Influence of Antibiotics on Functionality and Viability of Liver Cells In Vitro

Round 1

Reviewer 1 Report

This study uses HepG2/C3A as an in vitro model to compare and contrast the hepatotoxic potential of various antibiotics applied at clinically relevant concentrations. The results point to some biochemical perturbations using cell viability, albumin biosynthesis, CYP1A2 activities as read out. The key concern is the lack of a very clear correlation of the in vitro observation, back to the clinical manifestation of hepatotoxicity. Discrepancies are not clearly addressed, and the significance of the findings and the usefulness of the in vitro model is therefore diminished. Specific comments are as shown below:

 

11. In Figure 1, a proper dose response curve would provide a more wholesome profiling of the toxicity potential as compared to single concentration analysis. This will not help in the comparison of such potential across different compounds.

22. In Figure 2, it is not clear why cefuroxime and rifampicin were shortlisted for the experiment. What about the remaining compounds?

33. On page 6, the data on activity of mitochondrial dehydrogenase was described. This data should be included in either a table or figure for clearer appraisal of their effects

44. In Figure 3, there are compounds that show opposing results at different concentrations: e.g. ampicillin that decreased CYP1A2 activity at lower concentrations but increased at higher concentration. The reason for such observations should be discussed, or else this data appears to be purely descriptive and not alluding to any new understanding of antibiotics-induced hepatotoxicity.

55. Finally, the discussion of specific antibiotics did not allude the in vitro data derived from this study. Hence, the trends were not adequately explained and the validity of the in vitro model described here remains questionable.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this study, the authors tested in vitro the hepatotoxicity of different concentrations of various antibiotics frequently used in ICU. Authors used HepG2/C3A cells treated with the antibiotics and measured cell viability and functionality after 6 days.

Overall, this article shows the huge variability in antibiotics hepatotoxicity in vitro and the difficulties to predict clinical DILI. 

The article fits some of the main scopes of the journal: molecular cell biology, molecular pharmacology.

The article treats an actual problem (the idiosyncratic hepatotoxic effects of antibiotics in humans and the existing difficulties to predict DILI episodes) provides some evidence about the different mechanisms involved in liver injury caused by antibiotics. 

However, there is some missing information in the data and the introduction and discussion sections need to be clarified. Here, we provide some advice, and a number of major and minor points for revision are listed below.

 

Major points

1. The wording of the text is not totally correct. Please ensure that the paper has been checked by a native English speaker. 

2. The abstract includes some incorrect information that is not necessary. The authors did not use human hepatocytes, but human hepatoma cell lines. Please rewrite. Abstract also should indicate that selected antibiotics are chosen due to their frequent use in ICU.

3. Introduction:

-The introduction section is confusing. A short paragraph including information about differences between intrinsic and idiosyncratic DILI, differences between hepatocellular, cholestatic and mixed DILI, as well as how to diagnose this entity (causality scales, expert consensus…) is missing. Please rewrite.

4. Materials and methods section: 

-Why the authors use Cmax, 5Cmax and 10Cmax? Why therapeutic concentrations of antibiotics are not used?

-Why the authors expose the cells to the different antibiotics during 6 days?

- What is the difference between cell vitality and cell viability? Sometimes the authors use trypan blue and sometimes cell count. It is not clear

5. Results:

- It is surprising that the authors did not test amoxicillin hepatotoxicity since amoxicillin-clavulanate is the main causative drug in different DILI registries (Spanish DILI Registry (Stephens C, Robles-Diaz M, Medina-Caliz I, et al. Comprehensive analysis and insights gained from long-term experience of the Spanish DILI Registry. J Hepatol. 2021 Jul;75(1):86-97), US DILI Network (Chalasani N, Bonkovsky HL, Fontana R, et al. Features and Outcomes of 899 Patients With Drug-Induced Liver Injury: The DILIN Prospective Study. Gastroenterology. 2015 Jun;148(7):1340-52 e7), Latin DILI Network (Bessone F, Hernandez N, Mendizabal M, et al. When the Creation of a Consortium Provides Useful Answers: Experience of The Latin American DILI Network (LATINDILIN). Clin Liver Dis (Hoboken). 2019 Feb;13(2):51-57).

- A figure showing LDH levels after exposing cells to antibiotics should be included.

- A figure showing results of XTT test should be included.

6. Discussion:

- Discussion section is too long and heavy reading. Use more connectors or separate paragraphs indicating the antibiotic of interest. 

- The discussion section finishes with the phrase “The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted.”. However, the authors do not talk about the implications in the clinics or future research direction. Please complete the discussion with these topics. 

7. References:

- In general, references of the manuscript should be updated, in order to show information about antibiotics-DILI up to date. More recent references about DILI cases registries are needed. Some examples: Spanish DILI Registry (Stephens C, Robles-Diaz M, Medina-Caliz I, et al. Comprehensive analysis and insights gained from long-term experience of the Spanish DILI Registry. J Hepatol. 2021 Jul;75(1):86-97), US DILI Network (Chalasani N, Bonkovsky HL, Fontana R, et al. Features and Outcomes of 899 Patients With Drug-Induced Liver Injury: The DILIN Prospective Study. Gastroenterology. 2015 Jun;148(7):1340-52 e7), Latin DILI Network (Bessone F, Hernandez N, Mendizabal M, et al. When the Creation of a Consortium Provides Useful Answers: Experience of The Latin American DILI Network (LATINDILIN). Clin Liver Dis (Hoboken). 2019 Feb;13(2):51-57), Indian Network for DILI (Devarbhavi H, Joseph T, Sunil Kumar N, et al. The Indian Network of Drug-Induced Liver Injury: Etiology, Clinical Features, Outcome and Prognostic Markers in 1288 Patients. J Clin Exp Hepatol. 2021 May-Jun;11(3):288-298).

 

Minor points

1. Several abbreviations and their meanings are not indicated in the text.

2. Repeated phrases in lines 316-319 and 361-363. Moreover, these paragraphs are not well-written, ideas should be better expressed. 

3. The text has some typos. Please correct. 

 

Author Response

Please see to the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have addressed the concerns raised. I have no further question.

Author Response

Thank you very much for your re-review.