An In-Vitro Evaluation of Toxic Metals Concentration in the Third Molars from Residents of the Legnica-Głogów Copper Area and Risk Factors Determining the Accumulation of Those Metals: A Pilot Study

Round 1

Reviewer 1 Report

Abstract:

1. Why does the author only discuss this area?

2. “Two subgroups of patients were created according to the place of residence residents of the Legnica - Głogów Copper Area (49 people) and control group - residents of Wrocław (20 people). ” Are there too few people? Can't represent all?

 

Keywords

1. Keywords are too few and should be added.

 

Introduction

1. It is too brief and does not fully explain why this study discusses determine tissue concentration of toxic metals.

2. It is recommended to supplement this study mainly to monitor the impact of the environment on the human body.

3. There are also trace amounts of metal elements in the human body, how to prove the pollution from the environment?

 

Materials and Methods

1. The Declaration of Helsinki is not enough, there must be approval for human trials.

2. Please add how to measure metals in teeth and blood? “Atomic absorption spectrometry was used for the multi-element analysis of the dental material. ”

Too abstract.

3. Please describe the conditions of the subjects.

 

Results and Discussion

1. The authors should state who was the control group.

2. The author conducts a regional investigation and research, what is the number of mother groups? Confidence interval? Are the results representative of this region?

3. What does the author mainly want to discuss? Or what method do you want to improve?

4. How do the authors distinguish between these age gaps?

Conclusions

1. “The content of iron and lead was statistically significantly higher in patients living in L-G Copper Area than in residents of Wrocław, which may indicate greater environmental pollution in the L-G Cooper Area during the mineralization process.” The authors only evaluated teeth and blood. An analysis of the environment was not performed. So I think a more conservative assessment should be made.

 

It is suggested that the manuscript should be substantially revised.

Author Response

Dear Referee,

 

We would like to express our sincerest gratitude for your efforts put into criticizing the manuscript. We have taken into account all raised question here follows the detailed answers. Moreover, all changes we have made to the original manuscript, are marked in the red colour in the text.

 

Abstract:

1. Why does the author only discuss this area?

Answer: We would like to thank you for the comment. The abstract has been revised. The authors decided to reorganize the abstract to show the purpose of the study, present examined and control group and highlight the introduced novelties and results that are considered as statistically significant.

2. “Two subgroups of patients were created according to the place of residence residents of the Legnica - Głogów Copper Area (49 people) and control group - residents of Wrocław (20 people). ” Are there too few people? Can't represent all?

Answer: Thank you for the comment. The research material was collected in the period from June 2020 to June 2021, during the COVID-19 epidemic, therefore the number of patients in the control group, which was matched according age and gender to the study group, is small. We performed analyzes of test power for both groups and used statistical tests. Average results of these calculations are presented below:

 

	Test power
	Test for qualitative variable	Test for quantitative variable
L-G copper district group (n=49)	0.94	0.98
Control group (n=20)	0.65	0.70

Based on the calculations, we conclude that the number of people in the study group was correct, while the size of the control group was slightly lower than required. Comparative analyzes on predictors for toxic metal accumulation were performed in the study group (Table 3A, 3B, 4). Therefore, we added the phrase to the title of the article: “a pilot study”.

Keywords

1. Keywords are too few and should be added.

Answer: We would like to thank you for the comment. The manuscript has been revised and keywords have been added.

Introduction

1. It is too brief and does not fully explain why this study discusses determine tissue concentration of toxic metals.

 Answer: We would like to thank you for the comment. The manuscript has been revised.

2. It is recommended to supplement this study mainly to monitor the impact of the environment on the human body.

 Answer: We would like to thank you for the comment.The manuscript has been revised.

3. There are also trace amounts of metal elements in the human body, how to prove the pollution from the environment?

Answer: We would like to thank you for the comment. Indeed, trace elements are present in the human body, however in our own research we showed that the concentration of some toxic metals was higher from L-G Copper Area residents and was considered as statistically significant. Therefore, this may indicate the correlation with the environmental pollution.

Materials and Methods

1. The Declaration of Helsinki is not enough, there must be approval for human trials.

Answer: We would like to thank you for the comment. The manuscript has been revised. Prior to the study, the consent of the Bioethics Committee of the Wroclaw Medical University was obtained (consent number: KB-246/2019).

2. Please add how to measure metals in teeth and blood? “Atomic absorption spectrometry was used for the multi-element analysis of the dental material. ”

Too abstract.

Answer: We would like to thank you for the comment. The methodology section has been rearranged. Determination of Pb, Cd, Cr, Ni, Fe, Mn, Cu, Zn content was performed by atomic absorption spectrometry in an air-acetylene flame using the SpectraAA atomic absorption spectrometer with a V2 AA240FS flame attachment.

Vitamin D levels were measured from a capillary blood sample using the capillary method in the Vitality Health Check (VHC) Vitamind-D TEST system. 
After thoroughly washing, disinfecting and drying the blood collection site and obtaining blood circulation, a sterile disposable lancet was used to puncture the fingertip after which 10 µl of blood was collected using heparinized capillary tubes (BloodCollector - UniSampler device)
The blood was then placed in the CollectionTube, which had previously been used to obtain the buffer. After thorough mixing, the sample was placed using a micropipette on the Test Divice. After 15 minutes, the vitamin D value was read using the VHC Reader. More specific information has been added.

3. Please describe the conditions of the subjects.

Answer: We would like to thank you for the comment. Extracted teeth were stored at - 20 °C in boxes that were steriled to avoid chemical preparation of the samples before performing tests. More detailed information about conditions of the subject has been added.

Results and Discussion

1. The authors should state who was the control group.

Answer: Thank you for the comment. The manuscript has been revised. The control group consisted of  the Wrocław residents (n = 20). The research group, in contrast, consisted of the patients from Legnica-Głogów copper district (n = 49).

2. The author conducts a regional investigation and research, what is the number of mother groups? Confidence interval? Are the results representative of this region?

Answer: Thank you for the comment. In accordance with the statistical requirements, we conducted the study in a group drawn from the population of people living in the L-G copper district, and then we estimated the results of the study to the study population. Following the Reviewer's suggestion, we added the confidence interval values to the descriptive statistics of quantitative variables in Tables 1 and 2. There are no studies that we could compare with our results. The authors’ research is the first one, that evaluated the accumulation of heavy metals in impacted third molars from the patients living in Legnica – Głogów Copper Area. However, Bryła et. al. [1] examined the content of toxic metals in impacted third molars and adjacent bone tissue in patients from the city of Wroclaw (M) and the Wroclaw district (P) and we compared our results to this research.

3. What does the author mainly want to discuss? Or what method do you want to improve?

Answer: Thank you for the comment. In the results section the authors decided to emphasize results that are statistically significant. The methodology section has been revised in accordance with your recommendations. We improved the description of atomic absorption spectrometry and the storage of the samples after extraction of examined teeth.

4. How do the authors distinguish between these age gaps? - The authors performed the examination during the COVID-19 epidemic. The most common age of third molars extraction ranges between 18 - 25 years old. That is the reason why the biggest number of patients was from this age group.

Answer:

Conclusions

1. “The content of iron and lead was statistically significantly higher in patients living in L-G Copper Area than in residents of Wrocław, which may indicate greater environmental pollution in the L-G Cooper Area during the mineralization process.” The authors only evaluated teeth and blood. An analysis of the environment was not performed. So I think a more conservative assessment should be made. 

Answer: We would like to thank you for the comment. The manuscript has been revised. Indeed, the authors only evaluated teeth and blood, however there are scientific studies that prove that Legnica-Głogów Copper Area in Poland is district where the risk of heavy metal contamination is increased due to the industrial profile and structure and we wanted to show the correlation between the pollution of the environment and the content of toxic metals in impacted teeth.

It is suggested that the manuscript should be substantially revised.

Answer: We would like to thank you for the comment. The manuscript has been revised.

 

[1]. Bryła E., Dobrzyński, M., Konkol D., Kuropka P., Styczyńska M., Korczyński M. Toxic Metals Content in Impacted Third Molars and Adjacent Bone Tissue in Different Groups of Patients. Materials 2021, 14, 793. https://doi.org/10.3390/ma14040793.

 

 

 

 

Reviewer 2 Report

The reviewed manuscript aimed to measure the concentration of toxic metals (Mn, Cr, Ni, Cu, Fe, Cd, Pb, Zn) in third molars that were extracted. The study involved two groups of patients: 49 people who lived in the Legnica-Głogów Copper Area and a control group of 20 people who lived in Wrocław. The authors considered the correlation between level of Cr, Cu, Zn and age due to its significance effect. I am sure that the study deserves the attention of readers and can be published, however, some major concerns need to be addressed before accepting the paper for publication to improve the readability and clarity of the manuscript:

 

1-    The use of English language is reasonable, however, there are a number of punctuation and grammatical errors; that should be corrected and rephrased using academic English for a better flow of text for reader.

2-    Please consider reviewing the abstract and highlighting the novelty. I suggest reorganizing the abstract, highlighting the novelties introduced. The abstract should contain answers to some questions, what problem was studied and why is it important? and what conclusions can be drawn from the results? (Please provide specific results not generic ones). Please provide a brief introduction on the importance of such work.

3-    The introduction is poor and needs a lot of enhancement. The introduction is very short and brief and references are very old. Although we are now in 2023, there are no references in 2022. I think incorporating updated references show the interesting of researchers on the paper topic.

4-    At the end of the introduction, it is better to give more information about evaluation tests that will be used inside the work and what expected results that will be extracted from such tests.

5-     Results are merely described and is limited to comparing the experimental observation and describing results. The authors are encouraged to include a more detailed results and discussion section and critically discuss the observations from this investigation with existing literature.

6-    When discussing and comparing results, quantitative information should be given instead of only qualitative information. Did you carry out statistical analyses to clarify statistical significance?

7-    The conclusion section is very short, it doesn’t mention specific result. The authors repeated many parts of the introduction and didn’t consider their results.

 Please, read the text carefully before the next submission of the paper.

Author Response

Dear Referee,

 

We would like to express our sincerest gratitude for your efforts put into criticizing the manuscript. We have taken into account all raised question here follows the detailed answers. Moreover, all changes we have made to the original manuscript, are marked in the red colour in the text.

 

Comments and Suggestions for Authors

The reviewed manuscript aimed to measure the concentration of toxic metals (Mn, Cr, Ni, Cu, Fe, Cd, Pb, Zn) in third molars that were extracted. The study involved two groups of patients: 49 people who lived in the Legnica-Głogów Copper Area and a control group of 20 people who lived in Wrocław. The authors considered the correlation between level of Cr, Cu, Zn and age due to its significance effect. I am sure that the study deserves the attention of readers and can be published, however, some major concerns need to be addressed before accepting the paper for publication to improve the readability and clarity of the manuscript:

• The use of English language is reasonable, however, there are a number of punctuation and grammatical errors; that should be corrected and rephrased using academic English for a better flow of text for reader.

 Answer: Thank you for your attention. We have corrected the manuscript.

• Please consider reviewing the abstract and highlighting the novelty. I suggest reorganizing the abstract, highlighting the novelties introduced. The abstract should contain answers to some questions, what problem was studied and why is it important? and what conclusions can be drawn from the results? (Please provide specific results not generic ones). Please provide a brief introduction on the importance of such work.

Answer: We would like to thank you for the comment. The abstract has been revised. The authors added details about obtained results that are statistically significant. In addition, according to your recommendations, we included the information about the novelty and importance of our research.

• The introduction is poor and needs a lot of enhancement. The introduction is very short and brief and references are very old. Although we are now in 2023, there are no references in 2022. I think incorporating updated references show the interesting of researchers on the paper topic.

Answer: Thank you for your comment. The introduction has been revised. To our knowledge, our study is the first to assess the accumulation of toxic metals: Pb, Cd, Cr, Ni, Fe, Mn, Cu, Zn in third molars from residents of the Legnica-Głogów Copper Area. We have added more new references.

• At the end of the introduction, it is better to give more information about evaluation tests that will be used inside the work and what expected results that will be extracted from such tests.

Answer: Thank you for your attention. More information about performed tests has been added in the methodology section.

• Results are merely described and is limited to comparing the experimental observation and describing results. The authors are encouraged to include a more detailed results and discussion section and critically discuss the observations from this investigation with existing literature.

Answer: Thank you for the comment. The results section has been revised.

• When discussing and comparing results, quantitative information should be given instead of only qualitative information. Did you carry out statistical analyses to clarify statistical significance?

Answer: Thank you for your attention. We have corrected the text by adding descriptive data to the Discussion section.As we described in Material and Methods section, we performed statistical analysis of data with using adequate statistical tests, such as: chi-square test, Fisher exact test, Mann-Whitney U test, Kruskal-Wallis analysis with post-hoc Dunn’s test and Spearman correlation analysis. In the reviewed manuscript we added names of used statistical tests to each title of the table.

• The conclusion section is very short, it doesn’t mention specific result. The authors repeated many parts of the introduction and didn’t consider their results.

Answer: We would like to thank you for the comment. The conclusion has been revised.

Please, read the text carefully before the next submission of the paper.

Answer: Thank you for your comment. The manuscript has been revised.

 

 

Reviewer 3 Report

Dear Authors the is really interesting, but it is necessary to review some points in order to improve the quality of the paper: 
-First, i ask you to check the plagiarism of your article 

I suggest you to modify it and add the type of article.

-Please be sure to use only keywords accordingly to medical subject headings Mesh  

- The introduction section is very short and is needed to add other references to increase the quality of the manuscript. I suggest you to add some lines about the more frequent oral lesions, this paper are useful: [PubMed ID 29460529];  [doi: 10.11138/orl/2016.9.2.054] [doi: 10.1097/SCS.0000000000009103]

-You need to review the grammar and English of your article, with the help of a native English speaker (you can specify who helped you in reviewing English in the acknowledgements) or simply by using a site that can support you in English

 

Kind Regards

Author Response

Dear Referee,

 

We would like to express our sincerest gratitude for your efforts put into criticizing the manuscript. We have taken into account all raised question here follows the detailed answers. Moreover, all changes we have made to the original manuscript, are marked in the red colour in the text.

Dear Authors the is really interesting, but it is necessary to review some points in order to improve the quality of the paper: 
-First, i ask you to check the plagiarism of your article 

Answer: Thank you for the comment. The article has been checked.

I suggest you to modify it and add the type of article.

Answer: Thank you for the comment. The manuscript has been revised. We changed the title by adding a phrase - pilot study.

-Please be sure to use only keywords accordingly to medical subject headings Mesh  

Answer: Thank you for the comment. The keywords have been revised.

- The introduction section is very short and is needed to add other references to increase the quality of the manuscript. I suggest you to add some lines about the more frequent oral lesions, this paper are useful: [PubMed ID 29460529];  [doi: 10.11138/orl/2016.9.2.054] [doi: 10.1097/SCS.0000000000009103]

Answer: Thank you for your attention. The introduction has been improved.

-You need to review the grammar and English of your article, with the help of a native English speaker (you can specify who helped you in reviewing English in the acknowledgements) or simply by using a site that can support you in English.

Answer: Thank you for the comment. The manuscript has been changed according to your recommendations.

 

 

Reviewer 4 Report

The innovation and necessity of conducting the study is not well explained.  The volume of the study was small and it should have been mentioned in the limitations of the study.

Author Response

Dear Referee,

 

We would like to express our sincerest gratitude for your efforts put into criticizing the manuscript. We have taken into account all raised question here follows the detailed answers. Moreover, all changes we have made to the original manuscript, are marked in the red colour in the text.

The innovation and necessity of conducting the study is not well explained.  The volume of the study was small and it should have been mentioned in the limitations of the study.

Answer: Thank you for your comment. The manuscript has been revised.  We rearranged many sections of our research. The limitations of the study have been added.  The volume of the study was small because it was performed during the COVID-19 epidemic. We would also like to mention that it was a pilot study. We did not find other studies that would examine content of toxic metals in impacted third molars from the patients living in Legnica-Głogów Copper Area with a use of Atomic absorption spectrometry.

Reviewer 5 Report

Introduction

1. There are a few grammatical errors. Please correct them

2. Please write a null and alternative hypothesis

Methodology:

3. Explain the methodology in detail: Sample collection, Sample preparation, sample analysis, etc. Provide details of materials and methods used.

4. Please provide details of how the sample size was determined

5. Provide details of inclusion and exclusion criteria.

6. What was the basis of the selection of included individuals?

7. Studies reported that the presence of amalgam restoration in the oral cavity affect the concentrations of few metals. Was this point considered in the study?

8. Previously published studies have reported that mineralization of 3^rd molar starts at around 8-9 year of age and apex closure takes place at around 21-22 year of age. So major mineralization of these teeth takes place during this stage. A group of participants included in the current study was above this age. Will it be relevant to compare the levels of these heavy metals in the blood (which will reflect present levels) with the levels in teeth (which were mainly mineralized few years).

9. Participants included in the study were all natives of that particular area. Was data recorded to find out if they were residing in other areas during the time when the mineralization of teeth was taking place? Was this point considered in the study?

10. Please provide relevant pictures.

Results

11. As mentioned in Section 3.1 “This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn” Please arrange the results sections accordingly.

12. Please provide graphs for the data available in tables 3A and 3B (along with tables).

 Discussion

 

13. Add limitations of the study.

Author Response

Dear Referee,

 

We would like to express our sincerest gratitude for your efforts put into criticizing the manuscript. We have taken into account all raised question here follows the detailed answers. Moreover, all changes we have made to the original manuscript, are marked in the red colour in the text.

Introduction

1. There are a few grammatical errors. Please correct them.

Answer: Thank you for your attention. The introduction has been revised.

2. Please write a null and alternative hypothesis

Answer: We would like to thank you for the comment. We conducted many statistical comparative analyzes for qualitative and quantitative variables and for evaluating the type of distribution of quantitative variables in the study groups. For each of these analyses, a null and an alternative hypothesis should be formulated. In general, for comparative analyses, hypotheses H0 and H1 are as follows:

H0: there is no significant difference between the means obtained for the variable in the

study groups (Mi1=Mi2)

H1: there is significant difference between the means in the study groups (Mi1≠Mi2)

For correlations:

H0: there is no significant relationship between two variables

H1: there is significant relationship between two variables

For normal distribution of data:

H0: data are normally distributed

H1: data are not normally distributed

In our study distribution of the quantitative variable in one of the compared groups was usually significantly different from the normal distribution, therefore we used non-parametric tests to analyze the data.

Methodology:

3. Explain the methodology in detail: Sample collection, Sample preparation, sample analysis, etc. Provide details of materials and methods used.

Answer: We would like to thank you for the comment. The manuscript has been revised. All details that you mentioned  have been added to the study.

4. Please provide details of how the sample size was determined.

Answer: We would like to thank you for the comment. The manuscript has been revised. The sample consisted of the whole extracted tooth.

5. Provide details of inclusion and exclusion criteria.

Answer: We would like to thank you for the comment. The manuscript has been revised. The most important criterion for inclusion and exclusion is that the surveyed patients had lived since birth in Legnica-Głogów Copper Area or in Wrocław. The next inclusion criterion was the fact that patients were healthy and did not take any dietary supplements or medications.

6. What was the basis of the selection of included individuals?

Answer: We would like to thank you for the comment. The manuscript has been revised. The basis of the selection of included individuals was: sex, age, no use
of dietary supplements and permanent residence in Legnica- Głogów Copper Area or Wrocław.

7. Studies reported that the presence of amalgam restoration in the oral cavity affect the concentrations of few metals. Was this point considered in the study?

Answer: We would like to thank you for the comment. In the authors' study, we used teeth that were completely retained and that had no contact with the oral cavity environment. Thus amalgam restorations does not affect the concentration of heavy metals in impacted third molars.

8. Previously published studies have reported that mineralization of 3^rdmolar starts at around 8-9 year of age and apex closure takes place at around 21-22 year of age. So major mineralization of these teeth takes place during this stage. A group of participants included in the current study was above this age. Will it be relevant to compare the levels of these heavy metals in the blood (which will reflect present levels) with the levels in teeth (which were mainly mineralized few years).

Answer: We would like to thank you for the comment. The germs of completely retained third molars are like a trap - only during the mineralization process elements supplied with a blood can be incorporated into the apatite structure. Once the mineralization process is completed, it is not possible.

9. Participants included in the study were all natives of that particular area. Was data recorded to find out if they were residing in other areas during the time when the mineralization of teeth was taking place? Was this point considered in the study?

Answer: We would like to thank you for the comment. The surveyed patients had lived since birth in Legnica-Głogów Copper Area or in Wrocław and as we mentioned above this is the most important criterion for inclusion and exclusion. 

10. Please provide relevant pictures.

Answer: We would like to thank you for the comment. We added applicable pictures.

Results

11. As mentioned in Section 3.1 “This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn” Please arrange the results sections accordingly.

Answer: : We would like to thank you for the comment. The subheadings have been rearranged.

12. Please provide graphs for the data available in tables 3A and 3B (along with tables).

Answer: Thank you for the comment. The graphs have been provided.

Discussion

13. Add limitations of the study.

Answer: Thank you for the comment. The limitations of the study have been added.

 

 

Round 2

Reviewer 1 Report

The manuscript was revised appropriately.

I think it's acceptable.

Reviewer 2 Report

 

Many thanks for the revision and for incorporating all suggested changes to the manuscript that are nicely reflected. The authors did a good job of improving the article. I believe that the article has become much better, and now I recommend this article for publication.

Reviewer 4 Report

The article is well edited and all the points mentioned are done.

Reviewer 5 Report

The manuscript can be accepted in present form. Thank you