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. 2022 Dec 16:4:100079.
doi: 10.1016/j.fsirep.2022.100079. eCollection 2023 Dec.

Effects of elevated temperature on 8-OHdG expression in the American oyster (Crassostrea virginica): Induction of oxidative stress biomarkers, cellular apoptosis, DNA damage and γH2AX signaling pathways

Md Faizur Rahman  1 , Mohammad Maruf Billah  1 , Richard J Kline  1   2 , Md Saydur Rahman  1   2
Affiliations

Effects of elevated temperature on 8-OHdG expression in the American oyster (Crassostrea virginica): Induction of oxidative stress biomarkers, cellular apoptosis, DNA damage and γH2AX signaling pathways

Md Faizur Rahman et al. Fish Shellfish Immunol Rep. .

Abstract

Global temperature is increasing due to anthropogenic activities and the effects of elevated temperature on DNA lesions are not well documented in marine organisms. The American oyster (Crassostrea virginica, an edible and commercially important marine mollusk) is an ideal shellfish species to study oxidative DNA lesions during heat stress. In this study, we examined the effects of elevated temperatures (24, 28, and 32 °C for one-week exposure) on heat shock protein-70 (HSP70, a biomarker of heat stress), 8‑hydroxy-2'-deoxyguanosine (8-OHdG, a biomarker of pro-mutagenic DNA lesion), double-stranded DNA (dsDNA), γ-histone family member X (γH2AX, a molecular biomarker of DNA damage), caspase-3 (CAS-3, a key enzyme of apoptotic pathway) and Bcl-2-associated X (BAX, an apoptosis regulator) protein and/or mRNA expressions in the gills of American oysters. Immunohistochemical and qRT-PCR results showed that HSP70, 8-OHdG, dsDNA, and γH2AX expressions in gills were significantly increased at high temperatures (28 and 32 °C) compared with control (24°C). In situ TUNEL analysis showed that the apoptotic cells in gill tissues were increased in heat-exposed oysters. Interestingly, the enhanced apoptotic cells were associated with increased CAS-3 and BAX mRNA and/or protein expressions, along with 8-OHdG levels in gills after heat exposure. Moreover, the extrapallial (EP) fluid (i.e., extracellular body fluid) protein concentrations were lower; however, the EP glucose levels were higher in heat-exposed oysters. Taken together, these results suggest that heat shock-driven oxidative stress alters extracellular body fluid conditions and induces cellular apoptosis and DNA damage, which may lead to increased 8-OHdG levels in cells/tissues in oysters.

Keywords: 8-OHdG, 8‑hydroxy-2′-deoxyguanosine; BAX, bcl-2-associate X; BSA, bovine serum albumin; CAS-3, caspase-3; Caspase 3; DSBs, double-stranded breaks; EP, extrapallial; Extrapallial fluid; HSP70; HSP70, heat shock protein 70; Heat stress; Marine mollusks; PBS, Phosphate buffer saline; SSBs, single-stranded breaks; TUNEL, terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling; dsDNA breaks; dsDNA, double-stranded DNA; qRT-PCR, quantitative real-time polymerase chain reaction; ssDNA, single-stranded DNA; γ-H2AX, γ-histone family member X.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig 1
Fig. 1
Effects of one-week heat exposure on 8-hydroxy-2′-deoxyguanosine (8-OHdG) expression in gills of American oyster. (A-C) Histological appearance of representative photographs of gills collected from oysters exposed to control (CTL: 24 °C), medium temperature (MT: 28 °C), and high temperature (HT: 32 °C). Arrows indicate mucocyte (M) and mucus (Mu). (D-F) 8-OHdG expression in representative photographs of gills collected from oysters exposed to CTL, MT, and HT. Arrow indicates a higher 8-OHdG expression. (G) Negative control of 8-OHdG in gills of oyster. Scale bar = 100 µm. (H) Immunoreactive (IR) intensity of 8-OHdG in gills of oysters exposed to CTL, MT, and HT. Each value represents the mean ± SEM. Whiskers indicate the minimum and maximum values for each data set. The center line within the whisker box indicates the mean value. Data were analyzed with one-way ANOVA followed by Tukey's multiple comparison test. Different letters indicate significant differences (P<0.05) between experimental treatments (MT and HT) and the CTL.
Fig 2
Fig. 2
Effects of one-week heat exposure on 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels in gills of American oyster. (A) A representative photograph of 8-well assay strips containing serial dilutions of 8-OHdG standards and negative control (NC). (B) Standard curve of 8-OHdG. Each value represents the mean of duplicate determinations. (C) 8-OHdG levels. Each value represents the mean ± SEM. Whiskers indicate the minimum and maximum values for each data set. Center line within the whisker box indicates mean value. Asterisks indicate significant difference (Student t-test). Data were analyzed with one-way ANOVA followed by Tukey's multiple comparion test. Different letters indicate significant differences (P<0.05). CTL, control (24 °C); MT, medium temperature (28 °C), HT, high temperature (32 °C).
Fig 3
Fig. 3
Effects of one-week heat exposure on double-stranded DNA (dsDNA) expression in gills of American oyster. (A-C) dsDNA expression in representative photographs of gills collected from oysters exposed to control (CTL: 24 °C), medium temperature (MT: 28 °C), and high temperature (HT: 32 °C). Arrow indicates higher dsDNA expression. (D) Negative control of dsDNA expression in gills of oysters. Scale bar = 100 µm. (E) Immunoreactive (IR) intensity of dsDNA in gills of oysters exposed to CTL, MT, and HT. Each value represents the mean ± SEM. Whiskers indicate the minimum and maximum values for each data set. The center line within the whisker box indicates the mean value. Data were analyzed with one-way ANOVA followed by Tukey's multiple comparion test. Different letters indicate significant differences (P<0.05).
Fig 4
Fig. 4
Effects of one-week heat exposure on γ-histone family member X (γ-H2AX) protein expression in gills of American oyster. (A-C) γ-H2AX expression in representative photographs of gills collected from oysters exposed to control (CTL: 24 °C), medium temperature (MT: 28 °C), and high temperature (HT: 32 °C). Darker brown color (arrow) indicates higher γ-H2AX expression. (D) Negative control of γ-H2AX expression in gills of oysters. Scale bar = 100 µm. (E) Immunoreactive (IR) intensity of γ-H2AX in gills of oysters exposed to CTL, MT, and HT. Each value represents the mean ± SEM. Whiskers indicate the minimum and maximum values for each data set. The center line within the whisker box indicates the mean value. Data were analyzed with one-way ANOVA followed by Tukey's multiple comparion test. Different letters indicate significant differences (P<0.05).
Fig 5
Fig. 5
Effects of one-week heat exposure on heat shock protein 70 (HSP70) mRNA levels in gills of American oyster. Each value represents the mean ± SEM. Whiskers indicate the minimum and maximum values for each data set. Center line within the whisker box indicates mean value. Data were analyzed with one-way ANOVA followed by Tukey's multiple comparion test. Different letters indicate significant differences (P<0.05). Asterisks indicate significant difference (Student t-test). CTL, control (24 °C); MT, medium temperature (28 °C), HT, high temperature (32 °C).
Fig 6
Fig. 6
Effects of one-week heat exposure on heat shock protein-70 (HSP70) expression and apoptotic cells in gills of American oyster stained with immunohistochemistry and in situ TUNEL assay, respectively. (A-C) HSP70 expression in representative photographs of gills collected from oysters exposed to (A) control temperature (CTL: 24 °C), (B) medium temperature (MT: 28 °C), and (C) high temperature (HT: 32 °C). Arrow indicates higher HSP70 expression. (D-F) The presence of apoptotic nuclei shown as dark brown staining (arrow). (G) Negative control of HSP70 expression in gills of oysters. Scale bar = 100 µm. (H, I) Immunoreactive (IR) intensity of HSP70 and apoptotic nuclei in gills of oysters exposed to CTL, MT, and HT. Each value represents the mean ± SEM. Whiskers indicate the minimum and maximum values for each data set. The center line within the whisker box indicates the mean value. Data were analyzed with one-way ANOVA followed by Tukey's multiple comparion test. Different letters indicate significant differences (P<0.05).
Fig 7
Fig. 7
Effects of one-week heat exposure on Bcl-2-associated-X (BAX) protein expression in gills of American oyster. (A-C) BAX protein expression in representative photographs of gills collected from oysters exposed to control (CTL: 24 °C), medium temperature (MT: 28 °C), and high temperature (HT: 32 °C). Arrow indicates higher BAX expression. (D) Negative control of BAX expression in gills of oyster. Scale bar = 100 µm. (E) Immunoreactive (IR) intensity of BAX in gills of oysters exposed to CTL, MT, and HT. Each value represents the mean ± SEM. Whiskers indicate the minimum and maximum values for each data set. The center line within the whisker box indicates the mean value. Data were analyzed with one-way ANOVA followed by Tukey's multiple comparion test. Different letters indicate significant differences (P<0.05).
Fig 8
Fig. 8
Effects of one-week heat exposure on caspase-3 (CAS-3) mRNA levels in gills of American oyster. Each value represents the mean ± SEM. Whiskers indicate the minimum and maximum values for each data set. Center line within the whisker box indicates mean value. Data were analyzed with one-way ANOVA followed by Tukey's multiple comparion test. Different letters indicate significant differences (P<0.05). Asterisks indicate significant difference (Student t-test). CTL, control (24 °C); MT, medium temperature (28 °C), HT, high temperature (32 °C).
Fig 9
Fig. 9
Effects of one-week heat exposure on caspase-3 (CAS-3) protein expression in gills of American oyster. (A-C) CAS-3 protein expression in representative photographs of gills collected from oysters exposed to control (CTL: 24 °C), medium temperature (MT: 28 °C), and high temperature (HT: 32 °C). Arrow indicates higher HSP70 expression. (D) Negative control of CAS-3 expression in gills of oyster. Scale bar = 100 µm. (E) Immunoreactive (IR) intensity of CAS-3 in gills of oysters exposed to CTL, MT, and HT. Each value represents the mean ± SEM. Whiskers indicate the minimum and maximum values for each data set. The center line within the whisker box indicates the mean value. Data were analyzed with one-way ANOVA followed by Tukey's multiple comparion test. Different letters indicate significant differences (P<0.05).
Fig 10
Fig. 10
Effects of one-week heat exposure on extrapallial (EP) fluid glucose levels and protein concentrations of American oyster. Each value represents the mean ± SEM. Whiskers indicate the minimum and maximum values for each data set. The center line within the whisker box indicates mean value. Data were analyzed with one-way ANOVA followed by Tukey's multiple comparion test. Different letters indicate significant differences (P<0.05). CTL, control (24 °C); MT, medium temperature (28 °C), HT, high temperature (32 °C).
Fig 11
Fig. 11
A representative summary of the results of current study. Schematic diagram of heat stress and oxidative DNA damage and cellular apoptosis in gills of American oysters. Synthesis of 8-hydroxy-2′-deoxyguanosine (8-OHdG) and γ-histone family member X (γ-H2AX) proteins, and DNA double-stranded break (DSBs). Oligomerization of heat shock protein-70 (HSP70) and synthesis of apoptotic proteins (e.g., Bcl-2-associated-X (BAX), caspase-3, etc.) in gills of oysters.
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