Available at: http://digitalcommons.calpoly.edu/theses/1636
Date of Award
MS in Biological Sciences
Dr. Sean Lema
Recent evidence indicates that some of California’s coastal estuaries are contaminated with the chemical 4-nonylphenol (4-NP). Tissue burdens of 4-NP detected in the intertidal arrow goby (Clevelandia ios) in California are among the highest recorded worldwide, however, it remains unknown whether these fish are impacted by this 4-NP contamination. 4-NP is an established endocrine disrupting compound with estrogenic properties that can alter reproductive function. Furthermore, evidence that estrogens can modulate iono- and osmo-regulatory function in fishes implies that estuarine fishes exposed to 4-NP may also experience an impaired ability to maintain hydromineral balance. In Chapter 1 of this research, the time course of detectable xenoestrogen biomarker responses including gene transcripts encoding vitellogenins (vtgA and vtgC), choriogenins (chgL and chgHm), and estrogen receptors esr1 and esr2a were examined using quantitative real-time reverse transcription PCR (qRT-PCR) in adult male arrow gobies exposed to either 4-NP or E2. Specifically, adult gobies were treated with 4-NP at 10 μg/L (low 4-NP dose), or 4-NP at 100 μg/L (high 4-NP dose), ethanol vehicle (negative control), or 17β-estradiol (E2) at 50 ng/L (positive control) for 21 days. This 21 day exposure period was following by a 21 day depuration period to assess the time pattern of biomarker recovery. Results from these experiments indicated that 4-NP can induce increases in relative mRNA levels encoding vitellogenins, choriogenins, and estrogen receptor esr1 – but not esr2a – in the liver within 72 hrs, and that these transcriptional changes return to pre-exposure levels within 12 days of the termination of 4-NP or E2 exposure. In sum, these findings validate the use of mRNA levels for several estrogen-responsive genes as accurate biomarkers for short-term 4-NP exposure in the arrow goby. In Chapter 2, I evaluated the effects of 4-NP and E2 exposures on the osmoregulatory ability of C. ios. I exposed adult arrow gobies to 4-NP (10 μg/L or 100 μg/L) or E2 (50 ng/L) for 14 days, and then transferred the fish from a 33 ppt salinity (control) environment to either 20 ppt, or 5 ppt conditions. Whole body water content was then measured, and the relative mRNA levels for the ion channels Na+/K+/2Cl--cotransporter1 (nkcc1) and Na+/H+ exchanger-3 (nhe3), and the aquaporin water channel aquaporin-3 (aqp3) were quantified in the gill epithelium by qRT-PCR. Results showed that fish treated with 4-NP exhibited higher whole body water content, suggesting that 4-NP exposure results in excessive water uptake during hypoosmotic challenge. 4-NP treated gobies also exhibited elevated nkcc1 and reduced nhe3 and aqp3 mRNAs in the gill even prior to transfer of fish from the 33 ppt acclimation salinity. At 6 hrs after salinity transfer, transcripts encoding nkcc1 remained elevated in the gill epithelium of 4-NP treated gobies transferred to 20 ppt or maintained at 33 ppt (salinity control), while nhe3 and aqp3 mRNAs were still less abundant in gills of these fish. These findings point to impaired maintenance of water balance in gobies exposed to 4-NP, with those changes in fluid homeostasis possibly mediated in part by changes in gill ionic regulation. Taken as a entirety, the findings provided by this research reinforces accumulating data showing the potential for 4-NP to disrupt reproductive physiology in vertebrates, and points to the possibility that 4-NP may impair the ability of fish to regulate ion and water balance under changing salinity conditions.