Potential mechanisms of Na+/K+-ATPase attenuation by heat and pesticides co-exposure in goldfish: role of cellular apoptosis, oxidative/nitrative stress, and antioxidants in gills.

In this study, we examined the dose-dependent effects of an environmentally relevant pesticide cocktail (metalachlor, linuron, isoproturon, tebucanazole, aclonifen, atrazine, pendimethalin, and azinphos-methyl) and temperature change (22 vs. 32 °C for 4-week exposure) on Na+/K+-ATPase, 3-nitrotyrosine protein (NTP), dinitrophenyl protein (DNP), catalase (CAT), and superoxide dismutase (SOD) expressions in gills of goldfish (Carassius auratus). Histopathological analysis showed widespread damage to gill in elevated temperature (32 °C) and pesticide co-exposure groups, including fusion of secondary lamellae, club-shaped primary lamellae, rupture of epithelial layer, loss of normal architecture, and hemorrhaging. Immunohistochemical and qRT-PCR analyses showed significant decreases in Na+/K+-ATPase protein and mRNA expressions in gills exposed to higher temperature and pesticides; however, combined exposure to heat and pesticides significantly increases NTP, DNP, CAT, and SOD expressions. In situ TUNEL assay revealed elevated levels of apoptotic cells in response to combined exposure. Collectively, our results suggest the combined effects of heat and pesticide stress cause cellular damage, upregulate oxidative/nitrative stress biomarkers, and increase apoptotic cells, downregulate Na+/K+-ATPase expression in gills. This provides new evidence for oxidant/antioxidant-dependent mechanisms for downregulation of Na+/K+-ATPase expression in gills during combined exposure.

Elevated seasonal temperature disrupts prooxidant-antioxidant homeostasis and promotes cellular apoptosis in the American oyster, Crassostrea virginica, in the Gulf of Mexico: a field study.

One of the major impacts of climate change has been the marked rise in global temperature. Recently, we demonstrated that high temperatures (1-week exposure) disrupt prooxidant-antioxidant homeostasis and promote cellular apoptosis in the American oyster. In this study, we evaluated the effects of seasonal sea surface temperature (SST) on tissue morphology, extrapallial fluid (EPF) conditions, heat shock protein-70 (HSP70), dinitrophenyl protein (DNP, an indicator of reactive oxygen species, ROS), 3-nitrotyrosine protein (NTP, an indicator of RNS), catalase (CAT), superoxide dismutase (SOD) protein expressions, and cellular apoptosis in gills and digestive glands of oysters collected on the southern Texas coast during the winter (15 °C), spring (24 °C), summer (30 °C), and fall (27 °C). Histological observations of both tissues showed a notable increase in mucus production and an enlargement of the digestive gland lumen with seasonal temperature rise, whereas biochemical analyses exhibited a significant decrease in EPF pH and protein concentration. Immunohistochemical analyses showed higher expression of HSP70 along with the expression of DNP and NTP in oyster tissues during summer. Intriguingly, CAT and SOD protein expressions exhibited significant upregulation with rising seasonal temperatures (15 to 27 °C), which decreased significantly in summer (30 °C), leaving oysters vulnerable to oxidative and nitrative damage. qRT-PCR analysis revealed a significant increase in HSP70 mRNA levels in oyster tissues during the warmer seasons. In situ TUNNEL assay showed a significant increase in apoptotic cells in seasons with high temperature. These results suggest that elevated SST induces oxidative/nitrative stress through the overproduction of ROS/RNS and disrupts the antioxidant system which promotes cellular apoptosis in oysters.

Effects of elevated temperature on prooxidant-antioxidant homeostasis and redox status in the American oyster: Signaling pathways of cellular apoptosis during heat stress.

Increasing seawater temperature affects growth, reproduction, development, and various other physiological processes in aquatic organisms, such as marine invertebrates, which are especially susceptible to high temperatures. In this study, we examined the effects of short-term heat stress (16, 22, 26, and 30 °C for 1-week exposure) on prooxidant-antioxidant homeostasis and redox status in the American oyster (Crassostrea virginica, an edible and commercially cultivated bivalve mollusk) under controlled laboratory conditions. Immunohistochemical and real-time quantitative PCR (qRT-PCR) analyses were performed to examine the expression of heat shock protein-70 (HSP70, a biomarker of heat stress), catalase (CAT, an antioxidant), superoxide dismutase (SOD, an antioxidant), dinitrophenyl protein (DNP, a biomarker of reactive oxygen species, ROS), and 3-nitrotyrosine protein (NTP, an indicator of reactive nitrogen species, RNS), in the gills and digestive glands of oysters. In situ TUNEL assay was performed to detect cellular apoptosis in tissues. Histological analysis showed an increase in mucus secretion in the gills and digestive glands of oysters exposed to higher temperatures (22, 26, and 30 °C) compared to control (16 °C). Immunohistochemical and qRT-PCR analyses showed significant increases in HSP70, DNP and NTP protein, and mRNA expressions in tissues at higher temperatures. Cellular apoptosis was also significantly increased at higher temperatures. Thus, heat-induced oxidative and nitrative stress likely occur due to overproduction of ROS and RNS. Interestingly, expression of CAT and SOD increased in oysters exposed to 22 and 26 °C, but was at or below control levels in the highest temperature exposure (30 °C). Collectively, these results suggest that elevated seawater temperatures cause oxidative/nitrative stress and induce cellular apoptosis through excessive ROS and RNS production, leading to inhibition of the antioxidant defense system in marine mollusks.