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Immunodetection of heat shock protein 70 and cell death in liver of a neotropical fish acutely and chronically exposed to acetaminophen and propranolol

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Abstract

The use of pharmaceutical drugs ends frequently in their inappropriate disposal and treatment at waste water treatment plants, which is the cause of their widespread presence in the environment. Yet, there is limited understanding or knowledge of their effects to non-target aquatic organisms. The drugs acetaminophen (analgesic and antipyretic) and propranolol (β-blocker) are widely found in the aquatic environment, where they can interact with non-target exposed organisms, causing adverse effects. Heat shock proteins (namely HSP70) are molecular chaperones which help to refold misfolded cellular proteins, and the increase in their in vivo levels indicates a change in the cell to counteract the proteotoxic effects of the triggered stress, namely which is consequent to exposure to toxicants. The objective of this study was to quantify the levels of liver HSP70 proteins in individuals of the neotropical fish species Phalloceros harpagos, acutely and chronically exposed to concentrations of acetaminophen and propranolol, in the range of those already determined to occur in the wild. Fish acutely exposed to acetaminophen (concentrations of 8, 80, 800, and 8000 μg L−1) and to propranolol (levels of 1, 10, and 1000 μg L−1) evidenced increased intensity of HSP70 immunolabeling in liver cells. Similarly, animals chronically exposed to propranolol, at concentrations of 0.0625, 0.125, 0.25, and 0.5 μg L−1, showed a comparable trend. This finding suggests the triggering of a cytoprotective effect that was effective in preventing cell death in exposed groups in relation to the control group. In contrast, chronic exposure to acetaminophen caused a decrease in HSP70 labeling intensity in fish hepatocytes (animals exposed to 5, 10, 20, 40, and 80 μg L−1), with no induction of DNA fragmentation in the nuclei of hepatocytes of these fish. Some of the hepatic HSP70 responses observed in this study were obtained at levels already reported to occur in the wild. Finally, this study showed how levels of acetaminophen at microliter concentration can exert side effects on non-target organisms after chronic exposure, suggesting that environmentally exposed organisms may be subjected to adverse conditions that modify the typical response pattern of HSP70 levels.

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The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgments

Bruno Nunes is hired by ECO-R-pharmplast - Ecotoxicity of realistic combinations of pharmaceutical drugs and microplastics in marine ecosystems, Fundação para a Ciência e a Tecnologia, FCT (reference POCI-01-0145-FEDER-029203). Authors thank Dr. Osmar Malaspina (CEIS - Center for the Study of Social Insects) of UNESP in Rio Claro/SP, Brazil, for enabling the microtomy of the fish organs. Authors thank the PPGBMA for the use of the laser scanning confocal microscope (Pró-Equipamentos: 3420/2013-17, 2610/2014-90) and DBio (Departamento de Biologia da UFSCar, Sorocaba, SP, Brazil) by the infrastructures made available for the confocal microscopy. The first author thanks CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the scholarship during her master’s degree (PPGBMA) and for the PVE Program (CAPES).

Funding

This work was co-funded by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) - PVE Program (Process Number: CAPES 88881.068122/2014–01), CESAM (UIDB/50017/2020+UIDP/50017/2020), FCT/MEC and ERDF (PT2020 Partnership Agreement and Compete 2020).

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Authors and Affiliations

  1. Pós-Graduação em Biotecnologia e Monitoramento Ambiental, Universidade Federal de São Carlos, Rodovia João Leme dos Santos km 110, Itinga, Sorocaba, SP, 18052-780, Brazil

    Beatriz Vieira Ramos Pereira, Elaine Cristina Mathias da Silva-Zacarin & Monica Jones Costa

  2. Departamento de Biologia, Universidade Federal de São Carlos, Rodovia João Leme dos Santos km 110, Itinga, Sorocaba, SP, 18052-780, Brazil

    Elaine Cristina Mathias da Silva-Zacarin, Monica Jones Costa & André Cordeiro Alves dos Santos

  3. Departamento de Biologia, Universidade de Aveiro, Campus de Santiago, 3810-193, Aveiro, Portugal

    Bruno Nunes

  4. Centro de Estudos do Ambiente e do Mar (CESAM), Universidade de Aveiro, Campus de Santiago, 3810-193, Aveiro, Portugal

    Bruno Nunes

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  1. Beatriz Vieira Ramos Pereira
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Contributions

B.V. R. Pereira: collection of animals, conductor of the bioassays, microscopical analysis, statistical analysis, data interpretation, writing the article.

E.C.M. Silva-Zacarin: experimental design, microscopical analysis, data interpretation, writing the article.

M.J. Costa: experimental design, statistical analysis, data interpretation, text review of article.

A.C.A. dos Santos: experimental design, collection of animals, data interpretation, text review of article.

B. Nunes: experimental design, data interpretation, statistical analysis, writing the article.

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Correspondence to Elaine Cristina Mathias da Silva-Zacarin.

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Approval was obtained from the Ethics Committee on the Use of Animals of Federal University of São Carlos (UFSCar) according to Protocol CEUA/UFSCar: 2730071215, approved in 04/02/2016) and the collection of fish was authorized by the Biodiversity Information and Authorization System (SISBIO), number 52495-1.

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Pereira, B.V.R., da Silva-Zacarin, E.C.M., Costa, M.J. et al. Immunodetection of heat shock protein 70 and cell death in liver of a neotropical fish acutely and chronically exposed to acetaminophen and propranolol. Environ Sci Pollut Res 28, 11233–11244 (2021). https://doi.org/10.1007/s11356-020-11264-3

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