Genotoxicological Characterization of (±)cis-4,4′-DMAR and (±)trans-4,4′-DMAR and Their Association

Round 1

Reviewer 1 Report

General comment:

Lenzi et al. report cytotoxic and (possibly) genotoxic effects of an illicit psychostimulant which has been apparently illegally trafficked, traded and dealt for several years. I am not sure that it represents a great contribution to drug safety; I doubt that the criminals who synthesize and distribute the compound(s) in question follow IJMS and, even if they did, they would not care. Then again, any new compound with potential health (or other commercial) benefits has to undergo extensive testing and scrutinizing, first by the manufacturing company and then by a public regulator such as US FDA. The results obtained by Lenzi et al. would be enough to permanently kill any project involving the compounds. No respectable drug company would ever use the molecules as lead structures in any future project, even if it be more for legal than scientific reasons. In this sense, the present study might possibly be moderately useful. My opinion is based on my previous experience of being employed by a large international pharmaceutical company.

The MS is written in a style of variable quality. Some passages are acceptable, others are difficult to follow. I would insist on a thorough language check by a native speaker with good grounding in scientific writing (I will mention some examples of the text below, but these are not exhaustive by far).

Specific queries and comments

# 1      Line 33: It should probably be in plural: “stimulants”.

# 2      L 36: English sentences should start in capital letters. As “4” cannot be capitalised, I would suggest “4-Methyl..”

# 3      L 39: “related” or “correlated” or perhaps “linked to”?

# 4      L 52: This is an example of a statement hard to understand. I would start with “Drug users on internet discussion fora reported (approximate?) dosages producing a (characteristic?) sequence of psychological and behavioural effects; euphoria, increased sociability and verbal flow, feeling of physical energy as well as (general) mental stimulation.” and continue refining it until fully satisfied. I doubt that the users, with all due respect, reported “exact dose ranges” – these would have to be in micromol/kg to have any meaning.

# 5      L 116: 45 % of what? See also Table 1

# 6      L 120: Does this refer to Tab 1? If it does, then, may I observe that if you use more civilized concentration ranges you would probably obtain concentration dependence (and more meaningful data) for both cis- and trans- forms.

# 7      L 136: Is this "slight" difference statistically significant? Has it been tested?

# 8      L 159: What exactly is “MNi gate”? Also, MNi should be defined when it is used for the first time (and perhaps not used as an abbreviation in Abstract).

# 9      Discussion, the first paragraph: I have an (ethical) problem with this text. There are elements in it which could be misconstrued as providing some legitimacy for illegal drugs. Authors might think that it is their manuscript and their reputation which is at risk and they should decide what to write (and take their chances) but IJMS also has to consider its reputation (and possible legal consequences). As the text adds nothing in terms of scientific analysis of the present data, I would strongly argue that it be deleted, whole paragraph. Similar considerations potentially apply to lines 318 to 321; the expression “reassurance” could be gravely misconstrued in a wrong legal context.

# 10    Line 250: “determined” I would say “showed”, “displayed” or just “was more cytotoxic”

# 11     Line 315: Again, an example of a difficult-to-follow statement. Did you mean that the present results should be corroborated (or “followed up”) by in vivo studies?

# 12    Lines 318 t0 321: Quite a mouthful here – if you insist on retaining this statement (see # 9 above), consider splitting it into shorter, more digestible, sentences.

# 13    Another general comment: Authors seem to labour under the impression that the finding that very small changes in a molecular structure can have great consequences for the biological activity is an important discovery. For a medicinal chemist, it is practically trivial, almost an everyday observation. Moreover, cis and trans isomers are often very different in terms of electron density & charge distribution as well as other structural characteristics important for the configuration of the pharmacophores and, consequently, for the biological (pharmacological) activity. Consider modifying and toning down the relevant statements (e.g. in Abstract). The data actually do represent an interesting illustration of how cis- and trans- isomers of a compound can produce almost opposite biological effects, just write it in a more suitable manner.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

The article by Monia Lenzi et colleagues provides information about the genotoxicological characterization of (±)cis-4,4’-DMAR and 2 (±)trans-4,4’-DMAR and their association

The topic is of interest and the manuscript is well written. I recommended that this review study is acceptable for publication after revision.

Comments and suggestions:

The MS is difficult to follow and a scheme with all the steps of this study is missing.

Introduction section: add more data about synthetic stimulants

I suggest writing the chemical formulas for (±)cis-4,4’-DMAR and 2 (±)trans-4,4’-DMAR

Figure 6, images B and C: the letters are too small and difficult to read. Revise it.

What perspectives for human health does this MS have?

Consider revision accordingly.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The new version is much better. I only have two minor comments, see below. Additionally, may I suggest replacing “single” (line 334) with “..the racemates of cis- and trans-isomers..”  to make it sound clearer. Readers might also wonder whether you had any plans to test individual enantiomers, if they are/become available.

# 1    Line 150: Still very clumsy. Why don’t you say “..noticed that there was an apparent but not statistically significant increase in apoptosis (Fig.2)” Whether the increase was “slight”, or, greater than just “slight”, is a bit subjective so it is better to avoid such a term. I am suggesting “apparent” (if you wish to retain “increase” at all) because it looked like, or, “appeared” to be, an increase. In very strict terms, it does not matter what a result “looks” like; if it is not statistically significant, it is neither an “increase” nor “decrease”, just a random deviation - buy we are not always so strict.

# 2    Line 305   “..assumption..” does not make sense (or sounds a bit dissonant, if you like) in the context. I would expect something like “..simultaneous application..”

Author Response

The new version is much better. I only have two minor comments, see below. Additionally, may I suggest replacing “single” (line 334) with “..the racemates of cis- and trans-isomers..”  to make it sound clearer. Readers might also wonder whether you had any plans to test individual enantiomers, if they are/become available.

We corrected it as suggested by the Reviewer. 

# 1    Line 150: Still very clumsy. Why don’t you say “..noticed that there was an apparent but not statistically significant increase in apoptosis (Fig.2)” Whether the increase was “slight”, or, greater than just “slight”, is a bit subjective so it is better to avoid such a term. I am suggesting “apparent” (if you wish to retain “increase” at all) because it looked like, or, “appeared” to be, an increase. In very strict terms, it does not matter what a result “looks” like; if it is not statistically significant, it is neither an “increase” nor “decrease”, just a random deviation - buy we are not always so strict.

We corrected it as suggested by the Reviewer. 

# 2    Line 305   “..assumption..” does not make sense (or sounds a bit dissonant, if you like) in the context. I would expect something like “..simultaneous application..”

We corrected it as suggested by the Reviewer.