Arch Toxicol (2013) 87:2247–2248 DOI 10.1007/s00204-013-1155-9
LETTER TO THE EDITOR
Genotoxic damage in the oral mucosal cells of subjects carrying restorative dental fillings Giuseppa Visalli · Barbara Baluce · Sebastiano La Maestra · Rosanna T. Micale · Luciano Cingano · Silvio De Flora · Angela Di Pietro
Received: 24 October 2013 / Accepted: 28 October 2013 / Published online: 16 November 2013 © Springer-Verlag Berlin Heidelberg 2013
Five years ago, we demonstrated that a dose-dependent genotoxic damage is detectable by means of the single-cell gel electrophoresis (SCGE or comet assay) in peripheral blood lymphocytes of subjects carrying restorative dental fillings containing either Hg-based amalgams or methacrylates (Di Pietro et al. 2008). In in vitro, resin composite components have a lower toxicity than Hg from amalgams (Reichl et al. 2006), but several studies demonstrated that methacrylates are genotoxic in cultured human peripheral blood lymphocytes, as assessed by comet assay (Poplawski et al. 2009), in cultured human gingival fibroblasts, where they induce double-strand breaks, and in other test system (Blasiak et al. 2012). Later on, analyzing the oral mucosal
G. Visalli (*) · B. Baluce · A. Di Pietro Department of Hygiene, Public Health and Preventive Medicine, University of Messina, Via Consolare Valeria, 98125 Messina, Italy e-mail: [email protected] B. Baluce e-mail: [email protected] A. Di Pietro e-mail: [email protected] S. La Maestra · R. T. Micale · S. De Flora (*) Department of Health Sciences, University of Genoa, Via A. Pastore 1, 16142 Genoa, Italy e-mail: [email protected] S. La Maestra e-mail: [email protected] R. T. Micale e-mail: [email protected] L. Cingano Department of Surgical and Integrated Diagnostic Sciences, University of Genoa, Largo R. Benzi 10, 16132 Genoa, Italy e-mail: [email protected]
cells of 63 young subjects of both genders, we found that amalgam- and resin-based composite fillings are associated with a local genotoxic damage detectable by means of the micronucleus (MN) test and, more marginally, by the comet assay (Visalli et al. 2013). Recently, these conclusions were questioned by Rothfuss et al. (2013), who in a Letter to the Editor tried to challenge the validity of the data reported by Visalli et al. (2013). In our opinion, the only point about which they are right is that, due to a transcription mistake, we wrote 0.26 instead of 0.026 in the equation of the regression line relating the number of dental fillings (x) to the MN frequency (y). The correct equation is thus y = 0.295 + 0.026x and not y = 0.295 + 0.26x. In any case, it should be noted that (a) the slope of the regression line reported in Fig. 1 is correct, (b) the correlation between the number of restored surfaces and MN frequency is statistically significant, and (c) even by correcting 0.26 into 0.026, the effect is not negligible at all, since it means that each filling results on average in a ~10 % increase in MN frequency. The other points raised by Rothfuss et al. clearly reflect an attempt to lessen the validity of scientific data relative to materials manufactured by the industry, like they did previously in the case of other industrial products. Thus, the statement, “as members of the large population carrying restorative dental fillings we felt attracted by the subject… although none of us is actively involved in any work in this area” looks like an unsolicited justification. For instance, these authors criticize the fact that we used the term “biomonitoring” for our study, based on the fact that we did not make any measurements at different time points, preferably before and after the fillings, and that we did not check Hg concentrations in saliva. Clearly, it is right that any study can be improved, but it was already complicated to recruit tens of volunteers for this kind of analysis.
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Rothfuss et al. also questioned the fact that the results of the comet assay were statistically significant only in subjects carrying at least two fillings, when comparing the 90th and 95th percentile of TDNA% data. In this respect, it is known that for the tail length, the best performance for statistical analysis is obtained with the 90th percentile, capturing the upper tail of the distribution (Wiklund and Agurell 2003). The weak effect recorded when using the comet assay is due to the fast turnover of oral cells and to the fact that a pretreatment is needed to allow the lysis of these epithelial cells. In any case, we mainly based our conclusions on the results of the MN test, whereas we were well careful in interpreting the comet assay data both in the Abstract, where we specified “…inferred from the results of the MN test and, more marginally, from comet assay data”, and in the Discussion, where we reported that “a marginal and less consistent genotoxic response was also observed by using the comet assay…”. The statements that in the paper by Visalli et al. (2013), there is “lack of clear effect on DNA damage” and that “this also speaks against the plausibility of DNA damage in circulating lymphocytes that the authors reported previously (Di Pietro et al. 2008)” are erroneous, undocumented, and totally gratuitous. Note that the latter article was cited 46 times in less than 5 years. Similarly, the final sentence “We would like to suggest that toxicologists should be
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Arch Toxicol (2013) 87:2247–2248
more careful when suggesting such risks” is an arrogant lesson that we do not accept. As independent scientists, we report the results as they are. References Blasiak J, Synowiec E, Tarnawska J, Czarny P, Poplawski T, Reiter RJ (2012) Dental methacrylates may exert genotoxic effects via the oxidative induction of DNA double strand breaks and the inhibition of their repair. Mol Biol Rep 39:7487–7496 Di Pietro A, Visalli G, La Maestra S, Micale R, Baluce B, Matarese G, Cingano L, Scoglio ME (2008) Biomonitoring of DNA damage in peripheral blood lymphocytes of subjects with dental restorative fillings. Mutat Res 650:115–122 Poplawski T, Pawlowska E, Wisniewska-Jarosinska M, Ksiazek D, Wozniak K, Szczepanska J, Blasiak J (2009) Cytotoxicity and genotoxicity of glycidyl methacrylate. Chem Biol Interact 180:69–78 Reichl FX, Simon S, Esters M, Seiss M, Kehe K, Kleinsasser N, Hickel R (2006) Cytotoxicity of dental composite (co)monomers and the amalgam component Hg2+ in human gingival fibroblasts. Arch Toxicol 80:645–672 Rothfuss A, Vonk R, Schweinfurth H (2013) The evidence of genotoxic damage in the oral mucosa due to dental fillings is very questionable. Arch Toxicol 87:1155–1156 Visalli G, Baluce B, La Maestra S, Micale RT, Cingano L, De Flora S, Di Pietro A (2013) Genotoxic damage in the oral mucosa cells of subjects carrying restorative dental fillings. Arch Toxicol 87:179–187 Wiklund SJ, Agurell E (2003) Aspects of design and statistical analysis in the Comet assay. Mutagenesis 18:167–175
LETTER TO THE EDITOR
Genotoxic damage in the oral mucosal cells of subjects carrying restorative dental fillings Giuseppa Visalli · Barbara Baluce · Sebastiano La Maestra · Rosanna T. Micale · Luciano Cingano · Silvio De Flora · Angela Di Pietro
Received: 24 October 2013 / Accepted: 28 October 2013 / Published online: 16 November 2013 © Springer-Verlag Berlin Heidelberg 2013
Five years ago, we demonstrated that a dose-dependent genotoxic damage is detectable by means of the single-cell gel electrophoresis (SCGE or comet assay) in peripheral blood lymphocytes of subjects carrying restorative dental fillings containing either Hg-based amalgams or methacrylates (Di Pietro et al. 2008). In in vitro, resin composite components have a lower toxicity than Hg from amalgams (Reichl et al. 2006), but several studies demonstrated that methacrylates are genotoxic in cultured human peripheral blood lymphocytes, as assessed by comet assay (Poplawski et al. 2009), in cultured human gingival fibroblasts, where they induce double-strand breaks, and in other test system (Blasiak et al. 2012). Later on, analyzing the oral mucosal
G. Visalli (*) · B. Baluce · A. Di Pietro Department of Hygiene, Public Health and Preventive Medicine, University of Messina, Via Consolare Valeria, 98125 Messina, Italy e-mail: [email protected] B. Baluce e-mail: [email protected] A. Di Pietro e-mail: [email protected] S. La Maestra · R. T. Micale · S. De Flora (*) Department of Health Sciences, University of Genoa, Via A. Pastore 1, 16142 Genoa, Italy e-mail: [email protected] S. La Maestra e-mail: [email protected] R. T. Micale e-mail: [email protected] L. Cingano Department of Surgical and Integrated Diagnostic Sciences, University of Genoa, Largo R. Benzi 10, 16132 Genoa, Italy e-mail: [email protected]
cells of 63 young subjects of both genders, we found that amalgam- and resin-based composite fillings are associated with a local genotoxic damage detectable by means of the micronucleus (MN) test and, more marginally, by the comet assay (Visalli et al. 2013). Recently, these conclusions were questioned by Rothfuss et al. (2013), who in a Letter to the Editor tried to challenge the validity of the data reported by Visalli et al. (2013). In our opinion, the only point about which they are right is that, due to a transcription mistake, we wrote 0.26 instead of 0.026 in the equation of the regression line relating the number of dental fillings (x) to the MN frequency (y). The correct equation is thus y = 0.295 + 0.026x and not y = 0.295 + 0.26x. In any case, it should be noted that (a) the slope of the regression line reported in Fig. 1 is correct, (b) the correlation between the number of restored surfaces and MN frequency is statistically significant, and (c) even by correcting 0.26 into 0.026, the effect is not negligible at all, since it means that each filling results on average in a ~10 % increase in MN frequency. The other points raised by Rothfuss et al. clearly reflect an attempt to lessen the validity of scientific data relative to materials manufactured by the industry, like they did previously in the case of other industrial products. Thus, the statement, “as members of the large population carrying restorative dental fillings we felt attracted by the subject… although none of us is actively involved in any work in this area” looks like an unsolicited justification. For instance, these authors criticize the fact that we used the term “biomonitoring” for our study, based on the fact that we did not make any measurements at different time points, preferably before and after the fillings, and that we did not check Hg concentrations in saliva. Clearly, it is right that any study can be improved, but it was already complicated to recruit tens of volunteers for this kind of analysis.
13
2248
Rothfuss et al. also questioned the fact that the results of the comet assay were statistically significant only in subjects carrying at least two fillings, when comparing the 90th and 95th percentile of TDNA% data. In this respect, it is known that for the tail length, the best performance for statistical analysis is obtained with the 90th percentile, capturing the upper tail of the distribution (Wiklund and Agurell 2003). The weak effect recorded when using the comet assay is due to the fast turnover of oral cells and to the fact that a pretreatment is needed to allow the lysis of these epithelial cells. In any case, we mainly based our conclusions on the results of the MN test, whereas we were well careful in interpreting the comet assay data both in the Abstract, where we specified “…inferred from the results of the MN test and, more marginally, from comet assay data”, and in the Discussion, where we reported that “a marginal and less consistent genotoxic response was also observed by using the comet assay…”. The statements that in the paper by Visalli et al. (2013), there is “lack of clear effect on DNA damage” and that “this also speaks against the plausibility of DNA damage in circulating lymphocytes that the authors reported previously (Di Pietro et al. 2008)” are erroneous, undocumented, and totally gratuitous. Note that the latter article was cited 46 times in less than 5 years. Similarly, the final sentence “We would like to suggest that toxicologists should be
13
Arch Toxicol (2013) 87:2247–2248
more careful when suggesting such risks” is an arrogant lesson that we do not accept. As independent scientists, we report the results as they are. References Blasiak J, Synowiec E, Tarnawska J, Czarny P, Poplawski T, Reiter RJ (2012) Dental methacrylates may exert genotoxic effects via the oxidative induction of DNA double strand breaks and the inhibition of their repair. Mol Biol Rep 39:7487–7496 Di Pietro A, Visalli G, La Maestra S, Micale R, Baluce B, Matarese G, Cingano L, Scoglio ME (2008) Biomonitoring of DNA damage in peripheral blood lymphocytes of subjects with dental restorative fillings. Mutat Res 650:115–122 Poplawski T, Pawlowska E, Wisniewska-Jarosinska M, Ksiazek D, Wozniak K, Szczepanska J, Blasiak J (2009) Cytotoxicity and genotoxicity of glycidyl methacrylate. Chem Biol Interact 180:69–78 Reichl FX, Simon S, Esters M, Seiss M, Kehe K, Kleinsasser N, Hickel R (2006) Cytotoxicity of dental composite (co)monomers and the amalgam component Hg2+ in human gingival fibroblasts. Arch Toxicol 80:645–672 Rothfuss A, Vonk R, Schweinfurth H (2013) The evidence of genotoxic damage in the oral mucosa due to dental fillings is very questionable. Arch Toxicol 87:1155–1156 Visalli G, Baluce B, La Maestra S, Micale RT, Cingano L, De Flora S, Di Pietro A (2013) Genotoxic damage in the oral mucosa cells of subjects carrying restorative dental fillings. Arch Toxicol 87:179–187 Wiklund SJ, Agurell E (2003) Aspects of design and statistical analysis in the Comet assay. Mutagenesis 18:167–175
