Environmentally Relevant Concentrations of Triclosan Induce Cyto-Genotoxicity and Biochemical Alterations in the Hatchlings of Labeo rohita

Round 1

Reviewer 1 Report

Comments on manuscript entitled "Environmentally relevant concentrations of Triclosan induce cyto-genotoxicity and biochemical alterations in the hatchlings of Labeo rohita" by Sharma et. al.

The paper describes the genotoxic and biochemical changes in L. rohita hatchlings on exposure to TCS.

Following things need to be explained in the MS.

1. Figure 2: TCS induced nuclear and cellular abnormalities-The comparison of nuclear or cellular abnormalities are not clear compared to the normal cell (2a image). Normal cell image vs rest of the nuclear/cellular changes (2b-2r) captured are not clear which one is cell or nucleus? And as mentioned in the methodology these images were captured at 100X magnification (line 117-118)-doesn’t look like 100X magnification images. So, comparison of these minute differences of nucleus or cells as whole, is difficult to distinguish. Thus, it should be good to check the nuclear staining method-DAPI method vs cell staining method to check the real differences of cell vs nucleus.
2. Figure 3 and Table 3: The real tail length or tail moment is indistinguishable from Fig.3 on which table 3 is based. It would be nice to show the real DNA damage by western blotting which can support these values or images.
3. Also, immunohistochemistry showing DNA damage of these tissues would further strengthen the data.
4. The metabolic changes studied here makes no contribution to this MS because showing value decreased or increased (line 225-257) are meaningless unless the author describes clearly what is the significance of those studies and highlight why those metabolites are important to understand the pathway affected?
5. Needs proofreading by all authors-typos everywhere, e.g., line 281, line 295.

The paper is technically sound and well written. Comments above- will make the MS better.

Author Response

Reviewer 1

1. Figure 2: TCS induced nuclear and cellular abnormalities-The comparison of nuclear or cellular abnormalities are not clear compared to the normal cell (2a image). Normal cell image vs rest of the nuclear/cellular changes (2b-2r) captured are not clear which one is cell or nucleus? And as mentioned in the methodology these images were captured at 100X magnification (line 117-118)-doesn’t look like 100X magnification images. So, comparison of these minute differences of nucleus or cells as whole, is difficult to distinguish. Thus, it should be good to check the nuclear staining method-DAPI method vs cell staining method to check the real differences of cell vs nucleus.

Response: The quality and contrast of figure 2 has been improved to make the difference clear. Nucleus and cytoplasm have been marked in the image 2a and is same for other figures. These images were captured at 100X magnification under Olympus BX43 microscope, however, to adjust the images in the manuscript these were cropped. We used conventional Giemsa staining for micronucleus assay because it is cost effective and highlights similar frequency of MN compared to DAPI (Tian et al. 2006).

2. Figure 3 and Table 3: The real tail length or tail moment is indistinguishable from Fig.3 on which table 3 is based. It would be nice to show the real DNA damage by western blotting which can support these values or images.

Response: Contrast for figure 3 has been changed and it clearly shows concentration dependent increase in tail length now. Software Casp Lab was then used to analyze the comet images for measurement of DNA damage parameters i.e., tail length, tail moment, olive tail moment and percent tail DNA (Borrego-Soto et al., 2015) and the values obtained thereof were mentioned in Table 3. Comet assay is a simple, rapid and cheap method for measurement of DNA damage in majority of toxicological studies. Western blot is mainly used to monitor changes in the expression of DNA repair factors/ proteins while neutral comet assay is used to detect DNA double strand breaks (Gonzalo and Kreienkamp, 2016).

3. Also, immunohistochemistry showing DNA damage of these tissues would further strengthen the data.

Response: Immunohistochemistry is generally used to examine the expression of genotoxicity markers i.e., DNA damage/ repair proteins with the help of labelled antibodies. Additionally, this is a costlier and time-consuming approach along with it needs primary and secondary antibodies for specific genotoxicity markers. Larvae of the fish were very small i.e., 2.5 -3.0 mg only, therefore we have used comet assay.

4. The metabolic changes studied here makes no contribution to this MS because showing value decreased or increased (line 225-257) are meaningless unless the author describes clearly what is the significance of those studies and highlight why those metabolites are important to understand the pathway affected?

Response: Metabolic changes are generally used as stress markers in toxicology studies and has been mentioned in the discussion section that decrease and increase is an early indication of the damage to the cell or its components. Increase or decrease in these parameters is used to ascertain the health status of the organisms living in stressed habitats. Most of the xenobiotics are metabolic depressors as they affect the structure and function of biological molecules (Vijayavel and Balasubramanian, 2006). Increase or decrease in biochemical parameters varies according to the type of pollutant, species of fish and duration of exposure (Monteiro et al., 2005; Jee et al., 2005). Therefore, these parameters were included in the present study.

5. Needs proofreading by all authors-typos everywhere, e.g., line 281, line 295.

Response: Proofreading has been done.

Reviewer 2 Report

The manuscript contains a collection of experiments addressed to evaluate the effect of  triclosan on cyto-genotoxicity and biochemical alerations in hatchlings of Labeo rohita. It clearly appears that triclosan has some effects but the pattern remains obscure. 

Specific comments: 

The manuscript contains little typing imprecisions.

The figures are not sufficient explicative and the quality of some figures should be increased. 

Author Response

1. The manuscript contains little typing imprecisions.

           Response: The typing imprecisions have been corrected throughout the manuscript.

2. The figures are not sufficient explicative and the quality of some figures should be increased.

          Response: The quality of figures has been improved.

Reviewer 3 Report

Não encontro nenhuma referência aos comitês de ética.
Existe o procedimento ou é aprovado pelo Departamento de Zoologia, Guru Nanak Dev Universidade

Author Response

 

1. I can’t find any reference to ethics committees. Does the procedure exist or is approved by the department of zoology, Guru Nanak Dev University?

 

        Response: The experiments were conducted last year and till that time food fishes did not come under the purview of National ethics committee. All the experimental protocols in this study were approved by the internal animal Ethics Committee, Guru Nanak Dev University.

Reviewer 4 Report

The research is a contribution on the long-term toxicity of TCS, may be accepted with minor revision

In fig 1-3 ... the quality of the images should improve

Author Response

1. In fig 1-3 ... the quality of the images should improve.

 Response: The quality and contrast of all the images has been improved.

 

Reviewer 5 Report

A very well structured article, with a clearly described working protocol. Just minor corections, such as: 

• row 116, p3. ”Giemsa” with uppercase ”G”;
• row 281, p. 9, ”In the present study” with ”I” uppercase letter;
• row 347, please delete the full stop at the beginning of the row. 
•  

Author Response

 

1. row 116, p3. ”Giemsa” with uppercase ”G”.

Response: It has been done.

2. row 281, p. 9, ”In the present study” with ”I” uppercase letter.

Response: It has been done.

3. row 347, please delete the full stop at the beginning of the row. 

Response: It has been done.

Round 2

Reviewer 1 Report

Figure 2 showing nuclear and nucleo-cellular abnormalities-uncropped images-does not provide enough evidence that there are really such changes-For example: Look at figure 2a-normal cell image (uncropped)-that slide shows all types of images as shown in 2b, 2c, 2e, 2g, 2h, 2l, 2m, 2o, 2r. So, showing one cell/nucleus out of the 500 cells/replicate (as mentioned in the methods) is not providing enough evidence of that real change-that is why in my original comments-immunohistochemistry or western blotting experiments would have strengthen the data, if the changes are real.