Thallium(I) induces a prolonged inhibition of (6-4)photoproduct binding and UV damage excision repair activities in zebrafish (Danio rerio) embryos via protein inactivation.

Cyclobutane pyrimidine dimer (CPD) and (6-4)photoproduct (6-4 PP) are two major types of UV-induced DNA lesion and 6-4 PP is more mutagenic than CPD. Activated by lesion detection, nucleotide excision repair (NER) eliminates CPDs and 6-4 PPs. Thallium (Tl) is a toxic metal existing primarily as Tl+ in the aquatic environment. Ingestion of Tl+-contaminated foods and water is a major route of human poisoning. As Tl+ may inhibit enzyme activities via binding to sulfhydryl groups, this study explored if Tl+ could intensify UV mutagenicity by inactivating NER-linked damage recognition factors using zebrafish (Danio rerio) embryo as a model system. Incubation of Tl+ (as thallium nitrate) at 0.1-0.4 µg/mL with zebrafish extracts for 20 min caused a concentration-dependent inhibition of 6-4 PP binding activities as shown by a photolesion-specific band shift assay, while CPD binding activities were insensitive to Tl+. The ability of Tl+ to suppress 6-4 PP detection was stronger than that of Hg2+. Exposure of zebrafish embryos at 1 h post fertilization (hpf) to Tl+ at 0.4-1 µg/mL for 9 or 71 h also specifically inhibited 6-4 PP detection, indicating that Tl+ induced a prolonged inhibition of 6-4 PP sensing ability primarily via its direct interaction with damage recognition molecules. Tl+-mediated inhibition of 6-4 PP binding in embryos at distinct stages resulted in a suppression of NER capacity monitored by a transcription-based DNA repair assay. Our results revealed the potential of Tl+ to enhance UV mutagenicity by disturbing the removal of 6-4 PP through repressing the lesion detection step of NER.

Susceptibility of DNA damage recognition activities linked to nucleotide excision and mismatch repair in zebrafish (Danio rerio) early and mid-early embryos to 2.5 to 4.5 °C heat stress.

Fish at early life stages are sensitive to temperature change because of their narrower temperature tolerance ranges. Initiated by damage detection, DNA mismatch repair (MMR) and nucleotide excision repair (NER) maintain genome integrity respectively by eliminating mismatched nucleotides and helix-distorting DNA lesions. As discharge of heated effluent from power plants may elevate water temperatures to only 2 to 6 °C higher than ambient, this study explored if temperatures within this range affected MMR and NER-linked damage detection activities in fish embryos using zebrafish (Danio rerio) embryo as a model organism. Exposure of early embryos at 10 h post fertilization (hpf) to a warmer temperature at + 4.5 °C for 30 min enhanced damage recognition activities targeting UV-induced cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs) that distorted helical structures. Conversely, photolesions sensing activities were inhibited in 24 hpf mid-early embryos under the same stress conditions. A much higher temperature at + 8.5 °C imposed similar effects on UV damage detection. A mild heat stress at + 2.5 °C for 30 min, however, repressed both CPD and 6-4PP binding activities in 10 and 24 hpf embryos. Inhibition of damage recognition under mild heat stress impeded the overall NER capacity evidenced by a transcription-based repair assay. Warmer water temperatures at + 2.5 and + 4.5 °C also inhibited G-T mismatch binding activities in 10 and 24 hpf embryos, but G-T recognition was more sensitive to + 4.5 °C stress. Inhibition of G-T binding partially correlated with a downregulation of Sp1 transcription factor activity. Our results showed the potential of water temperature elevation within 2 to 4.5 °C to disturb DNA damage repair in fish at embryonic stages.

Aluminum (Al) causes a delayed suppression of nucleotide excision repair (NER) capacity in zebrafish (Danio rerio) embryos via disturbance of DNA lesion detection.

Aluminum (Al) is extensively used for making cooking utensils and its presence in the aquatic environment may occur through acid mine drainage and wastewater discharge. Al is known to induce genotoxicity in human cells, rodents, and fish. Nucleotide excision repair (NER) eliminates helix-twisting DNA lesions such as UV-induced dipyrimidine photoproducts. Because our earlier investigation revealed the operation of NER in zebrafish (Danio rerio) embryos, this study explored if inhibition of NER could be a mechanism of Al-induced genotoxicity using zebrafish embryo as a model system. An acute fish embryo toxicity test indicated that Al (as aluminum sulfate) at 2-15 mg/L were nonlethal to zebrafish embryos, yet exposure of embryos at 1 h post fertilization (hpf) to Al at 10-15 mg/L for 71 h significantly repressed their NER capacity monitored by a transcription-based DNA repair assay. Band shift analysis indicated a higher sensitivity of (6-4) photoproduct (6-4PP) than cyclobutane pyrimidine dimer (CPD) detecting activities to Al, reflecting the preferential influence of Al on the detection of strongly distorted DNA lesions. Time-course experiments showed a delayed response of NER to Al as repair machinery was unaffected by Al at 15 mg/L following a 35-h exposure, while Al treatment for the same period obviously inhibited 6-4PP binding activities although the gene expression of damage recognition factors remained active. Inhibition of 6-4PP detection blocked downstream lesion incision/excision detected by a terminal deoxy transferase-mediated end labeling assay. As the disturbance of damage sensing preceded that of the overall repair process, Al exposure was believed to downregulate NER capacity by inhibiting the activities of lesion detection proteins. Our results revealed the ability of Al to enhance its genotoxicity by suppressing NER capacity.

Heat stress upregulates G-T mismatch binding activities in zebrafish (Danio rerio) embryos preexposed and nonexposed to a sublethal level of cadmium (Cd).

G-T mispair frequently appears in eukaryotic DNA due to the spontaneous deamination of 5-methylcytosine paired with guanine and is therefore an important target for DNA mismatch repair (MMR). Our earlier studies showed the downregulation of G-T binding activities in cadmium (Cd)-exposed (Danio rerio) embryos. Since elevation of water temperature was reported to increase Cd toxicity in zebrafish, this study explored whether heat stress affected zebrafish mismatch binding capacity in the absence or presence of Cd. Heat stress (37 °C for 30 min) induced heat shock protein 70 mRNA expression in embryos at 10 and 24 h post fertilization (hpf). Heat stress weakly upregulated normal G-T sensing machinery and inhibited G-T recognition activity in embryos preexposed to 3 µM Cd for 9 h. Either heat shock or a 23-h Cd treatment alone caused a 1.7-fold stimulation of G-T binding capacity in 24 hpf embryos and heat stress of Cd-preexposed embryos further enhanced G-T binding activity to 2.5 fold of control. Normal and Cd-downregulated loop binding activities in 10 and 24 hpf embryos were almost unreactive to heat shock. Heat stress-upregulated G-T sensing in nonexposed, but not in Cd-preexposed, 24 hpf embryos correlated with stronger gene activities encoding MMR-linked mismatch detecting factors MutS homolog 2 and 6 plus a higher DNA binding activity of the transcription factor Sp1 that regulates msh2/msh6 expression. Our results suggested the importance of heat shock response in facilitating the correction of G-T mismatch in developing zebrafish even under Cd exposure.