Synthetic Cathinones Induce Cell Death in Dopaminergic SH-SY5Y Cells via Stimulating Mitochondrial Dysfunction
Round 1
Reviewer 1 Report
The authors show in this manuscript a study where the in-vitro toxicity and mechanistic pathways of butylone, pentylone and 3,4-methylenedioxypyrovalerone (MDPV) are investigated in SH-SY5Y cells. The text is clear and shows interesting results.
Consequently, this work should be suitable for publication in International Journal of Molecular Sciences. Minor changes have to take into account:
The Figure 1 is unclear because is difficult to distinguish between 6 and 24h curves, so it is difficult to relate the 24h curves to the EC shown in the table.
Author Response
Point 1: The Figure 1 is unclear because is difficult to distinguish between 6 and 24h curves, so it is difficult to relate the 24h curves to the EC shown in the table.
Response 1: The initial intention of showing the 6 h time point curve was to illustrate the time-dependent cytotoxicity of the drugs. However, in order to decrease the complexity of the figure, we have removed the 6 h time point data. 24 h exposure time is similar to those used for other in-vitro studies involving SCs.
Reviewer 2 Report
This article presents evidence of cell death and underlying cellular physiology changes due to rather high dose administration of a group of newly popular recreational drugs assocaited with neurologic pathology (butylone, pentylone and MDPV). Overall I found the study to be thoughtful, well-executed, and very well presented. However, my enthusiasm is diministed by two factors:
1) The doses are very high compared to what is known to be relevant in patients unless I am misunderstanding something. I do not really think this can be addressed, and I am willing to accept it as a limitation thoroughly acknowledged.
2) The authors have the opportunity to establish some meaningful causality by conducting rescue experiments for key elements of toxicity. Does a free radical scavenger prevent cell death, for instance? Do drugs that improve mitochondrial function help in this regard? Some further work on this would greatly improve the manuscript, I believe.
Detailed comments follow below.
Introduction
I think it is important to acknowledge that direct toxicity leading to cell death is only one of several mechanisms by which these drugs can be harmful to the nervous system. Also it is potentially important to consider their effects on non-neuronal cells. Both of these key points need to be mentioned in order to frame the study. Also related, is there any evidence in terms of human pathology that any of these drugs cause neuronal cell death?
Results
How were the dose ranges chosen? Almost any organic compound is neurotoxic in sufficient doses. How do these doses related to presumptive levels in brain tissue in humans who ingest them? Is there any data on this?
What is the rationale for the 6 an 24 hours time points?
It would seem to me that Figure 6 and Figure 7 should be a single figure, if 7 presents the quantitative results that 6 are an example of.
I think just showing the merged images in Figure 6 would actually be easier to interpret and would make the figure less busy.
In section 2.5 associated with Figure 8, it should be explicitly stated how caspase activity was measured.
Discussion
I do not think the authors have really proven a causal relationship between butylone, pentylone and MDPV and cell death due to the stated mechanisms of ROS production, mitochondrial dysfunction, and altered calcium homeostasis. To assert this some rescue experiments would be required.
The authors acknowledge that the doses they are studying may well be much in excess of what is clinically relevant. The fact that other authors in similar systems used such doses does not to my mind make it more valid. This is a key concern I see with this study.
Author Response
Response to Reviewer 2 Comments
Detailed comments follow below.
Introduction
I think it is important to acknowledge that direct toxicity leading to cell death is only one of several mechanisms by which these drugs can be harmful to the nervous system. Also it is potentially important to consider their effects on non-neuronal cells. Both of these key points need to be mentioned in order to frame the study. Also related, is there any evidence in terms of human pathology that any of these drugs cause neuronal cell death?
The point raised by the reviewer is valid and we acknowledge that other modes of toxicity are possible. To address this issue we have included this point in the introduction of the revised manuscript.
In terms of SCs stimulating neuronal death in humans, studies have documented the neurotoxic effects of synthetic cathinones in utero and early life exposure. References have been included in the introduction as well for example see Ref: 30 and 31 in the revised manuscript.
Results
How were the dose ranges chosen? Almost any organic compound is neurotoxic in sufficient doses. How do these doses related to presumptive levels in brain tissue in humans who ingest them? Is there any data on this?
The pathophysiological outcomes from this study are tempered by the drug dose required to elicit responses in this cell culture model. Unfortunately to the best of our knowledge, there is no scaling factor that has been reported for dose extrapolation between in-vitro, animals and humans. This is due to differences in route of administration, plasma protein binding, drug physicochemical properties, transport and metabolism, and oral bioavailability of the SCs.
What is the rationale for the 6 and 24 hours time points?
As indicated in the response to Reviewer 1 (point 1) the intention to include the 6 h time point curve was to illustrate the time-dependent cytotoxicity of the drugs. For simplicity, this data has been removed to highlight just the data at the 24 h time point. The selection of this 24 h exposure time is based on previous in-vitro studies involving SCs. We have now highlighted this in the Methods section 4.4 of the revised manuscript to provide justification for the selection of time point.
It would seem to me that Figure 6 and Figure 7 should be a single figure, if 7 presents the quantitative results that 6 are an example of.
I think just showing the merged images in Figure 6 would actually be easier to interpret and would make the figure less busy.
Thank you for your suggestions. We have reformatted Figure 6 to show only the merged images as suggested by the reviewer and now data in Figure 7 (quantitative result) has been added as part of Figure 6 in the revised manuscript and the Figure numbers adjusted accordingly.
In section 2.5 associated with Figure 8, it should be explicitly stated how caspase activity was measured.
Noted. We have added the following statement to section 2.5. “Caspase cleavage of proluminescent substrate liberates free aminoluciferin which is consumed by luciferase to generate a glow-type luminescent signal. The signal produced is proportional to the caspase 3 and 7 activity.”
Discussion
I do not think the authors have really proven a causal relationship between butylone, pentylone and MDPV and cell death due to the stated mechanisms of ROS production, mitochondrial dysfunction, and altered calcium homeostasis. To assert this some rescue experiments would be required.
The authors acknowledge that the doses they are studying may well be much in excess of what is clinically relevant. The fact that other authors in similar systems used such doses does not to my mind make it more valid. This is a key concern I see with this study.
The experiments conducted in this study is a basic neurotoxicity screening for possible preliminary cell death mechanisms arising from the exposure of these SCs.
Further experiments using a free radical scavenger is definitely useful to add value to existing findings. However, these additional experiments should be conducted in a primary cell line where lower dosage could be applied. Purposeful validation studies in the in-vivo setting should also be carried out to correlate these in-vitro findings. Comprehensive in-vivo evaluation conducted will be able to detect signs of neurological disorders, behavioural abnormalities and other sign of nervous system toxicity. However, these additional studies remain outside the scope of the current manuscript. The primary purpose of the current in-vitro findings is to complement the mechanistic information obtained from future in-vivo findings.
We agree with the reviewer that biologically relevant dose in human is necessary for the translational purpose of this study. However the “precise” concentrations in human obtained from the post-mortem data may not be reliable due to varied factors as discussed in the original manuscript in the limitation section. While it is unclear on how the actual dose of SCs used in this study reflects human usage of recreational SCs particularly at the time of administration, it remains important to understand the type of damages these drugs can elicit. This in turn may provide some understanding on the potential for similar doses in humans (if achievable) to cause neuronal cell damage via mechanisms that involve mitochondrial dysfunction leading to decreased cell viability.
For these reasons, the findings obtained in this study should only be viewed as a model for understanding the neurotoxic mechanisms that may play a role in the response of the human central nervous system to these SCs exposure.
Round 2
Reviewer 2 Report
I think the revisions have substantially improved this manuscript. My one lingering concern is that it is important to emphasize as a caveat that it is completely unclear what the actual concentrations of SCs are in brain tissue in vivo and thus it is unkown whether the dose ranges studies here are clinically relvant. This remains a weakness of the study, but as long as it is clearly acknowledged in the text, I do not think it should prevent publication.
Author Response
I think the revisions have substantially improved this manuscript. My one lingering concern is that it is important to emphasize as a caveat that it is completely unclear what the actual concentrations of SCs are in brain tissue in vivo and thus it is unknown whether the dose ranges studies here are clinically relevant. This remains a weakness of the study, but as long as it is clearly acknowledged in the text, I do not think it should prevent publication.
We agree with the reviewer and have included these comments under caveat in section 3. Discussion (Pg 11, line 347, 354-358) of the revised manuscript.
