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Comet assays to assess DNA damage and repair in grass shrimp embryos exposed to phototoxicants
Marine Environmental Research 54 (2002) 465–469 www.elsevier.com/locate/marenvrev
Comet assays to assess DNA damage and repair in grass shrimp embryos exposed to phototoxicants R. Leea,*, G.B. Kimb a
Skidaway Institute of Oceanography, University System of Georgia, 10 Ocean Science Circle, Savannah GA 31411, USA b Department of Marine Environmental Engineering, Gyeongsang Nat’l University, Tongyeong, South Korea 650-160
Abstract Exposure of grass shrimp (Palaemonetes pugio) embryos to four compounds (anthracene, pyrene, a-terthienyl, methylene blue) along with solar exposure resulted in extensive DNA strand damage using the comet assay. DNA tail moments of embryos exposed to these chemicals in the dark ranged from 1.8 to 4.3, while exposure to chemicals and solar resulted in tail moments of 14.3–15.3. Reduction of DNA tail moments when solar exposed embryos were transferred to the dark, suggested DNA repair systems were active. The comet assay can be used to follow both DNA damage and repair following exposure to phototoxic chemicals. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Comet; DNA; Embryos; Phototoxicant
The toxicity to marine biota of certain natural and anthropogenic compounds increases dramatically if after uptake there is sunlight exposure. This effect is referred to as phototoxicity or photoinduced toxicity and is shown by several polycyclic aromatic hydrocarbons, a-terthienyl, and methylene blue (Peachy & Crosby, 1996). At concentrations lower than those which cause mortality, there can be effects on growth and reproduction (Hall & Oris, 1991; Steinert, Montee, & Sastre, 1998). The sublethal effects of these photoactivated compounds is often due to direct or indirect damage to DNA (Gasparro, 1988; Gasper, & Schuster, 1997). One mechanism leading to DNA damage involves cation radicals which can be produced by the photoionization of aromatic hydrocarbons (Landrum, Giesy, Oris, & Allred, 1987; * Corresponding author. Tel.: +1-912-598-2494. E-mail address: [email protected] (R. Lee). 0141-1136/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(02)00128-9
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R. Lee, G.B. Kim / Marine Environmental Research 54 (2002) 465–469
Osborne & Crosby, 1987). These reactive radicals can react directly with DNA or act on oxygen to produce reactive oxygen species which are DNA damaging agents. In the present study, the comet assay was used to assess DNA damage in grass shrimp embryos exposed in a solar simulator to four phototoxic compounds. Embryos with photoinduced DNA damage were transferred to the dark and DNA repair was followed by comet assays. The comet assay, also called the single-cell gel electrophoresis assay, indicate the extent of DNA strand breaks in individual cells by measuring the migration of fragmented and relaxed DNA away from the nuclei immobilized in agarose gels. Developing embryos attached to the female grass shrimp, Palaeomonetes pugio, were removed by a cut at the stem attaching them to the female. Compounds to be tested (anthracene, pyrene, methylene blue, a-terthienyl) were dissolved in acetone and added (10 m volume) to 100 ml beakers containing 75 ml of filtered estuarine water (0.4-mm filter). Twenty stage 8 embryos (embryos go through 10 morphologically distinct stages before hatching) were added to each of the beakers. There were three replicates of each test with 20 embryos in each replicate. Embryos were kept in the dark with the chemicals for the first 3 h, followed by placement in a solar simulator (Suntest CPS, Atlas Instruments) for 1 or 2 h. The Suntest CPS solar simulator was adjusted to closely match the midday solar spectral irradiance (126 mW cm 2) at Athens, GA (34 N) on a clear June day, as described by Gao and Zepp (1998). Water temperatures in the solar simulator ranged from 25 to 27 C, which were the temperatures found in the estuary at the time of the study (May–June). Salinity of the water ranged from 25 to 28 ppt. For dark exposures dark taped breakers with embryos were placed in the solar simulator. The procedures for the comet assay were similar to those described by Singh, McCoy, Tice, and Schneider (1998), but with some modifications. Ten embryos were homogenized in 0.1M phosphate buffer (pH 7.5) followed by centrifuation of extracts and suspension of pellets in low melting DNA grade agarose. Fifteen microliters of the cell suspension were transferred onto slides previously coated with agarose and allowed to gel on an ice-chilled tray. After gelling, the slides were transferred from lysing solution and rinsed with distilled water. The slides were placed in a submarine gel electrophoreiss chamber, and the DNA allowed to unwind under alkaline conditions (pH 11) for 15 min. After unwinding, the samples were subjected to electrophoresis at 200 mA, 25V for 10 min. The slides were neutralized three times for 2 min each in 0.4 M Tris (pH 7.5). Slides were fixed in ethanol for 5 min and stained for DNA with ethidium bromide. Images of 50 randomly selected cells were analyzed in each slide using an epifluorescence microscope. The cell images were projected onto a high sensitivity CCD camera. A computerized image analysis system (Komet version 4.01, Kinetic Imaging Ltd) was used to determine tail moment which is the product of the percentage of DNA in the tail multiplied by the tail length. Three slides from each experiment were analyzed with the mean standard deviation reported Table 1 summarizes DNA damage produced by methylene blue, anthracene and a-terthienyl in the dark and light. DNA tail moments for embryos exposed to these compounds in the dark ranged from 1.8 0.7 to 4.7 1.2 while DNA tail moment
R. Lee, G.B. Kim / Marine Environmental Research 54 (2002) 465–469
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Table 1 DNA damage in grass shrimp embryos after exposure to various phototoxicants in solar simulator and darka Compound and concentration
Solar exposure
Transfer to dark after solar exposure
DNA tail moment
Methylene blue (1000 mg/l)
No Yes Yes
No No Yes
4.71.5 14.22.2 5.82.2
a-Terthienyl (50 mg/l)
No Yes Yes
No No Yes
4.31.2 15.54.1b 6.52.5
Anthracene (3 mg/l)
No Yes Yes
No No Yes
1.70.9 14.73.6b 6.71.9
None (control)
No Yes Yes
No No Yes
0.50.2 8.32.0 1.80.8
a For each compound there was an initial exposure of 120 embryos (stage 8) with a compound for 3 h in the dark. For solar exposure 60 embryos were transferred from the dark to the solar simulator for 1 h. For the transfer to dark after solar exposure experiment, there was transfer of 30 embryos from the solar simulator to the dark for 24 h. The mean DNA tail moment (percentage of DNA in tail multiplied by tail length)standard deviation is given for each exposure where three replicates (10 embryos per replicate) were counted with 50 cells counted on each slide. b Significantly different (P<0.01; ANOVA) from controls [no solar exposure or just solar (no chemicals)].
for the dark control was 0.3 0.2. Embryos exposed for 1 h to solar light, but no chemicals, showed significant DNA damage (P < 0.01; ANOVA) with a tail moment of 8.2 1.9, however tail moments were significantly higher than 8.2 when there was both uptake of the chemical and solar exposure (DNA tail moments ranged from 14.3 2.2 to 15.3 2.3). Embryos exposed to chemicals and solar and then transferred to the dark for 24 h had tail moments ranging from 5.8 2.8 to 6.5 2.7, indicating that DNA repair had taken place in the dark. Fig. 1 shows DNA damage by embryos exposed to both solar and pyrene. Exposure for 2 h to solar, pyrene in the dark, and pyrene in the dark plus 2 h to solar showed DNA tail moments of 8.5, 3.2, and 29.1, respectively. When embryos exposed to both solar and pyrene were transferred to the dark for 3 h there was a very large increase in DNA tail moment to 62. This large increase in DNA tail moment in the dark likely reflects incomplete excision repair at the time of lysis, since incomplete excision repair introduces single strand gaps in the DNA (Gedik, Ewen, & Collins, 1992). After 24 h in the dark the DNA tail moment was 23.4, indicating incomplete DNA repair. Earlier studies linked grass shrimp embryo development problems with DNA strand damage (Lee, Kim, Maruya, Steinert, & Oshima, 2000). Solar exposure of embryos to a number of
468
R. Lee, G.B. Kim / Marine Environmental Research 54 (2002) 465–469
Fig. 1. DNA strand damage (DNA tail moment) in grass shrimp embryos exposed to pyrene, solar light and darkness. Stage 8 embryos were exposed to the following conditions: dark for 2 h (D); dark for 2 h with pyrene [D(P)]; solar light for 2 h (L); solar light for 2 h followed by dark for 2 h (L–D); dark for 2 h with pyrene followed by solar light for 2 h followed by dark for 2 h (D–L–2D)(P); dark for 2 h with pyrene followed by solar light for 2 h followed by dark for 2 h (D–L–3D)(P); dark for 2 h with pyrene followed by solar light for 2 h followed by dark for 24 h (D–L–24D)(P). Each bar is the meanstandard deviation for three replicates. Pyrene concentration was 10 mg/l.
compounds known to be phototoxic resulted in DNA strand damage with evidence of DNA repair when transferred to the dark. The comet assay can be used to study DNA damage and repair in estuarine animals following exposure to phototoxic chemicals in both the laboratory and field.
Acknowledgements This work was supported by the NOAA Coastal Ocean Program through the South Carolina Sea Grant Consortium pursuant to National Oceanic and Atmospheric Administration Award No NA96PO113.
References Peachey, R. L., & Crosby, D. G. (1996). Marine Environmental Research, 42, 359–362. Hall, A. T., & Oris, J. T. (1991). Aquatic Toxicology, 19, 249–264.
R. Lee, G.B. Kim / Marine Environmental Research 54 (2002) 465–469
469
Steinert, S. A., Montee, R. S., & Sastre, M. P. (1998). Marine Environmental Research, 46, 355–358. Gasparro, F. P. (1998). In F. P. Gasparro (Ed.), Psoralen DNA photobiology (Vol. I; 136 pp.). Boca Raton: CRC Press. Gasper, S. M., & Schuster, G. B. (1997). Journal of the American Chemical Society, 119, 12762–12771. Landrum, P. F., Giesy, J. P., Oris, J. T., Allred, P. M. (1997). In J. H. Vandermeulen, & S. E. Hrudey (Eds.), Oil in freshwater: chemistry, biology, countermeasure technology (512 p.). New York: Pergamon Press. Osborne, M. R., & Crosby, N. T. (1987). Benzopyrenes (Vol. 5.). Cambridge: Cambridge University Press. Gao, H., & Zepp, R. G. (1998). Environmental Science and Technology, 32, 2940–2946. Singh, N. P., McCoy, M. T., Tice, R. R., & Schneider, E. L. (1988). Experimental Cell Research, 175, 184– 191. Gedik, C. M., Ewen, S. W. B., & Collins, A. R. (1992). International Journal of Radiation Biology, 62, 313–320. Lee, R., Kim, G. B., Maruya, K. A., Steinert, S. A., & Oshima, Y. (2000). Marine Environmental Research, 50, 553–557.
Comet assays to assess DNA damage and repair in grass shrimp embryos exposed to phototoxicants R. Leea,*, G.B. Kimb a
Skidaway Institute of Oceanography, University System of Georgia, 10 Ocean Science Circle, Savannah GA 31411, USA b Department of Marine Environmental Engineering, Gyeongsang Nat’l University, Tongyeong, South Korea 650-160
Abstract Exposure of grass shrimp (Palaemonetes pugio) embryos to four compounds (anthracene, pyrene, a-terthienyl, methylene blue) along with solar exposure resulted in extensive DNA strand damage using the comet assay. DNA tail moments of embryos exposed to these chemicals in the dark ranged from 1.8 to 4.3, while exposure to chemicals and solar resulted in tail moments of 14.3–15.3. Reduction of DNA tail moments when solar exposed embryos were transferred to the dark, suggested DNA repair systems were active. The comet assay can be used to follow both DNA damage and repair following exposure to phototoxic chemicals. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Comet; DNA; Embryos; Phototoxicant
The toxicity to marine biota of certain natural and anthropogenic compounds increases dramatically if after uptake there is sunlight exposure. This effect is referred to as phototoxicity or photoinduced toxicity and is shown by several polycyclic aromatic hydrocarbons, a-terthienyl, and methylene blue (Peachy & Crosby, 1996). At concentrations lower than those which cause mortality, there can be effects on growth and reproduction (Hall & Oris, 1991; Steinert, Montee, & Sastre, 1998). The sublethal effects of these photoactivated compounds is often due to direct or indirect damage to DNA (Gasparro, 1988; Gasper, & Schuster, 1997). One mechanism leading to DNA damage involves cation radicals which can be produced by the photoionization of aromatic hydrocarbons (Landrum, Giesy, Oris, & Allred, 1987; * Corresponding author. Tel.: +1-912-598-2494. E-mail address: [email protected] (R. Lee). 0141-1136/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(02)00128-9
466
R. Lee, G.B. Kim / Marine Environmental Research 54 (2002) 465–469
Osborne & Crosby, 1987). These reactive radicals can react directly with DNA or act on oxygen to produce reactive oxygen species which are DNA damaging agents. In the present study, the comet assay was used to assess DNA damage in grass shrimp embryos exposed in a solar simulator to four phototoxic compounds. Embryos with photoinduced DNA damage were transferred to the dark and DNA repair was followed by comet assays. The comet assay, also called the single-cell gel electrophoresis assay, indicate the extent of DNA strand breaks in individual cells by measuring the migration of fragmented and relaxed DNA away from the nuclei immobilized in agarose gels. Developing embryos attached to the female grass shrimp, Palaeomonetes pugio, were removed by a cut at the stem attaching them to the female. Compounds to be tested (anthracene, pyrene, methylene blue, a-terthienyl) were dissolved in acetone and added (10 m volume) to 100 ml beakers containing 75 ml of filtered estuarine water (0.4-mm filter). Twenty stage 8 embryos (embryos go through 10 morphologically distinct stages before hatching) were added to each of the beakers. There were three replicates of each test with 20 embryos in each replicate. Embryos were kept in the dark with the chemicals for the first 3 h, followed by placement in a solar simulator (Suntest CPS, Atlas Instruments) for 1 or 2 h. The Suntest CPS solar simulator was adjusted to closely match the midday solar spectral irradiance (126 mW cm 2) at Athens, GA (34 N) on a clear June day, as described by Gao and Zepp (1998). Water temperatures in the solar simulator ranged from 25 to 27 C, which were the temperatures found in the estuary at the time of the study (May–June). Salinity of the water ranged from 25 to 28 ppt. For dark exposures dark taped breakers with embryos were placed in the solar simulator. The procedures for the comet assay were similar to those described by Singh, McCoy, Tice, and Schneider (1998), but with some modifications. Ten embryos were homogenized in 0.1M phosphate buffer (pH 7.5) followed by centrifuation of extracts and suspension of pellets in low melting DNA grade agarose. Fifteen microliters of the cell suspension were transferred onto slides previously coated with agarose and allowed to gel on an ice-chilled tray. After gelling, the slides were transferred from lysing solution and rinsed with distilled water. The slides were placed in a submarine gel electrophoreiss chamber, and the DNA allowed to unwind under alkaline conditions (pH 11) for 15 min. After unwinding, the samples were subjected to electrophoresis at 200 mA, 25V for 10 min. The slides were neutralized three times for 2 min each in 0.4 M Tris (pH 7.5). Slides were fixed in ethanol for 5 min and stained for DNA with ethidium bromide. Images of 50 randomly selected cells were analyzed in each slide using an epifluorescence microscope. The cell images were projected onto a high sensitivity CCD camera. A computerized image analysis system (Komet version 4.01, Kinetic Imaging Ltd) was used to determine tail moment which is the product of the percentage of DNA in the tail multiplied by the tail length. Three slides from each experiment were analyzed with the mean standard deviation reported Table 1 summarizes DNA damage produced by methylene blue, anthracene and a-terthienyl in the dark and light. DNA tail moments for embryos exposed to these compounds in the dark ranged from 1.8 0.7 to 4.7 1.2 while DNA tail moment
R. Lee, G.B. Kim / Marine Environmental Research 54 (2002) 465–469
467
Table 1 DNA damage in grass shrimp embryos after exposure to various phototoxicants in solar simulator and darka Compound and concentration
Solar exposure
Transfer to dark after solar exposure
DNA tail moment
Methylene blue (1000 mg/l)
No Yes Yes
No No Yes
4.71.5 14.22.2 5.82.2
a-Terthienyl (50 mg/l)
No Yes Yes
No No Yes
4.31.2 15.54.1b 6.52.5
Anthracene (3 mg/l)
No Yes Yes
No No Yes
1.70.9 14.73.6b 6.71.9
None (control)
No Yes Yes
No No Yes
0.50.2 8.32.0 1.80.8
a For each compound there was an initial exposure of 120 embryos (stage 8) with a compound for 3 h in the dark. For solar exposure 60 embryos were transferred from the dark to the solar simulator for 1 h. For the transfer to dark after solar exposure experiment, there was transfer of 30 embryos from the solar simulator to the dark for 24 h. The mean DNA tail moment (percentage of DNA in tail multiplied by tail length)standard deviation is given for each exposure where three replicates (10 embryos per replicate) were counted with 50 cells counted on each slide. b Significantly different (P<0.01; ANOVA) from controls [no solar exposure or just solar (no chemicals)].
for the dark control was 0.3 0.2. Embryos exposed for 1 h to solar light, but no chemicals, showed significant DNA damage (P < 0.01; ANOVA) with a tail moment of 8.2 1.9, however tail moments were significantly higher than 8.2 when there was both uptake of the chemical and solar exposure (DNA tail moments ranged from 14.3 2.2 to 15.3 2.3). Embryos exposed to chemicals and solar and then transferred to the dark for 24 h had tail moments ranging from 5.8 2.8 to 6.5 2.7, indicating that DNA repair had taken place in the dark. Fig. 1 shows DNA damage by embryos exposed to both solar and pyrene. Exposure for 2 h to solar, pyrene in the dark, and pyrene in the dark plus 2 h to solar showed DNA tail moments of 8.5, 3.2, and 29.1, respectively. When embryos exposed to both solar and pyrene were transferred to the dark for 3 h there was a very large increase in DNA tail moment to 62. This large increase in DNA tail moment in the dark likely reflects incomplete excision repair at the time of lysis, since incomplete excision repair introduces single strand gaps in the DNA (Gedik, Ewen, & Collins, 1992). After 24 h in the dark the DNA tail moment was 23.4, indicating incomplete DNA repair. Earlier studies linked grass shrimp embryo development problems with DNA strand damage (Lee, Kim, Maruya, Steinert, & Oshima, 2000). Solar exposure of embryos to a number of
468
R. Lee, G.B. Kim / Marine Environmental Research 54 (2002) 465–469
Fig. 1. DNA strand damage (DNA tail moment) in grass shrimp embryos exposed to pyrene, solar light and darkness. Stage 8 embryos were exposed to the following conditions: dark for 2 h (D); dark for 2 h with pyrene [D(P)]; solar light for 2 h (L); solar light for 2 h followed by dark for 2 h (L–D); dark for 2 h with pyrene followed by solar light for 2 h followed by dark for 2 h (D–L–2D)(P); dark for 2 h with pyrene followed by solar light for 2 h followed by dark for 2 h (D–L–3D)(P); dark for 2 h with pyrene followed by solar light for 2 h followed by dark for 24 h (D–L–24D)(P). Each bar is the meanstandard deviation for three replicates. Pyrene concentration was 10 mg/l.
compounds known to be phototoxic resulted in DNA strand damage with evidence of DNA repair when transferred to the dark. The comet assay can be used to study DNA damage and repair in estuarine animals following exposure to phototoxic chemicals in both the laboratory and field.
Acknowledgements This work was supported by the NOAA Coastal Ocean Program through the South Carolina Sea Grant Consortium pursuant to National Oceanic and Atmospheric Administration Award No NA96PO113.
References Peachey, R. L., & Crosby, D. G. (1996). Marine Environmental Research, 42, 359–362. Hall, A. T., & Oris, J. T. (1991). Aquatic Toxicology, 19, 249–264.
R. Lee, G.B. Kim / Marine Environmental Research 54 (2002) 465–469
469
Steinert, S. A., Montee, R. S., & Sastre, M. P. (1998). Marine Environmental Research, 46, 355–358. Gasparro, F. P. (1998). In F. P. Gasparro (Ed.), Psoralen DNA photobiology (Vol. I; 136 pp.). Boca Raton: CRC Press. Gasper, S. M., & Schuster, G. B. (1997). Journal of the American Chemical Society, 119, 12762–12771. Landrum, P. F., Giesy, J. P., Oris, J. T., Allred, P. M. (1997). In J. H. Vandermeulen, & S. E. Hrudey (Eds.), Oil in freshwater: chemistry, biology, countermeasure technology (512 p.). New York: Pergamon Press. Osborne, M. R., & Crosby, N. T. (1987). Benzopyrenes (Vol. 5.). Cambridge: Cambridge University Press. Gao, H., & Zepp, R. G. (1998). Environmental Science and Technology, 32, 2940–2946. Singh, N. P., McCoy, M. T., Tice, R. R., & Schneider, E. L. (1988). Experimental Cell Research, 175, 184– 191. Gedik, C. M., Ewen, S. W. B., & Collins, A. R. (1992). International Journal of Radiation Biology, 62, 313–320. Lee, R., Kim, G. B., Maruya, K. A., Steinert, S. A., & Oshima, Y. (2000). Marine Environmental Research, 50, 553–557.