Immune, inflammatory, autophagic and DNA damage responses to long-term H2O2 exposure in different tissues of common carp (Cyprinus carpio)

https://doi.org/10.1016/j.scitotenv.2020.143831Get rights and content

Highlights

  • H2O2 exposure causes abnormal immune function in common carp.

  • TLRs and NF-kB are involved in H2O2-induced inflammatory response in the majority of tissues.

  • H2O2 exposure alters the autophagy process in different tissues of common carp.

  • Response mechanisms to H2O2 toxicity are in a dose-dependent and tissue-specific manner.

Abstract

Hydrogen peroxide (H2O2) is a stable reactive oxygen species (ROS) in aquatic environment, and high concentration of ambient H2O2 may directly or indirectly affect aquatic animal health. However, the response mechanism of fish to ambient H2O2 has not been well studied yet. Therefore, the aim of the study was to investigate the immune, inflammatory, autophagic and DNA damage responses to long-term H2O2 exposure in different tissues of common carp. The results showed that H2O2 exposure induced a significant immune response, with alterations in the levels of immune parameters including AKP, ACP, LZM, C3, HSP90 and HSP70 in different tissues. The inflammatory response evoked by H2O2 exposure was associated with the activations of TLRs and NF-κB (P65) in the majority of tested tissues. The autophagy process was significantly affected by H2O2 exposure, evidenced by the upregulations of the autophagy-related genes in liver, gills, muscle, intestines, heart and spleen and the downregulations in kidney. Meanwhile, the mRNA level of atm, a primary transducer of DNA damage response, was upregulated in liver, gills, intestines and spleen, and the DNA damage was evidenced by increased 8-OHdG level in intestines after H2O2 exposure. Moreover, cell cycle regulation-related genes, including cyclin A1, B and/or E1, highly expressed in all tested tissues except heart after H2O2 exposure. Interestingly, IBR analysis exhibited that immune, inflammatory, autophagic and DNA damage responses to H2O2 exposure were in a dose-dependent and tissue-specific manner. These data may contribute to understanding H2O2 toxicity for fish and assessing potential risk of H2O2 in aquatic environment.

Introduction

Hydrogen peroxide (H2O2) is a stable ROS (reactive oxygen species) with longer half-life in aquatic environment (Szymczak and Waite, 1988). The major source of H2O2 is generated by photochemical reaction with dissolved organic matter (DOM) in natural water (Cooper et al., 1988). It is also derived from atmospheric wet or dry deposition into aquatic ecosystem (Kang et al., 2002). In particle-rich eutrophic water, H2O2 is mainly originated from bacteria and algae secretion (Cory et al., 2016; Vermilyea et al., 2010). In addition, anthropogenic activities, such as the application of H2O2 in wastewater treatment, soil remediation and aquaculture, may directly lead to H2O2 accumulation in aquatic environment (Jawad et al., 2016; Overton et al., 2018). In natural water, H2O2 level varies considerably, ranging from 0.004 to 199 μM. Typical concentrations of H2O2 have been recorded in river (0.09–3.2 μM) (Cooper and Zika, 1983; Sinel'nikov, 1971), lake (0.00–5.31 μM) (Ndungu et al., 2019), estuary (0.01–0.35 μM) (Kieber and Helz, 1995), open ocean (0.06–0.45 μM) (Fujiwara et al., 1993) and rain (0–199 μM) (Sakugawa et al., 1990). It indicates that higher level of H2O2 is likely to occur in aquatic environment.

In aquaculture, H2O2, as an environment-friendly therapeutic compound, has been approved in many countries for the treatment of external diseases of fish (Burridge et al., 2010; Valenzuela-Muñoz et al., 2020; Yanong, 2008). It has been reported that up to 135 million kilogram of H2O2 was applied in salmon farms in Norway during 2009–2018 (Bechmann et al., 2019). H2O2 can effectively treat parasitic, fungal and bacterial infection in fish and fish eggs (Avendanoherrera et al., 2006; Montgomery-Brock et al., 2001; Wynne et al., 2020). It is also a pharmaceutical alternative for the control of sea lice in the salmon industry (Valenzuela-Muñoz et al., 2020). In recirculating aquaculture systems, low concentration of H2O2 improves water quality and controls parasitic loads (Pedersen et al., 2012; Pedersen and Pedersen, 2012). Moreover, H2O2 is considered as a feasible algaecide to eliminate cyanobacteria and cyanotoxins in aquatic environment (Wang et al., 2019a). Despite its low toxicity, the ecological risk related to application of H2O2, and its effects on animal, microorganism and zooplankton in aquatic environment have attracted much attention from researchers (Ndungu et al., 2019; Rach et al., 1998; Rosa et al., 2005).

As a strong oxidizing agent, high concentration of H2O2 in aquatic environment is harmful to the aquatic animals, which may result in abnormal physiological and behavioral changes, or even cause lethal and sub-lethal effects under certain condition. Early study showed that, in fish, the toxicity of H2O2 was associated with species, life stage and water temperature. The median lethal concentration (LC50, 1 h) of H2O2 was 183–260 ppm for rainbow trout (Oncorhynchus mykiss), 1640–2480 ppm for channel catfish (Ictalurus punctatus) and 1310–1630 ppm for bluegill (Lepomis machrochirus) at 22 °C (Rach et al., 1997). In rainbow trout, after 1–1.5 h of exposure, higher level of H2O2 (200 mg/L) caused gills damage and high mortality (Tort et al., 2002), while lower level of H2O2 (50 ppm) induced oxidative stress and lipid peroxidation in gills (Seker et al., 2015). Similarly, sub-lethal concentration of H2O2 (50 ppm, 1 h) resulted in physiological stress response, such as increased levels of plasma cortisol, lactate and glucose, in sea bass (Dicentrarchus labrax) (Ana et al., 2010). The adverse immune response was also observed in olive flounder (Paralichthys olivaeceus) (100–500 ppm) and seabream (Sparus aurata) (50 ppm) after H2O2 exposure for 1 h (Hwang et al., 2016; Mansour et al., 2020). Moreover, some species of fish were more sensitive to H2O2, and low tolerance concentration to H2O2 was seen in blue gourami (Trichogaster trichopterus) (<11.4 mg/L) and suckermouth catfish (Hypostomus plecostomus) (<6.6 mg/L) after 1 h of exposure (Yanong, 2008). Altogether, H2O2 (up to a certain concentration) exposure could lead to adverse effects on fish, and the existing studies mainly focused on its acute toxicity. However, the data regarding chronic toxicity of H2O2 on fish were very few.

This study aimed to investigate the physiological and molecular responses of fish to H2O2 and explore the underlying toxicity mechanism after long-term exposure. For this purpose, we selected common carp (Cyprinus carpio), a global farmed or wild species, as an animal model. Here, we exposed the common carp to lower concentrations of H2O2 (0.25–1 mM) for 14 days, and then observed the changes of immunity, inflammation, autophagy and DNA damage in different tissues. To the best of our knowledge, this is the first study to evaluate the potential response mechanism to H2O2 based on immune, inflammatory, autophagic and DNA damage responses in different tissues of fish after long-term exposure. These data provided valuable new insight into the response mechanism to H2O2 exposure in fish, which might contribute to the risk assessment of H2O2 in aquatic environment.

Section snippets

Animals and H2O2 exposure

A total of 160 FFRC carps, a new strain of common carp bred by Freshwater Fish Research Center (FFRC), were provided by a local farm (Wuxi, China). The average weight of the fish was 53 ± 3 g. Prior to the experiment, the fish were kept in an indoor recirculating aquaculture system, with 20 glass tanks (each tank 240 L), for two weeks to adapt to the experimental condition. In the aquaculture system, the temperature, dissolved oxygen and PH were maintained at 26 ± 2 °C, >6 mg/L and 7.4–8.1,

Changes of stress indices in serum

After 14 days, the physiological stress parameters including cortisol, lactic acid and glucose were clearly changed by H2O2 exposure (Fig. 1). The levels of cortisol and lactic acid linearly rose with the increase of H2O2 concentration, and significant difference was seen in cortisol in 1 mM H2O2 treatment group and lactic acid in 0.5 and 1 mM H2O2 treatment groups (Fig. 1A and B). The glucose level showed a significant increase under 0.25 mM H2O2 exposure, but an obvious decrease was seen

Discussion

This work is the first to report the complex response mechanism to H2O2 chronic exposure in different tissues of fish. Herein, we demonstrated immune, inflammatory, autophagic and DNA damage responses in liver, gills, muscle, intestines, heart, spleen and kidney of common carp under H2O2 exposure. Meanwhile, we compared the differences of response mechanism to H2O2 challenge among different tissues via IBR analysis. Further, we speculated the role of some signaling molecules, such as TLRs,

Conclusions

In the present study, we systematically investigated the potential response mechanism to H2O2 exposure in common carp. H2O2 exposure caused an immune response accompanied by elevated or suppressed immune parameters in different tissues of common carp. After H2O2 exposure, particularly 1 mM H2O2, the TLRs and NF-κB pathways were activated to induce inflammatory genes expression in the majority of tested tissues. We also found a promoted autophagic response in liver, gill, muscle, intestines,

CRediT authorship contribution statement

Rui Jia: Writing – original draft, Methodology. Jinliang Du: Validation, Resources. Liping Cao: Formal analysis. Wenrong Feng: Methodology, Investigation. Qin He: Data curation, Investigation. Pao Xu: Supervision, Funding acquisition. Guojun Yin: Conceptualization, Project administration, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by National Natural Science Foundation of China (NO.31702318), and Central Public-interest Scientific Institution Basal Research Fund, Freshwater Fisheries Research Center, CAFS (2019JBFM10, 11).

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