Title: Arsenic, asbestos and radon: Emerging players in lung tumorigenesis

Transcription

1 Author's response to reviews Title: Arsenic, asbestos and radon: Emerging players in lung tumorigenesis Authors: Roland Hubaux Daiana D. Becker-Santos Katey S.S. Enfield Stephen Lam Wan L. Lam Victor D. Martinez Version: 2 Date: 25 October 2012 Author's response to reviews: see over

2 Editorial Office Environmental Health Roland Hubaux, PhD BC Cancer Research Centre 675 West 10 th Avenue, Vancouver British Columbia, Canada V5Z 1L3 Phone: ext.7703, Fax: October 19, 2012 Ref: Impact of Arsenic, Asbestos and Radon Exposure on the Lung Cancer Genome Review Report Dear Editorial team, The authors would like to thank the reviewers for their time and valuable comments. We have addressed their comments as follows: COMMENTS CONCERNING MANUSCRIPT FORMATTING Note: We have not highlighted all the following changes in text for this section; since some of them involve the whole manuscript (e.g. remove line numbering). If changes are not highlighted in text, we have provided the page number where the modification was made, in order to facilitate Editor s labor. 1) Throughout the manuscript remove the line numbering. R: We have removed all line numbering from the manuscript. 2) On the title page, the title should include the study design, for example "A versus B in the treatment of C: a randomized controlled trial X is a risk factor for Y: a case control study". R: We have modified the original title Impact of arsenic, asbestos and radon exposure on the lung carcinogenesis to Arsenic, asbestos and radon: Emerging players in lung tumorigenesis. The new title is highlighted in yellow, page 1. 3) The text on the title page should be left justified and the superscript 2 and 3 changed to symbols. R: We have modified this according to the journal instructions. Added symbols are highlighted in yellow, on page 1. 4) The correspondence information should be substituted by the phrase *Corresponding author and all the other information removed. Insert the heading addresses and below list the addresses as author's initials: address e.g. JS:joe.shmoe@university.edu. Only the first letter of headings should be capitalized. R: We have added the required information from all authors on the first page of the manuscript. Information about authors is highlighted in yellow. Additionally, we have changed headers format (only first letter is capitalized) on pages 2, 3, 5, 7, 10, and 11 (new headings are highlighted in yellow). 5) Change italicized text to normal except where indicated in the References. R: We have changed all text to regular style. The following changes were made (page number refers to new manuscript). - p3: Arsenic heading was changed to regular text. - p4: Asbestos heading was changed to regular text. - p5: Radon heading was changed to regular text. - p5: Arsenic-induced DNA damage was modified to Arsenic and changed to regular text.

3 - p6: Asbestos and oxidative stress was modified to Asbestos and changed to regular text. - p7: DNA damage caused by the decay process of radon was modified to Radon and changed to regular text. - p7: Genetic alterations and chromosomal rearrangements was replaced to Molecular and genetic alterations and changed to regular text. - p9: Epigenetic alterations heading was changed to regular text. - p10: Aspergillus niger (2 nd paragraph on the Management strategies Chapter) was also changed to regular text, although we wonder if this should not be kept in italic. We have also changed all genes names to regular text. 6) The final section in the text should read Conclusions and should state clearly the main conclusions of the review and give a clear explanation of their importance and relevance. R: We have changed the heading text to Conclusions. Additionally, we have rewritten the last two paragraphs of this chapter. The added text is the following: Although tobacco smoke is the major cause of lung cancer, environmental carcinogens, such as arsenic, asbestos and radon play an increasingly important role in this disease, either independently or through additive or multiplicative effects [131, 132]. While the number of individuals exposed to these carcinogens is significant, the difficulty to associate tumor cases directly with exposure to these agents (mainly due to the long latency period between exposure and disease onset) may be highly underestimated. The growing interest in non-tobacco causes of lung cancer is reflected in the increasing number of reports describing molecular alterations correlated with exposure to these carcinogens. In this article, we have collected evidence of the involvement of specific molecular mechanisms that can lead to genetic and epigenetic aberrations in lung tumor genomes as a result of exposure to these agents. While sharing a few carcinogenic mechanisms, each agent may induce specific sets of alterations which might affect tumor biology and define tumor behaviour, presenting a unique opportunity for developing diagnostic and treatment options. Future research, including the integration of different genetic and epigenetic dimensions, will further the characterization of these etiologically distinct tumors and identify actionable candidates for therapeutic targets. 7) After the Conclusions insert the headings List of abbreviations, Competing interests and Authors' contributions. See the Instructions for authors and published articles for guidance how to word and organize this section. R: We have inserted the required information (highlighted in yellow on page 13) 8) The tables should come after the References. R: Tables1, 2, and 3, are now located on page 22, 23 and 24, following the References Section. We have also moved Figure legends, which are now located after the Tables. 9) In the References, remove the issue numbers and list up to 30 authors before using et al. Please also ensure that your revised manuscript conforms to the journal style ( ). R: We have reformatted all references in text using the endnote journal style. REVIWERS COMMENTS Reviewer 1: No changes required Reviwer 2: Major Compulsory Revisions

4 1) One of the major concerns is that the authors did not take into account enough the genetic effect of tobacco smoking, the major risk for lung cancer. When the authors summarize results on epigenetic and genetic effects of arsenic, asbestos and radon on lung cancer, it is not mentioned whether patients were smokers or nonsmokers. Response: Although our review was centered on the effects that can be attributed, at least in part, to arsenic, asbestos and radon exposure, we agree with the reviewer on the relevance of the genetic effects of tobacco smoking in lung cancer. We have addressed this comment as follows: - On the Genetic and epigenetic consequences in lung tumor genomes due to arsenic, asbestos and radon exposure section (page 7), we have separated the effects associated with each of these agents. We have also added the model (animal/cell human) used, and pointed out results specifically related to never smokers cases and smokers/never smokers cohorts, when information was available. The use of in vitro or animal models allows excluding some of the effects associated with tobacco smoke. - We have added information concerning the origin of cells and smoking status in Tables 2 and 3. 2) Another point that needs to be addressed more precisely is the genotoxic mechanisms of these carcinogens. The authors only focus on oxidative stress. Concerning asbestos, generation of reactive oxidative species is certainly involved, but other mechanisms could also explain the genetic and epigenetic effects of asbestos. For example, in cells in culture, asbestos fibers interfere with the mitotic spindle and thereby could induce numerical or structural chromosomal aberrations. Recently, a comprehensive review article, part of the Asbestos Workshop: A Science-Based Examination of the Mode of Action of Asbestos and Related Mineral Fibers funded by NIEHS, was published (Huang et al., 2011). In the present review at lines page 8, the authors address these issues only briefly and uncompletely. Authors should better describe the mechanism of asbestos carcinogenesis, which is not limited to oxidative stress induction, in page 7 (asbestos mechanism chapter). Consequently, they should modify the figure 3. Response: We would like to thank the Reviewer for providing this information. We have completed this section based on the comments of the reviewer and additional information (new text is highlighted in grey on the new manuscript). Additionally, we have added the direct physical interactions as mechanism of asbestos induced carcinogenicity on Figure 3. - We have changed the title of this section to Carcinogenic mechanisms induced by exposure to arsenic, asbestos, and radon (originally, Generation of reactive oxidative species is a major mechanism of lung carcinogenesis induced by arsenic, asbestos and radon). This was done in order to be more comprehensive, since we have included other mechanisms for carcinogenesis. - We have separated the mechanisms for each of the 3 carcinogens analyzed, giving some representative examples for the most commonly described. - We have updated the Figures and modified the text for Asbestos carcinogenesis (p6) as follows: Inhaled asbestos fibers longer than 5μm are not efficiently eliminated by phagocytosis. This can induce a cascade of molecular events that lead to fibrosis, inflammation and carcinogenesis (Figure 3) [73, 74]. On the other hand, fully phagocytized fibers can interfere with mitosis, leading to chromosomal missegregation [75]. The induction of reactive oxygen and nitrogen species upon incomplete phagocytosis of fibers plays an important role in DNA damage [76]. Asbestos induces the release of ROS, including O2- and H2O2 [73]. Such reactions can be catalyzed on the asbestos fiber surface, and asbestos fibers with high iron content, such as crocidolite and amosite, are capable of generating higher levels of ROS [77]. Similar to arsenic, asbestos also affects mitochondrial DNA and functional electron transport resulting in mitochondrial-derived ROS, which has been shown to induce base oxidation, single-strand breaks, micronuclei, and apoptosis in lung alveolar epithelial cells. [78, 79]. Therefore, asbestos carcinogenesis is suspected to occur through creation of an environment of chronic inflammation, and especially through the induction of oxidative stress, a well-known inducer of DNA damage [80]. Lesions at sites of fiber deposition and alterations in gene expression are other relevant mechanisms in asbestos-induced neoplasia in lungs and other target organs [81]. Asbestos mutagenicity has been also proposed as a mode of action for asbestos, although it role remains to be elucidated [81,82].

5 3) To illustrate the effects of asbestos, the authors quoted references 135, 142 and 151) that did not deal with lung cancer but with mesothelioma. So far, with the exception of CDKN2A gene locus, genetic alterations are different between these two tumors, indicating that asbestos effect at the genetic level could be cell type specific. The authors need to describe either asbestos effect in lung cancer alone or in both thoracic tumor including mesothelioma, but in a separate chapter. Response: Most of the cancer research related to asbestos concerns mesothelioma instead of lung cancer; and we agree with the reviewer that this could distract the focus of this review since we focus on mechanisms involved in lung cancer. Unless they are also concerning lung cancer, we have deleted all the references related to mesothelioma from the text and tables. 4) Important data on asbestos genetic effect on lung cancer are missing. Several studies searched for the signature of asbestos in thoracic cancers. They studied the link between KRAS, TP53 or CDKN2A mutations and asbestos exposure in lung cancers (Wang et al., 1995; Husgafvel-Pursiainen et al., 1999; Nelson et al., 1999; Andujar et al., 2010). Response: We have incorporated the information from the references above both in the text and in Table 2. Additionally, we have complemented the information about effects at genetic level with other references, such as: - Shukla A, Barrett TF, Nakayama KI, Nakayama K, Mossman BT, Lounsbury KM: Transcriptional upregulation of MMP12 and MMP13 by asbestos occurs via a PKCdelta-dependent pathway in murine lung. FASEB J 2006, 20(7): ) The authors should carefully check the genetic alterations in Table 2. For instance, in reference 131, it is shown that 14q11.2 was amplified in non-exposed asbestos patients and not in exposed patients. We have revised all Tables. In Table 2, we specifically removed regions for which similar alterations were also observed at higher rate in non-exposed patients. Minor Essential Revisions 1) Concerning some developed countries, several recent epidemiological studies suggest that mesothelioma incidence will not spike between 2020 and 2040, but reach recently a plateau (correction in page 5, lines ). Response: We agree with the Reviewer that the asbestos-related effects might have reached a plateau in some countries, and we have changed the last phrase on the asbestos (pages 4-5) section for the following text: Even though the current use and management of asbestos is under strict control in most countries, the high latency between exposure and asbestos-related disease development poses a significant public health threat [42] Since it is difficult to establish when the specific asbestos-related effects will appear, we would like to highlight that this issue will still need to be considered as a burden in the next following years. 2) As authors mentioned arsenic remediation, it should be interesting that the authors reported on asbestos remediation, that is under study especially in Italy. Response: On the Management strategies for radon, arsenic and asbestos exposure (3 rd paragraph, page 10) section, we have added some references concerning asbestos remediation plans recently occurring in Italy.

6 Discretionary Revisions 1) The sentence Asbestos fibers are not classical mutagens, since in most cases they are not fully metabolized by the organism and do not interact directly with DNA. is not appropriate (page 7, line 163). While metabolization is likely for chemical compounds such as PAH, solid compounds such as mineral fibers are unlikely metabolized. Moreover, as mentioned above, asbestos fibers could interact with chromosomes. Then, this sentence should be modified. Response: We agree with the comment. This sentence has been deleted. 2) Page 7, line 172: unformatted reference. Response: We have reformatted all references in the text. 3) If Zimbabwe production was banned in 2004, it should not be in yellow in Figure 1 or Authors are confident that asbestos production started again in Response: We have modified the color for Zimbabwe on Figure 1; however, we have kept the information concerning Zimbabwe on figure legend. We thank the reviewers again for their constructive input and hope to have satisfactory answered their comments. We also would like to thank the Editorial Team, and we look forward to your reply. Sincerely, Roland Hubaux, Ph.D. Post-doctoral Fellow Integrative Oncology British Columbia Cancer Agency