Plasticizer Di-(2-ethylhexyl) Phthalate and Its Metabolite Mono(2-ethylhexyl) Phthalate Inhibit Myogenesis in Differentiating Mouse and Human Skeletal Muscle Cell Models
, Ding-Cheng Chan 6, Rong-Sen Yang 7,* and Shing-Hwa Liu 4,8,9,*
Round 1
Reviewer 1 Report
The results of your research are valuable and worth publishing in the journal Applied Sciences, but some minor revisions need to be made:
- Lines 3 and 9: in the name of MEHP - mono(2-ethylhexy) phthalate, "l" is missing
- Lines 55 - 60: An explanation for mentioning an association between low-birth weight and phthalates in the context of myogenesis in this article is missing.
- The discussion of your results seems disorganized. I suggest reworking it, e.g. better link the results of epidemiological studies and other monitoring studies (L231-245) with your results (L245-249). Your study also lacks a comparison of your results with other studies regarding factors related to the association between myogenesis and phthalate exposure (L250-265).
Author Response
Reviewer 1
The results of your research are valuable and worth publishing in the journal Applied Sciences.
Response: We appreciate the reviewer's positive comment.
But some minor revisions need to be made:
- Lines 3 and 9: in the name of MEHP - mono(2-ethylhexy) phthalate, "l" is missing
Response: We appreciate the reviewer's comment. We have corrected these typing errors in this revised manuscript according to the suggestion of reviewer.
- Lines 55 - 60: An explanation for mentioning an association between low-birth weight and phthalates in the context of myogenesis in this article is missing.
Response: We appreciate the reviewer's comment. We have added an explanation for mentioning an association between low-birth weight and phthalates in the context of myogenesis in this revised manuscript according to the suggestion of reviewer.
These descriptions were shown in the Introduction:
The low-birth-weight is a marker of poor fetal development and growth [11]. An epidemiological study has shown that the prenatal di-n-butyl phthalate and DEHP exposures contribute to low-birth-weight in Chinese newborns [12]. A relationship between prenatal exposure to phthalates and preterm delivery in Chinese women has also been reported [13]. In very low birth weight infants, a significant negative correlation between the sum of DEHP metabolites and birth weight have been demonstrated [14]. These studies indicate that DEHP exposure may contribute to low-birth-weight. The reduction in myogenesis has been demonstrated to be reflected in the low-birth-weight of intrauterine growth-restricted (IUGR) fetal piglet [15] and sheep [16]. Hansen et al. have also found that myogenesis in human muscle stem cells cultured from the low-birth-weight individuals is retarded [17]. DEHP has been shown to inhibit myogenesis in C2C12 myoblasts [18]. However, it is still necessary to further explore the detailed effects and possible mechanisms of DEHP and its metabolite MEHP on skeletal myogenic differentiation.
- The discussion of your results seems disorganized. I suggest reworking it, e.g. better link the results of epidemiological studies and other monitoring studies (L231-245) with your results (L245-249). Your study also lacks a comparison of your results with other studies regarding factors related to the association between myogenesis and phthalate exposure (L250-265).
Response: We appreciate the reviewer's comment. We have re-organized the discussion of our results and added a comparison of our results with other studies in this revised manuscript according to the suggestion of reviewer.
These descriptions were shown in the Discussion:
Our data showed that there was no cytotoxicity in DEHP (1-1000 μM)-treated C2C12 myoblasts under differentiation medium for 96 hours. This result was con-sistent with the report by Chen et al. in which there was no cytotoxicity of DEHP (10-1000 μg/mL) on C2C12 cells [18]. Nevertheless, we further found that its metabolite MEHP displayed a greater cytotoxicity at concentrations of >100 μM. Our data also showed that non-cytotoxic-concentration DEHP (10-100 μM) and MEHP (10-50 μM) significantly suppressed the myoblast differentiation and myotube formation in C2C12 myoblasts and HSMPCs. These concentrations for DEHP and MEHP seem to be relevant to human exposure in bloods, which would be discussed later. The levels of DEHP in indoor air of Tokyo Metropolitan area have been shown to be up to 1000-fold higher than ambient levels in Japan [28]. It has been found that the concentrations of DEHP are 72±13 μg/mL and 668±96 μg/mL after 8 h and 24 h, respectively, in a cyclosporine solution stored in a PVC bag at room temperature [29]. In the replacement blood transfusions, the blood DEHP levels have been detected to be ranged from 10 to 650 μg/mL (about 25-1600 μM) [8]. A prospective and comparative clinical study showed that a mean maximum plasma level of DEHP was 8.3±5.7 µg/mL at any time in a group of 28 consecutive term infants during an extracorporeal membrane oxygenation therapy [30]. A study for newborn infant plasticizers exposure showed that the plasma DEHP levels were 3.4-1.1 μg/mL in the individual infants, and MEHP levels were 2.4-15.1 μg/mL in the corresponding samples [31]. Therefore, the test concentrations of DEHP/MEHP in the current study may be the blood concentrations to which human is likely to be exposed.
Reviewer 2 Report
Dear Authors,
The concerns of your study are listed below.
1. Abstract
1.1 Page 1 of 11, line 25: Please correct the word “Di(2-ethylhexyl)” to “di(2-ethylhexyl)”.
2. Introduction
2.1 Page 2 of 11, lines 64-71: I suggest you rewrite this section. It looks like the discussion.
3. Materials and Methods
3.1 Page 3 of 11, lines 110-111: Please inform the instrument, used for absorbance determination at 570 nm.
3.2 Page 3 of 11, lines 115-116: You may need to provide references for this staining method.
4. Results
4.1 I suggest you rewrite this section. Please write your result based on your ordering methods.
Author Response
Reviewer 2
Dear Authors,
The concerns of your study are listed below.
- Abstract
1.1 Page 1 of 11, line 25: Please correct the word “Di(2-ethylhexyl)” to “di(2-ethylhexyl)”.
Response: We appreciate the reviewer's comment. We have corrected these typing errors in this revised manuscript according to the suggestion of reviewer.
- Introduction
2.1 Page 2 of 11, lines 64-71: I suggest you rewrite this section. It looks like the discussion.
Response: We appreciate the reviewer's comment. We have rewrite this section in the Introduction of this revised manuscript according to the suggestion of reviewer.
These descriptions were shown in the Introduction:
The low-birth-weight is a marker of poor fetal development and growth [11]. An epidemiological study has shown that the prenatal di-n-butyl phthalate and DEHP exposures contribute to low-birth-weight in Chinese newborns [12]. A relationship between prenatal exposure to phthalates and preterm delivery in Chinese women has also been reported [13]. In very low birth weight infants, a significant negative correlation between the sum of DEHP metabolites and birth weight have been demonstrated [14]. These studies indicate that DEHP exposure may contribute to low-birth-weight. The reduction in myogenesis has been demonstrated to be reflected in the low-birth-weight of intrauterine growth-restricted (IUGR) fetal piglet [15] and sheep [16]. Hansen et al. have also found that myogenesis in human muscle stem cells cultured from the low-birth-weight individuals is retarded [17]. DEHP has been shown to inhibit myogenesis in C2C12 myoblasts [18]. However, it is still necessary to further explore the detailed effects and possible mechanisms of DEHP and its metabolite MEHP on skeletal myogenic differentiation.
- Materials and Methods
3.1 Page 3 of 11, lines 110-111: Please inform the instrument, used for absorbance determination at 570 nm.
Response: We appreciate the reviewer's comment. We have added the information for the instrument in this revised manuscript according to the suggestion of reviewer.
3.2 Page 3 of 11, lines 115-116: You may need to provide references for this staining method.
Response: We appreciate the reviewer's comment. We have added the reference for this staining method in this revised manuscript according to the suggestion of reviewer.
- Results
4.1 I suggest you rewrite this section. Please write your result based on your ordering methods.
Response: We appreciate the reviewer's comment. We have re-organized the Methods and Results sections that the results would be displayed based on the ordering methods in this revised manuscript according to the suggestion of reviewer.
Round 2
Reviewer 2 Report
Dear Authors,
I am satisfied with your attempt and your response to reviewer comments.
Sincerely.
