Inter- and intralaboratory variability of results is still a serious issue in the comet assay. There are several technical conditions of procedure, which may critically affect the results and electrophoresis terms were identified as main. A comparative assessment of the expected and actual field strength in five electrophoretic tanks and the contribution of the revealed differences to the variability in DNA damage carried out. Only for one tank, the measured field strength coincided with the expected 1 V/cm, while for four it ranged from 0.6 to 2.0 V/cm. The values of DNA damage assessed in the same samples of mouse kidney cells differed between tanks up to 4.7-fold for induced and up to 10-fold for spontaneous DNA damage. High local variations in the field strength and solution temperature across the platform as well as in %DNA in the tail of identical cell samples within electrophoresis runs also revealed. These variations were reduced by recirculation of electrophoresis solution. The results show that discrepancy between the estimated and the actual field strength can be reason of inter-laboratory variation of the comet assay results. Recirculation of the solution during electrophoresis will be useful to control of intra-laboratory and intra-assay variations.
Background:
NGF deficiency is one of the reasons for reduced β-cells survival in diabetes.
Our previous experiments revealed the ability of low-weight NGF mimetic, GK-2, to reduce hyperglycaemia
in a model of advanced diabetes. The increase in DNA damage in advanced diabetes was repeatedly
reported, while there were no data about DNA damage in the initial diabetes.
Aim:
The study aimed to establish whether DNA damage occurs in initial diabetes and whether GK-2
is able to overcome the damage.
Methods:
The early-stage diabetes was modelled in Balb/c mice by streptozotocin (STZ) (130 mg/kg,
i.p.). GK-2 was administered at a dose of 0.5 mg/kg, i.p., subchronically. The evaluation of DNA damage
was performed using the alkaline comet assay; the percentage of DNA in the tail (%TDNA) and
the percentage of the atypical DNA comets (“ghost cells”) were determined.
Results:
STZ at this subthreshold dose produced a slight increase in glycemia and MDA. Meanwhile,
pronounced DNA damage was observed, concerning mostly the percentage of “ghost cells” in the pancreas,
the liver and kidneys. GK-2 attenuated the degree of hyperglycaemia and reduced the % of “ghost
cells” and %TDNA in all the organs examined; this effect continued after discontinuation of the therapy.
Conclusion:
Early-stage diabetes is accompanied by DNA damage, manifested by the increase of
“ghost cells” percentage. The severity of these changes significantly exceeds the degree of hyperglycaemia
and MDA accumulation. GK-2 exerts an antihyperglycaemic effect and attenuates the degree
of DNA damage. Our results indicate that the comet assay is a highly informative method for search of
antidiabetic medicines.
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