Heat Shock Proteins and Cytochrome P450 1a1 Expression as Biomarkers of Exposure to Bisphenol A in Catla catla

been used as biomarker of exposure in various fish species and other aquatic organisms (Vijayan et al., 1998; Chen et al., 2011; Jin et al., 2010). The results of these studies showed that heat shock proteins are sensitive markers of chemical exposure and use of molecular approaches towards this end can be very promising.

Gene expression studies are now increasingly used to determine protein and mRNA levels and can be good prognostic parameters for predicting protein metabolism (Chen et al., 2011). Moreover, measuring mRNA level is sensitive method and provide mechanistic approach to evaluate toxic endpoints. Therefore, in the present study, cytochrome P4501A1 and heat shock protein 70 and 90 expression was studied as potential biomarkers of exposure to BPA in a commercial edible fish, Catla catla.

A stock solution of BPA was prepared in ethanol (2mg/ml), exposure concentrations (10, 100, 1000µg/l) were prepared by dissolving stock solution in aquarium water. Female C. ctala were purchased from a commercial fish farm. After acclimatization for two weeks, fish were divided into four groups (n=3) in replicate and exposed to various concentrations (10, 100 and 1000 µg/l) of BPA for 14 days. Toxicant solution was replaced every other day. At the end of exposure period, fish were anesthetized with clove oil and length and weight were measured. Fish were humanely dissected and liver was removed, snap frozen in liquid nitrogen and stored at -80oC until further analysis.

RNA was isolated from frozen tissues using Trizol reagent, cDNA was prepared with 1µg of total RNA using Reverse aid MMLV cDNA synthesis kit (Thermo Fischer Scientific). Geometric mean of tata box binding protein and glyceraldehyde-3-phosphate was used as reference control following Faheem et al. (2018). Gene sequence was obtained from National Center for Biotechnology Information (NCBI) and primers for all genes were designed using primer 3plus software. Primer sequence, gene accession number and product size is listed in Table I. Real time q-PCR was performed on CFX 96 (Bio-Rad, Hercules, CA, USA) with Syber green fluorescent label. A melt curve analysis was performed at the end of reaction to ensure amplification of the single product and PCR product was run on 1.5 % agarose gel. Ct values generated by software were converted to fold change relative to control using 2-∆∆Ct method as described by Livak and Schmittgen (2001).

Gene expression analysis is expressed as mean±S.E.M. Data was checked for homogeneityof varience through Shapiro-Wilk test. To evaluate significant difference among groups (p<0.05), one way analysis of variance was used followed by Tukey’s post hoc test. All statistical analysis was performed using IBM SPSS (Version: 20).

Primers of genes were specific in binding and yielded a single band of desired product size (Fig. 1). Expression of cytochrome P450 1a1 gene relative to control is shown in Figure 2. The cytochrome P450 enzymes metabolize toxic compounds and CYP1A1 is most important as it is involved in metabolism of estrogens (Whyte et al., 2000). In the present study, effect of BPA was studied on mRNA expression of cyp1a1 in liver of Catla catla which is endemic to Pakistan and commercially important species. Graded concentration of BPA (10,100,1000µg/l) for 14 days resulted in marked decrease in cyp1a1 transcript level in a concentration dependent manner. Similar decreases in cyp1a1 level were reported in Atlantic cod exposed to 50 µg/l of BPA for 21 days (Olsvik et al., 2009). Similarly, a dietary exposure of bisphenol A (5 and 50 mg/kg bw) for 21 days in Sparus aurata juveniles resulted in significant down regulation of cyp1a1 level. In present study, down-regulation of cyp1a1 recorded in all treatment groups (Fig. 2A) is in agreement with available data on the cross-talk between the reproductive system and the detoxification pathway (Jeong et al., 2001) and is supported by studies showing an inverse relationship between plasma E2 levels and EROD activity during the reproductive cycle (Arukwe and Goksøyr, 1997). Faheem et al. (2017) recently reported that exposure of same concentrations of BPA to Catla catla females resulted in increased plasma estradiol concentration. The decrease in cyp1a1 transcript level may be the cause of increased E2 levels reported by Faheem et al. (2017), as elevated E2 concentrations are known to be associated with decreases in CYP1A1 activity (Whyte et al., 2000). These studies and results of present study confirmed that estrogenic compounds like BPA inhibits the detoxification process and therefore, mRNA level of key gene (cyp1A1) can be used as potential biomarker to confirm presence of estrogenic compounds in water bodies.

Heat shock proteins (HSPs) are highly conserved cellular proteins present in all organisms studied to date and are known to function as stress proteins or molecular chaperones (Sung et al., 2011; Dang et al., 2018). HSP levels are mostly studied to assess the toxicity in organism both in laboratory conditions and at polluted environmental sites and therefore used as biomarkers to evaluate toxicity and overall ecological status of a water body (de Pomerai, 1996). In the present study, mRNA level of HSP 70 and HSP 90 was evaluated after 14 days’ exposure to gradient concentrations of BPA. Expression of heat shock protein 70 and 90 (Fig. 2B). Exposure of 10µg/l BPA for 14 days caused 3 folds’ significant increase in mRNA level of HSP 70 while 2.4-fold increase was recorded in fish exposed to 1000µg/l BPA for 14 days (Fig. 2B). Exposure of 10,100 and 1000µg/l BPA resulted in significantly higher expression of HSP 90 in liver of Catla catla. Pufferfish (Takifugu obscurus) exposed to 600 µg/l of bisphenol-A and 300 µg/l of 4-tert-octylphenol for 96 h resulted in significant upregulation of hsp 70 and hsp 90 after 24 h till 96 h (Kim et al., 2013). Sparus aurata juveniles fed with octylphenol for 21 days resulted in significant increased HSP70 mRNA expression compared to control (Traversi et al., 2014). Male and female black goby injected with nonylphenol and estradiol resulted in significantly increased expression of hsp70 mRNA after 72 hours (Maradonna and Carnevali, 2007). Many earlier studies also confirmed the elevated expression of HSP mRNA in fish exposed to pollutants hence, it can be used as a biomarker of exposure in fish (Rajeshkumar et al., 2013).

In conclusion, the inhibition of cyp1a1 and elevation of hsp transcript result in decreased detoxification ability which may lead to compromised health of organisms living in areas contaminated with environmental estrogens such as BPA. Our results offer baseline information which can be used for further research related to the EDCs and use of native carps like Catla catla as a bio-indicator for monitoring environmental health status.

The authors have declared no conflict of interest.