Reviewer's report
Title:Neoplastic transformation of rat liver epithelial cells is enhanced by
non-transferrin-bound iron.
Version: 1Date:5 September 2007
Reviewer: Hans Goldenberg
Reviewer's report:
General
-------------------------------------------------------------------------------
Major Compulsory Revisions (that the author must respond to before a decision
on publication can be reached)
The authors want to show the tumor-promoting properties of
non-transferrin-bound iron on liver cells. This is a very
relevant question for iron-overload-diseases. However, their
experimental setup cannot really answer it in a meaningful
way. Their central point is the effect of a form of freely
soluble ferric iron (ferric ammonium citrate) at a
concentration of 200µM on tumor promotion in the cell as
determined by assay of colony formation. This concentration
is far off any amounts to be expected in even severe iron
overload, where the reported iron concentrations are around
10 to 20µM, maybe maximal 50. Though for the formation of
colonies (the central message of the paper) there are also data
for lower concentrations of iron, these are not adequately
controlled. The effect of the carcinogen MNNG is not assayed
for over the long term that is necessary to obtain a significant
increase in the number of colonies.
Methods: The assay of labile iron is not performed correctly
(interestingly, there is no reference to the original method,
either). Loading cells with calcein-AM to use calcein as intracellular iron sensing
indicator and leaving them in this
state for days may yield misleading result due to its degradation under certaing
conditions The intracellular
degradation has been described(see also : Hasinoff, BB., 2003, J.Inorg.
Biochem.), and fluorescent dyes are
inherently unstable.
Results: The figure displaying the iron content of the cells has
no scale and no error bars are shown. Moreover, the control cells seem to be
completely
devoid of iron. This is unlikely, every cell needs at least some
iron. It is not really difficult to obtain quantitative data on iron
content, e.g., by atomic absorption spectrometry. A similar
reservation applies to the ferritin concentration, which also
looks likebeing zero in control cells. Of course, there might be
a big difference in the order of magnitude, but why did the
authors not try to determine the increase of cellular ferritin
over time and with different concentrations. That would enable
the experimenter to document the increase, and not just the
appearance of iron and ferritin out of nothing.
The data on the antiproliferative effect of iron display a
significant effect only at a concentration of 500µM. This is
physiologically irrelevant, it may only be interesting for
protein-free cell culture, which is not the purpose of this
study.
In figure 3, the scale seems misleading. A closer look reveals
that ammonium citrate together with MNNG displays a
proliferative effect. Whether it is significant, cannot be judged
from the figure, the standard deviation bars are too small, and
statistical numbers are missing (a general flaw of this study),
but it cannot be excluded to the viewer.
Thus, the degree of sloppiness in the experimental setup
renders this manuscript as not acceptable for publication in a
scientific journal.
Reference: Hasinoff, BB, J.Inorg.Biochem. 2003; 95: 157-164
-------------------------------------------------------------------------------
Minor Essential Revisions (such as missing labels on figures, or the wrong use of
a term, which the author can be trusted to correct)
-------------------------------------------------------------------------------
Discretionary Revisions (which the author can choose to ignore)
What next?: Reject because scientifically unsound
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: Yes, but I do not feel adequately qualified to assess the
statistics.
Declaration of competing interests:
no
Reviewer's report
Title:Neoplastic transformation of rat liver epithelial cells is enhanced by
non-transferrin-bound iron.
Version: 1Date:5 September 2007
Reviewer: Hans Goldenberg
Reviewer's report:
General
-------------------------------------------------------------------------------
Major Compulsory Revisions (that the author must respond to before a decision
on publication can be reached)
The authors want to show the tumor-promoting properties of
non-transferrin-bound iron on liver cells. This is a very
relevant question for iron-overload-diseases. However, their
experimental setup cannot really answer it in a meaningful
way. Their central point is the effect of a form of freely
soluble ferric iron (ferric ammonium citrate) at a
concentration of 200µM on tumor promotion in the cell as
determined by assay of colony formation. This concentration
is far off any amounts to be expected in even severe iron
overload, where the reported iron concentrations are around
10 to 20µM, maybe maximal 50. Though for the formation of
colonies (the central message of the paper) there are also data
for lower concentrations of iron, these are not adequately
controlled. The effect of the carcinogen MNNG is not assayed
for over the long term that is necessary to obtain a significant
increase in the number of colonies.
Methods: The assay of labile iron is not performed correctly
(interestingly, there is no reference to the original method,
either). Loading cells with calcein-AM to use calcein as intracellular iron sensing
indicator and leaving them in this
state for days may yield misleading result due to its degradation under certaing
conditions The intracellular
degradation has been described(see also : Hasinoff, BB., 2003, J.Inorg.
Biochem.), and fluorescent dyes are
inherently unstable.
Results: The figure displaying the iron content of the cells has
no scale and no error bars are shown. Moreover, the control cells seem to be
completely
devoid of iron. This is unlikely, every cell needs at least some
iron. It is not really difficult to obtain quantitative data on iron
content, e.g., by atomic absorption spectrometry. A similar
reservation applies to the ferritin concentration, which also
looks likebeing zero in control cells. Of course, there might be
a big difference in the order of magnitude, but why did the
authors not try to determine the increase of cellular ferritin
over time and with different concentrations. That would enable
the experimenter to document the increase, and not just the
appearance of iron and ferritin out of nothing.
The data on the antiproliferative effect of iron display a
significant effect only at a concentration of 500µM. This is
physiologically irrelevant, it may only be interesting for
protein-free cell culture, which is not the purpose of this
study.
In figure 3, the scale seems misleading. A closer look reveals
that ammonium citrate together with MNNG displays a
proliferative effect. Whether it is significant, cannot be judged
from the figure, the standard deviation bars are too small, and
statistical numbers are missing (a general flaw of this study),
but it cannot be excluded to the viewer.
Thus, the degree of sloppiness in the experimental setup
renders this manuscript as not acceptable for publication in a
scientific journal.
Reference: Hasinoff, BB, J.Inorg.Biochem. 2003; 95: 157-164
-------------------------------------------------------------------------------
Minor Essential Revisions (such as missing labels on figures, or the wrong use of
a term, which the author can be trusted to correct)
-------------------------------------------------------------------------------
Discretionary Revisions (which the author can choose to ignore)
What next?: Reject because scientifically unsound
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: Yes, but I do not feel adequately qualified to assess the
statistics.
Declaration of competing interests:
no
Please review this paper.
user
Reviewer's report
Title:Neoplastic transformation of rat liver epithelial cells is enhanced by
non-transferrin-bound iron.
Version: 1Date:5 September 2007
Reviewer: Hans Goldenberg
Reviewer's report:
General
-------------------------------------------------------------------------------
Major Compulsory Revisions (that the author must respond to before a decision
on publication can be reached)
The authors want to show the tumor-promoting properties of
non-transferrin-bound iron on liver cells. This is a very
relevant question for iron-overload-diseases. However, their
experimental setup cannot really answer it in a meaningful
way. Their central point is the effect of a form of freely
soluble ferric iron (ferric ammonium citrate) at a
concentration of 200µM on tumor promotion in the cell as
determined by assay of colony formation. This concentration
is far off any amounts to be expected in even severe iron
overload, where the reported iron concentrations are around
10 to 20µM, maybe maximal 50. Though for the formation of
colonies (the central message of the paper) there are also data
for lower concentrations of iron, these are not adequately
controlled. The effect of the carcinogen MNNG is not assayed
for over the long term that is necessary to obtain a significant
increase in the number of colonies.
Methods: The assay of labile iron is not performed correctly
(interestingly, there is no reference to the original method,
either). Loading cells with calcein-AM to use calcein as intracellular iron sensing
indicator and leaving them in this
state for days may yield misleading result due to its degradation under certaing
conditions The intracellular
degradation has been described(see also : Hasinoff, BB., 2003, J.Inorg.
Biochem.), and fluorescent dyes are
inherently unstable.
Results: The figure displaying the iron content of the cells has
no scale and no error bars are shown. Moreover, the control cells seem to be
completely
devoid of iron. This is unlikely, every cell needs at least some
iron. It is not really difficult to obtain quantitative data on iron
content, e.g., by atomic absorption spectrometry. A similar
reservation applies to the ferritin concentration, which also
looks likebeing zero in control cells. Of course, there might be
a big difference in the order of magnitude, but why did the
authors not try to determine the increase of cellular ferritin
over time and with different concentrations. That would enable
the experimenter to document the increase, and not just the
appearance of iron and ferritin out of nothing.
The data on the antiproliferative effect of iron display a
significant effect only at a concentration of 500µM. This is
physiologically irrelevant, it may only be interesting for
protein-free cell culture, which is not the purpose of this
study.
In figure 3, the scale seems misleading. A closer look reveals
that ammonium citrate together with MNNG displays a
proliferative effect. Whether it is significant, cannot be judged
from the figure, the standard deviation bars are too small, and
statistical numbers are missing (a general flaw of this study),
but it cannot be excluded to the viewer.
Thus, the degree of sloppiness in the experimental setup
renders this manuscript as not acceptable for publication in a
scientific journal.
Reference: Hasinoff, BB, J.Inorg.Biochem. 2003; 95: 157-164
-------------------------------------------------------------------------------
Minor Essential Revisions (such as missing labels on figures, or the wrong use of
a term, which the author can be trusted to correct)
-------------------------------------------------------------------------------
Discretionary Revisions (which the author can choose to ignore)
What next?: Reject because scientifically unsound
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: Yes, but I do not feel adequately qualified to assess the
statistics.
Declaration of competing interests:
no
assistant