Research Studies
Study Tracker
Mixture effect of arsenic and fluoride at environmentally relevant concentrations in zebrafish (Danio rerio) liver: Expression pattern of Nrf2 and related xenobiotic metabolizing enzymes.
| Aquatic Toxicology | Mondal P, Shaw P, Bandyopadhyay A, Dey Bhowmik A, Chakraborty A, Sudarshan M, Chattopadhyay A. |
Zebrafish Arsenic Chemical Co-Exposures Liver Mechanisms of Cellular Toxicity Oxidative Stress Animal Study

Abstract

Original absract online at
https://www.sciencedirect.com/science/article/abs/pii/S0166445X19302942

Highlights

  • Arsenic and fluoride caused oxidative stress both individually and in combination.
  • Co-exposure caused less alteration in GSH level, MDA content and CAT activity.
  • Expression of Nrf2 and XMEs showed least sensitivity towards the ‘mixture effect’.
  • An antagonistic relationship between arsenic and fluoride was evident.

Nrf2 is a crucial transcription factor that regulates the expression of cytoprotective enzymes and controls cellular redox homeostasis. Both arsenic and fluoride are potent toxicants that are known to induce Nrf2. They are reported to coexist in many areas of the world leading to complex mixture effects in exposed organisms. The present study investigated the expression of Nrf2 and related xenobiotic metabolizing enzymes along with other stress markers such as histopathological alterations, catalase activity, reduced glutathione content and lipid peroxidation in zebrafish liver as a function of combined exposure to environmentally relevant concentrations of arsenic (37.87  gL-1 or 5.05 × 10-7 M) and fluoride (6.8 mg L-1 or 3.57 × 10-4 M) for 60 days. The decrease in the total reduced glutathione level was evident in all treatment conditions. Hyperactivity of catalase along with conspicuous elevation in reactive oxygen species, malondialdehyde content and histo-architectural anomalies signified the presence of oxidative stress in the treatment groups. Nrf2 was seen to be induced at both transcriptional and translational levels in case of both individual and co-exposure. The same pattern was observed in case of its nuclear translocation also. From the results of qRT-PCR it was evident that at each time point co-exposure to arsenic and fluoride seemed to alter the gene expression of Cu/Zn Sod, Mn Sod, Gpx and Nqo1 just like their individual exposure but at a very low magnitude. In conclusion, this study demonstrates for the first time the differential expression and activity of Nrf2 and other stress response genes in the zebrafish liver following individual and combined exposure to arsenic and fluoride.

Introduction

Organisms are concurrently exposed to multiple hazardous chemicals due to complexity of the surrounding environment. These lead to elicitation of unique, complex biological response. Efforts to identify the ‘mixture effects’ of such co-occurring contaminants might lead to a clear understanding on organisms’ respond to their complex environment. Arsenic (As) and fluoride (F) are two notable toxicants known to coexist in nature. Their co-occurrence in water are reported in many areas of Latin America (Argentina, Bolivia, Chile, Colombia, Peru and Mexico) (González-Horta et al., 2015; Alarcón-Herrera et al., 2013), Asia (India, China, Japan, Korea and Pakistan) (NRC, 2006; Dutta, 2013) and Africa (Ethiopia, Ghana, Nigeria and Tanzania) (Mahlangu et al., 2012; Yang et al., 2013). According to WHO, 2011, the permissible limits of As and F are 10 ?gL?1 (1.33?×?10-7 M) and 1.5?mg?L-1 (0.789?×?10-4 M) respectively. Besides groundwater, significantly high concentrations of As or F are also reported in rice, a staple food in India and many other South Asian countries, through which such contamination can spread to areas where groundwater is not the primary source (Mondal and Polya, 2008; Bhattacharya et al., 2017). Arora and Bhateja (2014) observed a significant relation between mean F concentration of the soil and mean F concentration of rice and wheat.

Extensive research have been conducted in recent years on the genotoxic, hepatotoxic and neurotoxic potential of As or F exposure alone in various organisms particularly in mammals and fish (Liu et al., 2007; Sarkar et al., 2017; Chattopadhyay et al., 2011; Mukhopadhyay et al., 2015; Podder et al., 2010) whereas reports on the biological response to their combined effects are rare and ambiguous. Some reports suggest that simultaneous exposure to As and F tend to neutralize each other exhibiting antagonism (Flora et al., 2009) though reports on their synergistic effects are also available (Tiwari and Rao, 2010; Jhala et al., 2008). Therefore more in depth studies are required on this aspect particularly on the modulation of cellular antioxidant system. The present study aims to address this issue focusing on the role of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), the master regulator of intracellular oxidative stress response, and related xenobiotic metabolizing enzymes (XMEs). Nrf2 belongs to the basic leucine zipper nuclear transcription factor family with Cap ‘n’ collar (CNC) structure in its C-terminal region (Ma, 2013). In the state of redox homeostasis, it remains bound to cytoskeleton-associated kelch-like ECH-associated proteins 1 (Keap1) that prevents its nuclear translocation and promotes cytoplasmic degradation (Itoh et al., 1999; Jaiswal, 2004). In stressed cells, the formed reactive oxygen species (ROS) interacts with Keap1, leading to release of Nrf2 and subsequent translocation into the nucleus. Inside the nucleus, Nrf2 binds to response elements on DNA; known as antioxidant response elements (AREs) located within the 5?-flanking promoter/enhancer region of its target genes and allow their transcriptional activation (Wasserman and Fahl, 1997). In addition to monitor the expression pattern of Nrf2, our work also attempted to study the expression of some important Nrf2 dependent XMEs like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and NAD(P)H:quinone oxidoreductase 1 (NQO1) that play indispensable role in maintaining cellular redox homeostasis. SODs, belonging to the first line of cellular defense against oxidative stress destroys superoxide radicals (O2?), the precursor of other reactive oxygen radicals (Ighodaro and Akinloye, 2018). On the basis of sub-cellular occurrence and requirement of co-factor, they are broadly classified into two types; cytosolic or copper/zinc SOD (Cu/Zn SOD) and mitochondrial or manganese SOD (Mn SOD) (Bhattacharya, 2015). They actively convert the O2– to hydrogen peroxide (H2O2) and molecular oxygen (O2), among which the former itself is a highly toxic free radical responsible for the formation of another deleterious ROS, hydroxyl radical (OH?) through Fenton reaction. CAT and GPX rescue the cell from such condition by breaking down H2O2 into water and molecular oxygen (Ighodaro and Akinloye, 2018). NQO1 is a Phase II enzyme that plays a crucial role in protection against endogenous and exogenous quinones and is considered as an important biomarker of toxicological stress.

The present study used zebrafish as the model organism because they respond with great sensitivity to the changes in the aquatic environment (Sarkar et al., 2017) and also share more than 70% orthologous genes with the human genome (Howe et al., 2013). We attempted to evaluate the combinatorial effect of As and F in fish liver because it is the major organ responsible for detoxification (Mukhopadhyay and Chattopadhyay, 2014).

Excerpts

Discussion

Previous studies about individual exposure of As and F suggested that their toxic effects are manifested by ROS formation due to electron leakage, enhanced mitochondrial activity and increased electron chain activity (Costantini et al., 2000). In our study too, ROS production, when measured through DCFDA method after 30 days of exposure, confirmed extensive production of ROS as a function of both individual and co-exposure to As and F. Interestingly co-exposed groups showed ROS production that

Conclusion

The present study provided the first experimental evidence where ‘mixture effects’ of environmentally relevant concentrations of As and F was evaluated with emphasis on the Nrf2-Keap1-ARE pathway after chronic exposure. We conclude that both the said toxicants when present together exert a much subdued effect on activation and functioning of the above mentioned cytoprotective pathway leading to less pronounced alteration in expression of XMEs. The probable cause of such an observation is that

Conflict of interest

The authors declare that there is no conflict of interest.

Acknowledgements

The authors express their gratitude to UGC-DAE-CSR (Grant no. UGC-DAE-CSR-KC/CRS/15/IOP/03), Kolkata, India for the financial assistance. PM and AB are grateful to Council of Scientific & Industrial Research, India for Junior Research Fellowship. PS and ADB acknowledge the Meritorious Fellowship and non-NET Fellowship from the University Grants Commission, India.