance liquid chromatographic analysis. J. Chromtogr. Sci. 42, 4953.
Tielemans, C., Gastaldello, K., Goldmann, M., and Vanherweghem, J. L. (1996). Acute haemodialysis membrane-associated reactions. Nephrol. Dial. Transplant. 11 (Suppl.), 112115.
Tielemans, C., Madhoun, P., Lenaers, M., Schandene, L., Goldmann, M., and Wanherweghem, J. L. (2000). Anaphylactoid reactions during hemodialysis on AN69 membranes in patients receiving ACE inhibitors. Kidney Int. 38, 992998.[CrossRef]
Tipple, M. A., Shusterman, N., Bland, L. A. McCarthy, M. A., Favero, M., S., Arduino, M. J., Reid, M. H., and Jarvis, W. R. (1991). Illness in hemodialysis patients after exposure to chloramine contaminated dialysate. ASAIO Trans. 37, 588591.[Medline]
Van der Velden, J. (1998). Correlation between contractile protein composition and energetic and mechanical properties of the heart. In Printpartners Ipskamp, Enchede, The Netherlands.
Wiles, D. M., Tovborg Jensen, J. P., and Carlsson, D. J. (1983). Polymer stabilization by hindered amines. Pure Appl. Chem. 55, 16511659.[ISI]
Zhuchkova, N. I., Shwalev, W. N., and Stropus, R. A. (1984). Qualitative analysis of the density of adrenergic nervous plexuses of the heart in sudden cardiac death. In: Pathomechanism and Prevention of Sudden Cardiac Death due to Coronary Insufficiency. (L. Szekeres, L. Gy. Papp, and L. Takáts, Eds.), pp. 269274. Akademia Press, Budapest, Hungary.
Zimmer, H. G. (1997). Catecholamine-induced cardiac hypertrophy: significance of proto-oncogene expression. J. Mol. Med. 57, 849859.[CrossRef]
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