A Study of Carry-Over and Histopathological Effects after Chronic Dietary Intake of Citrinin in Pigs, Broiler Chickens and Laying Hens

Round 1

Reviewer 1 Report

A study concerning the mycotoxin citrinin (CIT) is presented. The authors investigated the intake of this toxin by different kinds of livestock, its accumulation and histopathological effects on kidneys, liver, jejunum and duodenum and possible transfer into edible tissue.

In their introduction the authors describe the relevance of citrinin as a toxin and possible ways into the food chain. They give a good overview of previous studies and other relevant literature. In detail, literature reported effects of this compound at various levels on different animals are summarized. Also, bioavailability and residence studies are mentioned, further reinforcing the necessity of the presented study.

In the results section, first the validation of the UHPLC-MS/MS method used to quantify citrinin and its metabolite dihydrocitrinone in different tissue samples is described briefly. This includes LOD, LOQ, linearity, goodness-of-fit coefficients, injection carry-over and apparent recovery. Judging by this validation the methods appears to be robust and suitable for this study.

Carry-over and depletion studies are then presented. Animals were fed with contaminated feedstuff for three weeks. CIT and HO-CIT concentrations in plasma were monitored. After those three weeks CIT concentration where measured in plasma, muscle, skin + fat, kidney, liver, egg yolk and egg white for several consecutive days.

Finally, toxicity studies of kidneys, liver, duodenum and jejunum utilizing Light Microscopy and Transmission Electron Microscopy are presented, which found no severe toxic effects. However, it could be confirmed that mitochondrial damage appears to play a vital role in the mechanism of nephrotoxicity.

In summary, this work demonstrates that carry-over of CIT to edible tissues of livestock and thereby exposure of humans after meat consumption is possible, although only to a very limited extent. Carry-over ratios were found to be low, but some accumulation of the toxin in edible tissue of chickens was observed. The highest carry-over ratio was found for liver tissues. Carry-over ratio was also found to decrease with increasing exposure levels. The discussion section and comparison to other works therein is very well done.

In the Materials and Method section the execution of this study is described in sufficient detail, so that it could be repeated in principle. The only issue that is missing are the chromatographic and mass spectrometric parameters and instrumental details used in UHPLC-MS/MS experiments. If this is a previously publish method, this may be fair enough, but this was not apparent from the manuscript and needs to be clarified.

In general, this work is a very elaborated study of the dietary intake of CIT in livestock. The results are relevant for the field of research and in assessing the dangerousness of CIT in the food chain. This could influence legislative action and the presented results are a valuable source of data for other researchers.

 

Minor Comment:

The title of this manuscript is formulated in a rather unusual way. It states a short (but incomplete) summary/conclusion of the presented study. I would recommend to change it to a statement of the objective like: Investigation of….

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Major comment

This manuscript is difficult to follow as it is not easy to understand whether the purpose of the study was to investigate the carry-over of CIT into edible tissues or whether the purpose of the study was to make a toxicological study of CIT.

Concerning the analysis of carry-over, the concentration of CIT used in feed (1000 µg/kg in pig and 100 to 3,500 µg/kg) seems very high for a study on carry-over at realistic exposure level of CIT in feed because, according to the authors, the highest detected CIT concentration in feed was 3.9 μg/kg feed (L43).

Concerning the study of the chronic effect of CIT in pig, broilers chickens and laying hens, only histopathological analysis are reported in this manuscript and nothing was said about effect on performences, organs weight at autopsy and no reasult on biochemistry/haematology were reported, which seems difficult to accept for a study in toxicology. May be the authors planed to publish those results in another manuscript, but this should be said.

Detailled comments

Toxicology

Authors said in the title that the concentration of CIT used in feed has « no Histopathological Effects in Kidney, Liver and Intestines » , also L172-174,  but how results of histopathologic analysis were interpreted is very confusing. Authors reported change in the mithochondria at the highest doses (L177-180, Fig 3 to 5) and said that « the number of degenerated mitochondria differed significantly between the control pigs and pigs receiving CIT » (L200-2011) and that similar results were observed in chickens at the high dose (204-205). Also, the authors found « degenerated mitochondria in (A), implying recovery from oxidative stress » (L214) and discussed the histopathological changes L316-320 as « apoptosis due to oxidative stress caused by CIT ». The conclusion of authors was « the present study confirms at ultrastructural level that mitochondrial damage appears to play a vital role in the mechanism of nephrotoxicity and changes are already visible at relatively low doses of CIT. »

Because on the results found in these analysis and because CIT is knwon for its nephrotoxicity, and because of the key roles of oxidative damages and mithochodria damages in toxicity it seems very difficult to accept that what was observed in this study haa no toxicological signification! At least functional measures should be done in plasma and urine to say that the damages observed are without toxicological significance, and even with these measures, it would be difficult to agree with this conclusion. Also, I think a few analysis of variables used to reveal oxidative damage should be done, to stregthen the hypothesis made concerning the effect of the high doses, some variables are very easy to measure.

Carry-over

L117-150 : clarify wether CIT and HO-CIT were measured in controls (not exposed to CIT), and results observed in these groups

L118-120 and Table1-3 : clarify in text and Tables the time at which analysis were performed after the last meal contaning the contaminated feed was given to the animals (what was the starvation period before the analysis of CIT and OH-CIT in tissues).

L144-150 : it seems difficult to say the steady state was reached or not as measures were only performed one time at 5 hours after the meal was given. More-over, what permitted to say that the steady-state was reached on day 4in pigs ? Data provided in Fig 1 seems to reveal no difference in CIT concentration on days 6 to 21, may be a significant difference was observed on day 0.5 and day 4, but no result of statistic analysis was provided.

From data provided in Fig 2, because the first result was obtained on day 7, it is absolutly impossible to conclude that the steady state was reached within 1.5 days in broiler chickens

Depletion studies and Figure 2 : The presentation of results as the mean +/- SD in fig 2 is very confusing when n=2. It seems from the SD in broiler of days 22, 23 and in laying hens on day 24 that the dispersion of results is very high.

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Thank you for your answers and clarifications to your manuscript. I understand that your aim was not to do a full toxicological study and I understand that using lower doses of Citrinine would make the determination of the carry-over impossible for analytical reasons. However, I think strong care should be taken in the interpretation of the lesions of mitochondria observed. As I understand it, you do not have the results to say whether functional alterations have occurred in this study and you rely on a comparison with the results previously obtained in the literature. As you point out, citrinin is close in its mechanism of action to OTA. Were mitochondrial lesions similar to those you observe described in the OTA toxicity studies? If so, how these lesions were judged in terms of toxicological significance? A new EFSA scientific opinion on OTA in March 2020 highlight the contribution of non-genotoxic mechanisms in the overall toxicity of OTA (https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2020.6113), I think this new opinion should be discussed. Also, there is a relative scientist consensus on the key role swelling of mitochondria plays in cell death (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752577/). Even this point was already discussed in the manuscript, I think the way the title of the manuscript was written suggests that the lesions observed are of minor significance and I think this confusion should be avoided, may be by omitting reference to the lesions observed in the title or by clearly indicating what the lesions observed are.

Author Response

Dear reviewer, 

Thank your for your valuable comments and for providing interesting references of EFSA and mitochondrial swelling. We agree that we cannot draw a firm conclusion with respect to the observed mitochondrial changes. However, as mitochondrial changes related to CIT were described in other studies, we cannot neglect the potential relevance and the link between ultrastructural changes and oxidative stress. Therefore, we described the observations, and described the repeatedly reported toxicological effects of CIT, but we clearly mentioned that the observations cannot be fully interpreted in the revised manuscript. Mitochondrial degeneration and swelling are not enough to conclude toxic effects, as other measurements are necessary as you have mentioned in revision round 1. We have also included references concerning similarities with OTA. As other toxic effects of OTA were reported (tRNA synthetase inhibition, DNA adduct formation), we did not go into detail as the mechanisms of toxicity cannot be fully compared. Also, the recent scientific opinion of EFSA on OTA does mention mitochondrial effects, but no histopathological changes were described. Furthermore, the title was changed to avoid any further confusion: A Study of Carry-over and Histopathological Effects after Chronic Dietary Intake of Citrinin in Pigs, Broilers Chickens and Laying hens.

We hope that our revision is considered acceptable.

 

Further revisions were done:

Line 308: clear statement that changes were only visually observed and full interpretation is not possible without any further confirmation

Line 319: one of the mechanisms of OTA toxicity related to mitochondria was provided (EFSA reference). 

Line 320: references to similar histopathological effects of OTA (quail and laying hens) were included