What the Erythrocytic Nuclear Alteration Frequencies Could Tell Us about Genotoxicity and Macrophage Iron Storage?

Erythrocytic nuclear alterations have been considered as an indicative of organism's exposure to genotoxic agents. Due to their close relationship among their frequencies and DNA damages, they are considered excellent markers of exposure in eukaryotes. However, poor data has been found in literature concerning their genesis, differential occurrence and their life span. In this study, we use markers of cell viability; genotoxicity and cellular turn over in order to shed light to these events. Tilapia and their blood were exposed to cadmium in acute exposure and in vitro assays. They were analyzed using flow cytometry for oxidative stress and membrane disruption, optical microscopy for erythrocytic nuclear alteration, graphite furnace atomic absorption spectrometry for cadmium content in aquaria water, blood and cytochemical and analytical electron microscopy techniques for the hemocateretic aspects. The results showed a close relationship among the total nuclear alterations and cadmium content in the total blood and melanomacrophage centres area, mismatching reactive oxygen species and membrane damages. Moreover, nuclear alterations frequencies (vacuolated, condensed and blebbed) showed to be associated to cadmium exposure whereas others (lobed and bud) were associated to depuration period. Decrease on nuclear alterations frequencies was also associated with hemosiderin increase inside spleen and head kidney macrophages mainly during depurative processes. These data disclosure in temporal fashion the main processes that drive the nuclear alterations frequencies and their relationship with some cellular and systemic biomarkers

Percentage of erythrocytes labeled by PI in <i>in vivo</i> assay.

<p>Values followed by different letters differ by Mann-Whitney test (p<0.05).</p

Single macrophages of spleen and head kidney of <i>O</i>. <i>niloticus</i> stained by Pearls’ histochemical technique.

<p>(A, B) Pigment-containing granules predominantly labeled with hemosiderin (blue). (C, D) Percentage of labeling for hemosiderin submitted to different treatments. Values followed by different letters differ by ANOVA and Bonferroni post test (p <0.05).</p

Spleen and head kidney MMCs.

<p>(A) Surface images obtained by secondary electrons from spleen and (D) head kidney tissue sections. The corresponding backscattered electron images of electron-dense granules from the red dots were identified in the respective images (B, E). The spectra obtained by X-ray microanalysis of the granule in spleen (C) and head kidney (F), showing characteristics peaks of C, O, P, S, Ca and Fe and C, O, P, S and Ca, respectively.</p

Melanomacrophage centres in spleen and head kidney of <i>O</i>. <i>niloticus</i> stained by Pearls’ histochemical technique.

<p>(A, B) MMC exhibiting lipofuscin (yellow-brownish) and melanin (black) pigments. (C, D) Percentage of the volumetric data melanomacrophage centres in tilapia submitted to different treatments. Values followed by different letters differ by Fisher test (p <0.05).</p

Percentages of erythrocytes labeled by PI in <i>ex vivo</i> assay.

<p>(A) Exposure to 2 μg L^-1 and 0.2 μg L^-1 in 24 and (B) 48 hours. On the right, PI fluorescence histograms 24 and 48 hours respectively. Values followed by different letters differ by Fisher test (p <0.05).</p

Spearman’s correlation coefficients (R) matrix between nuclear alterations and PI positive ERP.

<p>BL—blebbed, BU—bud, CO- nuclear condensed, NO—notched, LO—lobed, MN—micronuclei, VA—nuclear vacuolated and PI—PI-positive cells.</p><p>* Indicates (p <0.05) and</p><p>** indicates (p<0.005).</p><p>Spearman’s correlation coefficients (R) matrix between nuclear alterations and PI positive ERP.</p

Concentration (means ± S.D.) of cadmium in blood obtained from animals of the <i>in vivo</i> assay.

<p>Values followed by different letters differ by ANOVA and Bonferroni tests (p<0.05).</p

Total number of ENAs in <i>in vivo</i> assay.

<p>Values followed by different letters differ by Chi-square test (p < 0.05).</p