- Email: [email protected]
Oxidative damage to DNA: an immunohistochemical approach for detection of 7,8-dihydro-8-oxodeoxyguanosine in marine organisms
MARINE ENVIRONMENTAL RESEARCH Marine Environmental Research 58 (2004) 725–729 www.elsevier.com/locate/marenvrev
Oxidative damage to DNA: an immunohistochemical approach for detection of 7,8-dihydro-8-oxodeoxyguanosine in marine organisms Nicola Machella
a,b,* ,
Francesco Regoli a, Antonio Cambria c, Regina M. Santella b
a
b
Istituto di Biologia e Genetica, Universita Politecnica delle Marche, Via Ranieri 65, Monte d’Ago, 60100 Ancona, Italy Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, 701 West 168th Street, New York, NY 10032, USA c Dipartimento di Scienze Chimiche, Universita di Catania, V.le A.Doria, 95125 Catania, Italy
Abstract The modified nucleoside 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-dG) is an index of oxidative DNA damage. An immunohistochemical approach based on the use of monoclonal antibody 1F7 against 8-oxo-dG was investigated in marine organisms with immunoperoxidase and immunofluorescent detection. Relative staining intensity as a measure of the 8-oxo-dG level was microscopically assessed. After laboratory exposures to benzo[a]pyrene (B[a]P), higher levels of oxidative DNA damage were clearly detected in all treated animals compared to controls. While this method eliminates DNA extraction reducing the processing of biological samples, absolute values are not provided. Further, the method requires only small amounts of tissue and potentially discriminates susceptibility to oxidative damage in different cell types. These results suggest that the assay should have practical applications in marine ecotoxicology. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: 7,8-Dihydro-8-oxodeoxyguanosine; Oxidative DNA damage; Monoclonal antibody 1F7; Genotoxicity; Mussel; Eel; Benzo[a]pyrene
*
Corresponding author. Tel.: +39-071-2204613; fax: +39-071-220-4609. E-mail address: [email protected] (N. Machella).
0141-1136/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2004.03.022
726
N. Machella et al. / Marine Environmental Research 58 (2004) 725–729
8-Oxo-dG, a major oxidative DNA product formed by hydroxyl radicals or prooxidant chemicals, is a marker of genotoxic damage. Since a number of environmental pollutants induce oxidative stress (Winston & Di Giulio, 1991), its measurement is increasingly being used in marine ecotoxicology (L opez-Barea & Pueyo, 1998). Increased levels of 8-oxo-dG have been documented within target tissues of aquatic vertebrates and invertebrates after environmental or laboratory exposures to environmental pollutants (Canova et al., 1998; Malins & Gunselman, 1994). In recent years, attention has focused on measurement of 8-oxo-dG levels and although several methods are currently available, controversy still remains about the actual levels within cells and tissues (ESCODD, 2002). Incomplete DNA hydrolysis and artificial oxidation of native guanine to 8-oxo-dG are considered the main drawbacks that may occur during the analysis (Halliwell, 1999). Also, depending on the extraction protocol, DNA isolation can influence the background level of 8-oxodG in cultured human cells (Ravanat et al., 2002). A general consensus still has not been reached concerning the basal levels of 8-oxo-dG within standard biological samples (ESCODD, 2002; Halliwell, 1999). Despite this, improvement and integration of methodologies to assess oxidative DNA damage would be of great relevance for marine ecotoxicology in order to better understand the environmental impact of genotoxins. In this regard, the monoclonal antibody 1F7, previously developed and used to detect 8-oxo-dG in oral cells and nasal biopsies (Santella, 1999), was tested for the first time in Mediterranean mussel (Mytilus galloprovincialis) and European eel (Anguilla anguilla). Immunoperoxidase and immunofluorescence staining were done in eel liver sections, while only immunoperoxidase staining was used on mussel digestive gland and haemocytes. In order to induce oxidative DNA damage, animals were treated under laboratory conditions with different doses of benzo[a]pyrene, a model genotoxic compound. Mussels were exposed in artificial seawater to concentrations of B[a]P ranging from 100 to 1000 ppb in 0.0001% DMSO for 10 days, while controls did not receive the solvent. Eels were intraperitoneally injected with 0, 0.1, 1, 10, 50 mg/kg of B[a]P (dissolved in corn oil) and sacrificed after seven days. The immunohistochemical stainings were performed with modifications of the protocols standardized in humans by Yarborough, Zhang, Hsu, and Santella (1996) and Motykiewicz et al. (2002). Proteinase K digestion and DNA denaturation times were reduced to preserve the morphological integrity of the tissue samples. Quantification of 8-oxo-dG was made on five randomly chosen fields, counting ten cells per field. For the immunoperoxidase method, a Cell Analysis System 200 microscope (Cell Measurement Program Software Package) was used and the damage was expressed as Relative Staining Intensity (RSI). The fluorescence intensity, expressed as Average Gray Value Average (AGVA), was assessed by a Nikon Eclipse-600 microscope, equipped with a Hamamatsu CCD digital camera (C4742-95) and quantified by the MetaView Imaging System Software. The relative damage (RD) level was calculated as the ratio between the mean staining intensity values of treated and control samples. Control slides of cultured human lymphoblastoid cells, treated with 5 mM H2 O2 for 30 min and untreated, were used to confirm the staining
N. Machella et al. / Marine Environmental Research 58 (2004) 725–729
727
Fig. 1. M. galloprovincialis and A. anguilla. Representative immunohistochemical stainings for 8-oxo-dG detection. Immunoperoxidase method: (a) digestive gland sections of control mussel and (b) mussel exposed to 500 ppb of B[a]P (200X). (c) Haemocytes of control mussel and (d) mussel exposed to 500 ppb of B[a]P (1000X). (e) Liver sections of control eel and (f) eel exposed to 50 mg/kg of B[a]P (400X). Immunofluorescence method: (g) liver sections of control eel and (h) eel exposed to 50 mg/kg of B[a]P (1000X).
specificity. Additional controls included marine sample slides that were stained after omitting the primary antibody or digesting tissues with DNAse before the staining.
728
N. Machella et al. / Marine Environmental Research 58 (2004) 725–729
Fig. 2. Levels of 8-oxo-dG detected in A. anguilla with the immunoperoxidase and immunofluorescence method, expressed as RD, compared to controls ðp < 0:05Þ.
In mussels, immunoperoxidase staining revealed increased levels of damage, both in digestive gland and haemocytes of treated samples (Fig. 1). Higher levels of damage were observed in haemocytes compared to digestive gland, and no dose response relationship was obtained in either tissue (data not shown). Marked increases in relative staining intensity were also evident in treated eels, using either the immunoperoxidase or immunofluorescence methods, as indicated by Fig. 1. However, while immunoperoxidase revealed similar increments of 8-oxo-dG, at different doses, the effects measured by the immunofluorescence detection generally increased with a linear trend at higher B[a]P doses (Fig. 2). The differences between the immunoperoxidase and the immunofluorescence detection methods were confirmed by the magnitude of RD values: quite homogeneous and lower in the former, more variable and higher in the latter (Fig. 2). These results suggested a greater sensitivity of the fluorescence method even though immunoperoxidase detection may be required when there is natural cell autofluorescence. The main advantages of the immunohistochemical assessment of DNA damage include a limited processing of samples (liquid nitrogen freezing, cryostat sections, brief fixation and staining), no DNA isolation or nuclear lysis (thus limiting a source of artificial oxidation) and the possibility to work on previously frozen samples without the need of fresh tissues from living organisms. Further, the method is potentially able to identify differential susceptibility of cells within different tissues to genotoxic insult, with a rapid and relatively inexpensive procedure. The disadvantages include potential antibody cross-reactivity with other modified nucleotides and the semi-quantitative nature of the data that does not provide absolute 8-oxo-dG levels.
N. Machella et al. / Marine Environmental Research 58 (2004) 725–729
729
Although these results must be considered preliminary, laboratory exposures were helpful in investigating the consistency of the approach and the efficacy of the use of antibody 1F7 in these marine organisms. To further assess the potential use of this approach in marine ecotoxicology, future analyses will be necessary to validate the detection of DNA damage in field conditions.
Acknowledgements This work was partially supported by NIEHS grant P30ES09089 and represents part of the Doctoral Thesis of N. Machella in Biochemical and Biomolecular Sciences.
References Canova, S., Degan, P., Peters, L. D., Livingstone, L. D., Voltan, R., & Venier, P. (1998). Mutation Research, 399, 17–30. ESCODD (European Standards Committee on Oxidative DNA Damage) (2002). Carcinogenesis 23, 2129–2133. Halliwell, B. (1999). Mutation Research, 443, 37–52. L opez-Barea, J., & Pueyo, C. (1998). Mutation Research, 399, 3–15. Malins, D. C., & Gunselman, G. S. (1994). Proceedings of the National Academy of Sciences USA, 91, 13038–13041. Motykiewicz, G., Faraglia, B., Wang, L. W., Terry, M. B., Senie, R. T., & Santella, R. M. (2002). Environmental and Molecular Mutagenesis, 40, 93–100. Ravanat, J. L., Douki, T., Duez, P., Gremaud, E., Herbert, K., Hofer, T., Lasserre, L., Saint-Pierre, C., Favier, A., & Cadet, J. (2002). Carcinogenesis, 23, 1911–1918. Santella, R. M. (1999). Cancer Epidemiology Biomarkers and Prevention, 8, 733–739. Winston, G. W., & Di Giulio, R. T. (1991). Aquatic Toxicology, 19, 137–161. Yarborough, A., Zhang, Y. J., Hsu, M. T., & Santella, R. M. (1996). Cancer Research, 56, 683–688.
Oxidative damage to DNA: an immunohistochemical approach for detection of 7,8-dihydro-8-oxodeoxyguanosine in marine organisms Nicola Machella
a,b,* ,
Francesco Regoli a, Antonio Cambria c, Regina M. Santella b
a
b
Istituto di Biologia e Genetica, Universita Politecnica delle Marche, Via Ranieri 65, Monte d’Ago, 60100 Ancona, Italy Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, 701 West 168th Street, New York, NY 10032, USA c Dipartimento di Scienze Chimiche, Universita di Catania, V.le A.Doria, 95125 Catania, Italy
Abstract The modified nucleoside 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-dG) is an index of oxidative DNA damage. An immunohistochemical approach based on the use of monoclonal antibody 1F7 against 8-oxo-dG was investigated in marine organisms with immunoperoxidase and immunofluorescent detection. Relative staining intensity as a measure of the 8-oxo-dG level was microscopically assessed. After laboratory exposures to benzo[a]pyrene (B[a]P), higher levels of oxidative DNA damage were clearly detected in all treated animals compared to controls. While this method eliminates DNA extraction reducing the processing of biological samples, absolute values are not provided. Further, the method requires only small amounts of tissue and potentially discriminates susceptibility to oxidative damage in different cell types. These results suggest that the assay should have practical applications in marine ecotoxicology. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: 7,8-Dihydro-8-oxodeoxyguanosine; Oxidative DNA damage; Monoclonal antibody 1F7; Genotoxicity; Mussel; Eel; Benzo[a]pyrene
*
Corresponding author. Tel.: +39-071-2204613; fax: +39-071-220-4609. E-mail address: [email protected] (N. Machella).
0141-1136/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2004.03.022
726
N. Machella et al. / Marine Environmental Research 58 (2004) 725–729
8-Oxo-dG, a major oxidative DNA product formed by hydroxyl radicals or prooxidant chemicals, is a marker of genotoxic damage. Since a number of environmental pollutants induce oxidative stress (Winston & Di Giulio, 1991), its measurement is increasingly being used in marine ecotoxicology (L opez-Barea & Pueyo, 1998). Increased levels of 8-oxo-dG have been documented within target tissues of aquatic vertebrates and invertebrates after environmental or laboratory exposures to environmental pollutants (Canova et al., 1998; Malins & Gunselman, 1994). In recent years, attention has focused on measurement of 8-oxo-dG levels and although several methods are currently available, controversy still remains about the actual levels within cells and tissues (ESCODD, 2002). Incomplete DNA hydrolysis and artificial oxidation of native guanine to 8-oxo-dG are considered the main drawbacks that may occur during the analysis (Halliwell, 1999). Also, depending on the extraction protocol, DNA isolation can influence the background level of 8-oxodG in cultured human cells (Ravanat et al., 2002). A general consensus still has not been reached concerning the basal levels of 8-oxo-dG within standard biological samples (ESCODD, 2002; Halliwell, 1999). Despite this, improvement and integration of methodologies to assess oxidative DNA damage would be of great relevance for marine ecotoxicology in order to better understand the environmental impact of genotoxins. In this regard, the monoclonal antibody 1F7, previously developed and used to detect 8-oxo-dG in oral cells and nasal biopsies (Santella, 1999), was tested for the first time in Mediterranean mussel (Mytilus galloprovincialis) and European eel (Anguilla anguilla). Immunoperoxidase and immunofluorescence staining were done in eel liver sections, while only immunoperoxidase staining was used on mussel digestive gland and haemocytes. In order to induce oxidative DNA damage, animals were treated under laboratory conditions with different doses of benzo[a]pyrene, a model genotoxic compound. Mussels were exposed in artificial seawater to concentrations of B[a]P ranging from 100 to 1000 ppb in 0.0001% DMSO for 10 days, while controls did not receive the solvent. Eels were intraperitoneally injected with 0, 0.1, 1, 10, 50 mg/kg of B[a]P (dissolved in corn oil) and sacrificed after seven days. The immunohistochemical stainings were performed with modifications of the protocols standardized in humans by Yarborough, Zhang, Hsu, and Santella (1996) and Motykiewicz et al. (2002). Proteinase K digestion and DNA denaturation times were reduced to preserve the morphological integrity of the tissue samples. Quantification of 8-oxo-dG was made on five randomly chosen fields, counting ten cells per field. For the immunoperoxidase method, a Cell Analysis System 200 microscope (Cell Measurement Program Software Package) was used and the damage was expressed as Relative Staining Intensity (RSI). The fluorescence intensity, expressed as Average Gray Value Average (AGVA), was assessed by a Nikon Eclipse-600 microscope, equipped with a Hamamatsu CCD digital camera (C4742-95) and quantified by the MetaView Imaging System Software. The relative damage (RD) level was calculated as the ratio between the mean staining intensity values of treated and control samples. Control slides of cultured human lymphoblastoid cells, treated with 5 mM H2 O2 for 30 min and untreated, were used to confirm the staining
N. Machella et al. / Marine Environmental Research 58 (2004) 725–729
727
Fig. 1. M. galloprovincialis and A. anguilla. Representative immunohistochemical stainings for 8-oxo-dG detection. Immunoperoxidase method: (a) digestive gland sections of control mussel and (b) mussel exposed to 500 ppb of B[a]P (200X). (c) Haemocytes of control mussel and (d) mussel exposed to 500 ppb of B[a]P (1000X). (e) Liver sections of control eel and (f) eel exposed to 50 mg/kg of B[a]P (400X). Immunofluorescence method: (g) liver sections of control eel and (h) eel exposed to 50 mg/kg of B[a]P (1000X).
specificity. Additional controls included marine sample slides that were stained after omitting the primary antibody or digesting tissues with DNAse before the staining.
728
N. Machella et al. / Marine Environmental Research 58 (2004) 725–729
Fig. 2. Levels of 8-oxo-dG detected in A. anguilla with the immunoperoxidase and immunofluorescence method, expressed as RD, compared to controls ðp < 0:05Þ.
In mussels, immunoperoxidase staining revealed increased levels of damage, both in digestive gland and haemocytes of treated samples (Fig. 1). Higher levels of damage were observed in haemocytes compared to digestive gland, and no dose response relationship was obtained in either tissue (data not shown). Marked increases in relative staining intensity were also evident in treated eels, using either the immunoperoxidase or immunofluorescence methods, as indicated by Fig. 1. However, while immunoperoxidase revealed similar increments of 8-oxo-dG, at different doses, the effects measured by the immunofluorescence detection generally increased with a linear trend at higher B[a]P doses (Fig. 2). The differences between the immunoperoxidase and the immunofluorescence detection methods were confirmed by the magnitude of RD values: quite homogeneous and lower in the former, more variable and higher in the latter (Fig. 2). These results suggested a greater sensitivity of the fluorescence method even though immunoperoxidase detection may be required when there is natural cell autofluorescence. The main advantages of the immunohistochemical assessment of DNA damage include a limited processing of samples (liquid nitrogen freezing, cryostat sections, brief fixation and staining), no DNA isolation or nuclear lysis (thus limiting a source of artificial oxidation) and the possibility to work on previously frozen samples without the need of fresh tissues from living organisms. Further, the method is potentially able to identify differential susceptibility of cells within different tissues to genotoxic insult, with a rapid and relatively inexpensive procedure. The disadvantages include potential antibody cross-reactivity with other modified nucleotides and the semi-quantitative nature of the data that does not provide absolute 8-oxo-dG levels.
N. Machella et al. / Marine Environmental Research 58 (2004) 725–729
729
Although these results must be considered preliminary, laboratory exposures were helpful in investigating the consistency of the approach and the efficacy of the use of antibody 1F7 in these marine organisms. To further assess the potential use of this approach in marine ecotoxicology, future analyses will be necessary to validate the detection of DNA damage in field conditions.
Acknowledgements This work was partially supported by NIEHS grant P30ES09089 and represents part of the Doctoral Thesis of N. Machella in Biochemical and Biomolecular Sciences.
References Canova, S., Degan, P., Peters, L. D., Livingstone, L. D., Voltan, R., & Venier, P. (1998). Mutation Research, 399, 17–30. ESCODD (European Standards Committee on Oxidative DNA Damage) (2002). Carcinogenesis 23, 2129–2133. Halliwell, B. (1999). Mutation Research, 443, 37–52. L opez-Barea, J., & Pueyo, C. (1998). Mutation Research, 399, 3–15. Malins, D. C., & Gunselman, G. S. (1994). Proceedings of the National Academy of Sciences USA, 91, 13038–13041. Motykiewicz, G., Faraglia, B., Wang, L. W., Terry, M. B., Senie, R. T., & Santella, R. M. (2002). Environmental and Molecular Mutagenesis, 40, 93–100. Ravanat, J. L., Douki, T., Duez, P., Gremaud, E., Herbert, K., Hofer, T., Lasserre, L., Saint-Pierre, C., Favier, A., & Cadet, J. (2002). Carcinogenesis, 23, 1911–1918. Santella, R. M. (1999). Cancer Epidemiology Biomarkers and Prevention, 8, 733–739. Winston, G. W., & Di Giulio, R. T. (1991). Aquatic Toxicology, 19, 137–161. Yarborough, A., Zhang, Y. J., Hsu, M. T., & Santella, R. M. (1996). Cancer Research, 56, 683–688.