33 Annales Universitatis Paedagogicae Cracoviensis Studia Naturae, 3 (supplement): 33–38, 2018, ISSN 2543-8832 DOI: 10.24917/25438832.3supp.4 Łukasz M. Kołodziejczyk*, Magdalena Puzik, Agnieszka Greń, Marta Batoryna, Grzegorz Formicki, Edyta Kapusta Department of Animal Physiology and Toxicology, Institute of Biology, Pedagogical University of Cracow, Podbrzezie 3, 31-054 Kraków, Poland; *lukasz.kolodziejczyk@up.krakow.pl Does benzo[a]pyrene affect the embryonic development of the heart? Introduction �e chicken embryo and the avian in ovo model are one of the prominent experi- mental procedures in several tests for toxins and the preclinical testing of drugs. It is also the oldest known embryological protocol of basic developmental research (Davey, Tickle, 2007). It is well documented that many milestone discoveries on the embryol- ogy of vertebrates and on the organogenesis of brains or hearts were made using this model organism (Le Douarin, 1998; Pardanaud et al., 2001). Benzo[a]pyrene is a polycyclic aromatic hydrocarbon that is a well-known car- cinogen, teratogen, and neurotoxin widely present in urban air pollution, cigarette smoke, and certain kinds of foods, i.e. smoked �sh, smoked meat, etc. �e problem of intoxication with this substance is actually one of the most urgent in big develop- ing cities, such as Cracow (European Environment Agency…, 2016). �ere are many reports con�rming its toxicity for mice and other mammals; however, knowledge on its impact on birds is still limited. Whereas, birds are an important element of the typical urbicenosis, and basic knowledge on the biology of this group of vertebrates suggests that they may be a useful biotest for this stress factor (Lee, Shim, 2007). �e e�ect of benzopyrenes on the developing heart of vertebrates is unknown and requires e�ective update. �e embryos of birds exhibit several opportunities to perform physi- ological experiments on the heart (Tazawa et al., 1994). �e aim of presented paper is to determine the main action of benzo[a]pyrene on selected parameters of the heart muscle of chicken embryos in the in ovo develop- mental model, with special attention to the antioxidative defence mechanisms and the bioelectric properties of heart rhythm. Łu ka sz M . K oł od zi ej cz yk , M ag da le na P uz ik , A gn ie sz ka G re ń, M ar ta B at or yn a, G rz eg or z Fo rm ic ki , E dy ta K ap us ta 34 Material and methods We used chicken embryos of the race ‘Ross 308’ to verify the in�uence of benzo[a] pyrene on physiological and biochemical parameters of the heart muscle. Fertilised chicken eggs were obtained from a certi�ed farm (Łężkowice, Poland) and incubated in an automated incubator (HEKA, Germany) at 37.5oC. �e benzo[a]pyrene in an organic oil solution (Sigma Aldrich, USA) was injected in ovo on the 6th day of the incubation into the yolk at the following doses of 1 mg/kg weight of eggs; 0.5 mg/kg w. e. and 0.1 mg/kg w. e. �e intact eggs and eggs injected with the organic oil were used as control groups. On the 14th day of the incubation, eggs were opened in order to examine embryos and achieve tissues for further analyses. We performed the electrocardiography of embryos using AsCARD AMBER equipment (Aspel, Poland) and 4 extremital copper electrodes. We also estimated the weight of hearts post mortem using a laboratory balance (Rad Wag, Poland). Finally, we determined the concentration of reduced glutathione (GSH) according to Ellman’s method and malonyldialdehyde (MDA) using TBARS MDA Assay in the heart tis- sue. To perform all spectrophotometric measurements, we used a Sunrise Absorbance Reader (Tecan, Austria). �e quantitative data was analysed statistically using Shapiro-Wilk tests and Stu- dent tests with the signi�cance level at p < 0.05. Results and discussion �e electrocardiography performed on the 14th day of incubation does not show any important changes in the heart rate and rhythm in relation to controls (Fig. 1). In the electrocardiogram basal sinus rhythm without evident QRS intervals was visualised, which is typical for immature bird hearts (Yoshiyama, Kanke, 2005). �e increased weight of the heart muscle was observed in the embryos treated with a dose of 1 mg/kg w. e. In this group, the average heart weight accounted 211 mg. In contrast, the average heart weight in control groups was approximately equal to 120 mg. (Fig. 2A). �is result may suggest that the higher doses of benzo[a]pyrene increase blood retention in the systemic circulation, which results in the heart hypertrophy. A  similar e�ect was described in several pathologies connected with increased vascu- lar resistance (Pardanaud et al., 2001). In groups contaminated with lower doses of ben- zo[a]pyrene, heart weight did not di�er signi�cantly from the control and intact eggs. We determined a statistically signi�cant increase of the GSH concentration in the heart tissue from the group injected with 1 mg/kg w. e. In the case of the lower ben- zo[a]pyrene doses, the level of GSH was similar to the controls (Fig. 2B). 35 D oes benzo[a]pyrene affect the em bryonic developm ent of the heart? Fig. 1. Examples of electrocardiograms of chicken embryos on the 14th day of incubation. C – control; BaP – an individual contaminated with benzo[a]pyrene Di�erences in the MDA concentrations in all experimental groups were not statis- tically signi�cant in relation to the controls (Fig. 2C). Łu ka sz M . K oł od zi ej cz yk , M ag da le na P uz ik , A gn ie sz ka G re ń, M ar ta B at or yn a, G rz eg or z Fo rm ic ki , E dy ta K ap us ta 36 Fig. 2. A – the e�ects of benzo[a]pyrene on the heart weight post mortem of chicken embryos; B – the e�ects of benzo[a]pyrene on the concentration of reduced glutathione in the heart tissue; C – the e�ects of benzo[a]pyrene on the concentration of malonyldialdehyde in the heart tissue; INT – intacts; C – con- trol; B1 – 1 mg/kg w. e. of benzo[a]pyrene; B0.5 – 0.5 mg/kg w. e. of benzo[a]pyrene; B0.1 – 0.1 mg/kg w. e. of benzo[a]pyrene; signi�cant di�erences between the experimental and control groups are indicated with asterisks (*p < 0.05, **p < 0.01); the error bars denote the standard deviation of the mean value; n = 6 37 �ese results may suggest that benzo[a]pyrene is a toxicological stress factor, which activates the glutathione synthesis in the organism in response to the acute poisoning and may activate other mechanisms of the glutathione-dependent antiox- idative defence. It has been proven that some neurotoxins (i.e. acrylamide) involve varied disturbances and defence responses in the antioxidative system of the chicken embryo’s brain (Batoryna et al., 2017; 2018). Conclusion We conclude that the subacute dose of benzo[a]pyrene is a stress factor, which strong- ly activates the glutathione-dependent antioxidative defence and probably do not af- fect the heart conducting system of the chicken embryo; however, the in�uence of this substance on the morphology and biochemistry of the developing heart requires further examination. References Batoryna, M., Lis, M.W., Formicki, G. (2017). Acrylamide-induced disturbance of the redox balance in the chick embryonic brain. Journal of Environmental Science and Health, Part B, 52(8), 600–606. DOI: 10.1080/03601234.2017.1316158. Batoryna, M., Lis, M.W., Formicki, G. (2018). Antioxidant defense in the brain of 1-d-old chickens exposed in ovo to acrylamide. British Poultry Science, 59(2), 198–204. DOI: 10.1080/00071668.2017.1415427. Davey, M.G., Tickle, C. (2007). �e chicken as a model for embryonic development. Cytogenetic and Genome Research, 117, 231–239. European Environment Agency (2016). Air quality in Europe – 2016 report, EEA Report No 28/2016. Publications O�ce of the European Union, Luxembourg. Le Douarin, N.M. (1998). Les chimères de caille et de poulet pour étudier l’embryogenèse. Pour la Science, 252, 46–54. [In French]. Lee, B.M., Shim, G.A. (2007). Dietary exposure estimation of benzo[a]pyrene and cancer risk assessment. Journal of Toxicology and Environmental Health, Part A, 70(15–16), 1391–1394. Pardanaud, L., Moyon, D., Eichmann, A. (2001). L’embryologie des vaisseaux. Médecine sciences, 5(17), 543–551. [In French] Tazawa, H., Watanabe, W., Burggren, W.W. (1994). Embryonic heart rate in altricial birds, the Pigeon (Columba domestica) and the Bank Swallow (Riparia riparia). Physiological Zoology, 67(6), 1448– 1460. Yoshiyama, Y., Kanke, M. (2005). Toxic interactions between �uconazole and disopyramide in chick em- bryos. Biological and Pharmaceutical Bulletin, 28(1), 151–153. Abstract Benzo[a]pyrene is a polycyclic aromatic hydrocarbon with a well-proven toxic e�ect on animal cells and tissues. We used a chicken in ovo developmental model to verify its in�uence on selected parameters of the heart rhythm and on the antioxidative defence in the heart tissue. We determined that the dose of 1 mg/kg weight of eggs of benzo[a]pyrene strongly activates the glutathione-dependent antioxidative system, but it did not signi�cantly a�ect the heart conducting system of the chicken embryo. We postulate that further D oes benzo[a]pyrene affect the em bryonic developm ent of the heart? Łu ka sz M . K oł od zi ej cz yk , M ag da le na P uz ik , A gn ie sz ka G re ń, M ar ta B at or yn a, G rz eg or z Fo rm ic ki , E dy ta K ap us ta 38 study on the benzo[a]pyrene action during embryonic development of birds is recommended. Key words: benzo[a]pyrene, ecg, embryo, GSH, heart, MDA Received: [2018.07.11] Accepted: [2018.12.10] Czy benzo[a]piren wpływa na rozwój zarodkowy serca? Streszczenie Benzo[a]piren jest wielopierścieniowym węglowodorem aromatycznym o  dobrze znanym toksycznym działaniu na komórki i tkanki zwierząt. Wykorzystaliśmy model rozwoju zarodków kury in ovo do wery- �kacji wpływu tej substancji na rytm serca oraz wybrane parametry układu antyoksydacyjnego w mięśniu sercowym. Wykazaliśmy, że dawka 1 mg/kg masy jaj silnie aktywuje zależny od glutationu mechanizm anty- oksydacyjny, ale jak się wydaje nie wpływa znacząco na układ przewodzący serca zarodków kury. Konieczne są dalsze badania wpływu benzo[a]pirenu na rozwój zarodkowy ptaków. Słowa kluczowe: benzo[a]piren, EKG, zarodek, GSH, serce, MDA Information on the authors Łukasz M. Kołodziejczyk He is a PhD student at the Department of Animal Physiology and Toxicology in the Institute of Biology, Pedagogical University of Cracow. His scienti�c interests are developmental biology and comparative anatomy. Magdalena Puzik She is a graduate of bioinformatics in the Institute of Biology, Pedagogical University of Cracow. Agnieszka Greń https://orcid.org/0000-0003-2383-1096 She is a professor at the Department of Animal Physiology and Toxicology in the Institute of Biology, Pedagogical University of Cracow. Marta Batoryna She is a PhD student at the Department of Animal Physiology and Toxicology in the Institute of Biology, Pedagogical University of Cracow. Grzegorz Formicki https://orcid.org/0000-0001-9964-6132 He is a professor at the Department of Animal Physiology and Toxicology in the Institute of Biology, Pedagogical University of Cracow. Edyta Kapusta https://orcid.org/0000-0002-4350-5514 She is a laboratory specialist at the Department of Animal Physiology and Toxicology in the Institute of Biology, Pedagogical University of Cracow.