Elsevier

Aquatic Toxicology

Volumes 118–119, 15 August 2012, Pages 108-115
Aquatic Toxicology

Characterization of a bystander effect induced by the endocrine-disrupting chemical 6-propyl-2-thiouracil in zebrafish embryos

https://doi.org/10.1016/j.aquatox.2012.04.001Get rights and content

Abstract

This study was conducted to evaluate possible bystander effects induced by the model chemical 6-propyl-2-thiouracil (PTU) on melanin synthesis. Zebrafish (Danio rerio) embryos were treated with PTU by either microinjection exposure, via waterborne exposure or indirectly through bystander exposure. Melanin content, related mRNA and protein expression were examined at the end of exposure (36 h post-fertilization). Direct exposure to PTU decreased the melanin content, up-regulated mRNA expressions of oculocutaneous albinism type 2 (OCA2), tyrosinase (TYR), dopachrometautomerase (DCT), tyrosinase-related protein 1 (TYRP1) and silver (SILV), and increased the protein expressions of TYR and SILV. Bystander exposure also up-regulated mRNA and protein expressions of TYR and SILV but increased melanin contents. Correlation analysis demonstrated that mRNA expressions of OCA2, TYR, DCT, TYRP1, SILV and protein expressions of TYR and SILV in bystander exposure groups were positively correlated with corresponding expressions in microinjection exposure groups. The results might have environmental implications and highlight the need to consider the bystander effects when assessing potential risks of endocrine-disrupting chemicals.

Highlights

► The study is one of the first to evaluate bystander effects of EDCs in living organisms. ► Characterizing the effect is important to understand the impact of EDCs on unexposed animals. ► The results highlight the need to consider the bystander effects when assessing risks of EDCs.

Introduction

Some chemicals can modulate the endocrine system either directly as hormone mimics or indirectly by affecting various signal transduction pathways or enzyme systems (Gracia et al., 2006, Solomon et al., 2008, Zhang et al., 2005, Zhang et al., 2008a, Zhang et al., 2008b). Such chemicals are now commonly referred to as endocrine-disrupting compounds (EDCs). There have been reports of abnormalities in the endocrine systems of wildlife and humans that have been attributed to exposure to EDCs (Guillette and Gunderson, 2001, Rasier et al., 2006), which made US unveil new law that required Environmental Protection Agency (EPA) to develop and validate test systems to identify the EDCs in food and water (EDSP; U.S. EPA, 2006).

In fish, the endocrine system contains a number of hormones and signal transduction pathways as well as hundreds of pheromones. Intra-specific communication by means of pheromones, behavior and some metabolic products is well accepted in fish (Bergman and Moore, 2003, Giaquinto and Volpato, 1997, Liley, 1982, Rowland, 1999). Pheromones, behavior or related metabolites could cause altered physiology, metabolism or behaviors to maintain a constant degree of coordination between organisms (Bergman and Moore, 2003, Giaquinto and Volpato, 1997, Liley, 1982, Rowland, 1999). Therefore, an EDC that can disrupt endocrine function in one individual might cause a corresponding effect in individuals with which it comes into contact that were not originally exposed to the EDC. This is defined as the “bystander effect” in unexposed organisms. This effect is not caused by chemical(s) released from exposed animals, but is a result of exposed animals experiencing some behavioral/physiological (e.g., pheromones) alteration due to exposure, which is then having an impact on the bystanders, resulting in bystander effect.

Different in vivo and in vitro testing systems are currently used to assess the risks of exposure to EDCs already in the environment and of new chemicals that might be released in the future. One of the in vivo testing methods is the fish toxicity bioassay, in which individuals are exposed to constant concentrations of chemicals in static renew or continuous flow exposure systems. However, in the real world, concentrations of chemicals fluctuate, and avoidance and migration behaviors of fish result in that animals have different exposure histories. It is common that exposed and unexposed individuals spend at least some time in each other's proximity. Therefore, in the present study, a new exposure approach, “bystander exposure”, was developed to investigate potential interactions between organisms exposed to different concentrations of chemicals. Specifically, in the present study, the model chemical 6-propyl-2-thiouracil (PTU) was chosen as a reference agent to study possible bystander effects on the melanin synthesis pathways of zebrafish embryos. This pathway was chosen because the results of previous study had demonstrated that color patterns in fish are multicomponent signals, which can be used to communicate between and among individual fish by physiological regulation or behavioral change (Price et al., 2008). In addition, this pathway allows simple observation without complicated experimental procedures. Zebrafish embryos have been suggested as an appropriate model for screening of melanogenic regulatory compounds (Choi et al., 2007). PTU is an inhibitor of tyrosinase that is used routinely to inhibit synthesis of melanin in zebrafish (Choi et al., 2007, Dryja et al., 1978, Peterson et al., 2000). It was hypothesized that PTU exposure could cause some behavioral/physiological (e.g., pheromones) alteration, which is then having an impact on the bystanders, subsequently causing the induction of bystander effects on melanin synthesis pathways.

Section snippets

Materials and reagents

TRIzol regent and M-MLV reverse transcription kits were obtained from Invitrogen (New Jersey, NJ, USA) and Promega (Madison, WI, USA), respectively. SYBR Green kits and PTU were purchased from Toyobo (Tokyo, Japan) and Sigma (St. Louis, MO, USA), respectively. Primary antibodies against tyrosinase (TYR) and silver (SILV) were obtained from Epitomics (Burlingame, CA, USA) and Everest Biotech Ltd (Oxfordshire, United Kingdom), respectively.

PTU exposure protocol

Adult zebrafish (25-week-old) were maintained according

Melanin content

No significant effects on survival rates were observed after three different methods of exposure to PTU. Survival rates were >90% in all the exposure groups including controls. Waterborne exposure to 18 mg PTU/L resulted in significantly less melanin content (53.3%) in zebrafish embryos compared with the control, while exposure to lesser concentrations (0.018, 0.18 or 1.8 mg PTU/L) did not change melanin production (Fig. 2). Waterborne exposure to 0.075, 0.75 and 7.5 μg/L PTU did not change melanin

Discussion

The model chemical PTU is a TYR inhibitor and exposure to PTU could significantly decrease melanin production in zebrafish (Choi et al., 2007). The results reported here were consistent with previous observations. Both waterborne and microinjection exposure of embryos to PTU resulted in significantly less production of melanin. Furthermore, mRNA and protein expression analysis demonstrated that PTU exposure significantly up-regulated OCA2, TYR, DCT, TYRP1 and SILV mRNA expression and increased

Acknowledgements

The research was supported by a grant from Major State Basic Research Development Program (No. 2008CB418102) and a grant from Nanjing University Talent Development Foundation. This work was also supported by the National Nature Science Foundation of China (NSFC) Grant 20890113 and 31000249 to B.-S. Zhou and J.-H. Wang, respectively. The research was supported, in part, by a Discovery Grant from the National Science and Engineering Research Council of Canada (Project # 326415-07). The authors

References (49)

  • M. Saaristo et al.

    Sand goby (Pomatoschistus minutus) males exposed to an endocrine disrupting chemical fail in nest and mate competition

    Hormones and Behavior

    (2009)
  • M. Saaristo et al.

    An endocrine disrupting chemical changes courtship and parental care in the sand goby

    Aquatic Toxicology

    (2010)
  • H.B. Schonthaler et al.

    A mutation in the silver gene leads to defects in melanosome biogenesis and alterations in the visual system in the zebrafish mutant fading vision

    Developmental Biology

    (2005)
  • X. Zhang et al.

    Real-time PCR array to study effects of chemicals on the hypothalamic-pituitary-gonadal axis of the Japanese medaka

    Aquatic Toxicology

    (2008)
  • G.T. Ankley et al.

    Dynamic nature of alterations in the endocrine system of fathead minnows exposed to the fungicide prochloraz

    Toxicological Sciences

    (2009)
  • R. Asur et al.

    Bystander effects induced by chemicals and ionizing radiation: evaluation of changes in gene expression of downstream MAPK targets

    Mutagenesis

    (2010)
  • D.A. Bergman et al.

    Field observations of intraspecific agonistic behaviour of two crayfish species, Orconectes rusticus and Orconectes virilis, in different habitats

    Biological Bulletin

    (2003)
  • S. Boonanuntanasarn et al.

    Melicular cloning, gene expression in albino mutants and gene knockdown studies of tyrosinase mRNA in rainbow trout

    Pigment Cell Research

    (2004)
  • I. Braasch et al.

    Evolution of pigment synthesis pathway by gene and genome duplication in fish

    BMC Evolutionary Biology

    (2007)
  • E. Camp et al.

    Tyrosinase gene expression in zebrafish embryos

    Development Genes and Evolution

    (2001)
  • T.Y. Choi et al.

    Zebrafish as a new model for phenotype-based screening of melanogenic regulatory compounds

    Pigment Cell Research

    (2007)
  • V.W.Y. Choi et al.

    Radioadaptive response induced by alpha-particle-induced stress communicated in vivo between zebrafish embryos

    Environmental Science & Technology

    (2010)
  • H. Dabrowska et al.

    Dietary uptake efficiency of 2,2′,4,4′,5,5′-hexachlorobiphenyl in yellow perch and rainbow trout: role of dietary and body lipids

    Environmental Toxicology and Chemistry

    (1999)
  • I.A. Drummond

    Kidney development and disease in the zebrafish

    Journal of the American Society of Nephrology

    (2005)
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