Earthworms Repair H₂O₂-Induced Oxidative DNA Adducts without Removing UV-Induced Pyrimidine Dimers

Materials and Methods

Earthworms and chemicals. The earthworms (Metaphire posthuma) were purchased from an earthworm supply store, maintained in their native soil, and fed with peels of tangerine under dark and moist conditions before their sacrifice. Healthy adult earthworms with a well-developed clitellum were scarified and their testis cells carefully removed for UV radiation and comet assay.

All chemicals and solvents used throughout this study were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and Aldrich Chemical Co. (Milwaukee, WI, USA). T4 UV endonuclease V was purchased from Epicentre Technologies (Madison, WI, USA). Formamidopyrimidine-DNA glycosylases (Fpg) and endonuclease III were purchased from Trevigen (Gaithersburg, MD, USA).

UV density measurement and UV exposure. A UV light crosslinker (Spectrolinker XL-1000; Spectronics Co., Westburg, NY, USA) was used; and the UV 254 nm dose was measured by a sensor in the UVC light box. All cells were washed with phosphate-buffered saline (PBS), drained in a dish, and exposed to UVC radiation at a dose-rate of 0.5 J/m² of UVC on ice, and the comet assay was performed immediately. As for the H₂O₂ treatment, the indicated doses of H₂O₂ were added directly into the medium and cells were exposed to the indicated time.

Efficiency of earthworm testis cells at excising UVC- and H₂O_2-induced DNA adducts. Cells were pretreated with 5 mM hydroxyurea plus 50 μM cytosine-β-D-arabinofuranoside (H/A) for 0.5 h, then irradiated with 6 J/m² UVC, or co-treated with H/A and 80 μM H₂O₂ for 0.5 h, and then re-incubated for 0-6 h in H/A before slide making and comet assay.

Efficiency of earthworm testis cells at rejoining UVC- and H₂O_2-induced DNA strand breaks after cellular excision step. Cells were pretreated with H/A for 0.5 h, then irradiated with 6 J/m² UVC, or co-treated with H/A and 80 μM H₂O₂ for 0.5 h, and then re-incubated for 6 h in H/A to allow the excision step. H/A was then removed and the cells were allowed to rejoin the DNA strand breaks for the indicated time (0-6 h) before slide making and comet assay.

Comet assay. The standard comet assay without enzyme digestion is described as follows: Agarose gels were made using PBS. A glass microscope slide was placed on a hot plate at 60°C, and 100 μl of 1% normal-melting point agarose at 65°C was applied to cover an area of 6.0×2.5 cm. A coverslip was applied immediately and the slide placed on ice to set the gel. Coverslips were then removed by dipping slides into water containing ice. To each gel surface, a second layer of agarose containing cells in a total volume of 80 μl was applied. This second layer was prepared by mixing 400 μl of 1.5% low-melting point agarose at 40°C with 200 μl of PBS containing earthworm cells (10³ cells/μl). Coverslips were applied immediately and slides were placed on ice to set the gel of second layer. Then the coverslips were removed again for the third layer. In a similar manner as the first layer, a 100 μl of 1.5% low-melting-point agarose was applied as the third layer. After removing coverslips, slides were immersed in 4°C cell lysis solution for 18 h or more time as indicated. Cell lysis solution contained 2.5 M NaCl, 100 mM EDTA and 10 mM Tris; the pH was adjusted to 10.0 with NaOH and 1% N-laurylsarcosine, 1% Triton X-100, and 10% dimethylsulfoxide added immediately before use. DNA was then denatured by incubating in 0.3 M NaOH, 1 mM EDTA (pH 13.4) for 20 min. Electrophoresis was performed at 25 V, 300 mA for 25 min.

Slides were washed briefly in distilled water, blotted, and transferred to 0.4 M Tris-HCl, pH 7.5. DNA was stained by adding 12.5 μl of 200× Sybr green I to slides. A coverslip was applied and comets were observed under a fluorescence microscope (wavelength 450-490, farb teiler (FT) 510, long pass filter (LP) 520). The image of 50 cells per treatment was recorded with a digital camera (Kodak DCS-420). The migration of DNA from the nucleus of each cell was measured with a computer program using the parameter of comet moment. The comet moment was calculated using the formula ∑0→n [(amount of DNA at distance X) × (distance X)]/total DNA. This protocol is referred to as the standard comet assay.

For enzyme digestion, as performed in previous work (11, 12), after lysis treatment, slides were washed with distilled water then incubated at 37°C for 30 min in enzyme reaction buffer [in the experiments, Trevigen; 10 mM HEPES-KOH (pH 7.4), 100 mM KCl; 10 mM EDTA, 0.1 mg/ml bovine serum albumin as Fpg digestion buffer; 10 mM HEPES-KOH (pH 7.4), 100 mM KCl, and 10 mM EDTA as endonuclease III digestion buffer]. Then 0.5 unit of UV endonuclease V (for UVC-induced CPDs), or 1 unit of Fpg followed by 1 unit of endonuclease III (for H₂O₂-induced oxidative adducts) in 10 μl of enzyme reaction buffer was added to the center of each half of the gel. A coverslip was applied and the slides were incubated at 37°C for 2 h in a sealed box containing wet tissue paper. Slides were prepared for electrophoresis as described above.

The enzyme-incorporated earthworm comet assay system was established, using UV endonuclease V to identify UV-induced CPD (13), and Fpg plus endonuclease III to identify H₂O₂-induced oxidative DNA adducts (14, 15).

Statistical analysis. Data are expressed as the mean±SEM and analysis of variance (ANOVA) was employed for statistical analysis. Student's t-test was used in two-sample comparisons. P<0.05 was considered to be statistically significant.