## 3 Results and Discussion
### Histopathology
In order to assess the consequences of the toxic effects of metal-dependent organ injury and the consequent activation of a reparatory mechanism, the expression of PCNA  
Figure 5: The effects of specific tissue heavy metal bioaccumulation on inflammatory pathway activation. (**A**) Immunohistochemical expression of TNF-\(\alpha\) in the gills, kidney, and liver of _Barbus__Jbus__Jbus__(BB), _Chondrostoma__nosus_ (CN), and _Squalins__cphalus__(SC); (**B**) Western blot analysis of TNF-\(\alpha\) protein levels in in the gills, kidney, and liver of _Barbus__Jbus__Jbus__(BB), _Chondrostoma__nosus_ (CN), and _Squalins__cphalus__(SC); (**C**) correlations of the histopathological lesion grade (ranging from 1 to 3) in the gills, kidneys, and liver of _Barbus__Jbus__(BB), _Squalins__cphalus__(SC), and _Chondrostoma__nosus_ (CN) with TNF-\(\alpha\) level determined in the same tissues of all three analyzed species. Statistical results mark the calculated significance between the TNF-\(\alpha\) concentration and the lesion grade in the respective organ per each species: *** significant difference at \(p<0.001\); *** significant difference at \(p<0.01\); * significant difference at \(p<0.05\). was analyzed. A large number of PCNA immunopositively cells were observed in gills distributed both along the filaments and lamellae, followed by the kidney, in comparison with the liver of all three analyzed species (Figure 6A). The immunohistochemical results are consistent with the Western blot analysis outcomes (Figure 6B). Figure 6: The effects of specific heavy metal bioaccumulation in tissues regarding the activation of repair mechanisms. (**A**) Immunohistochemical expression of PCNA in the gills, kidney, and liver of _Barbus barbus_ (BB), _Chondrastoma nasus_ (CN), and _Squalius cephalus_ (SC); (**B**) Western blot analysis of PCNA protein levels in the gills, kidney, and liver of _Barbus barbus_ (BB), _Chondrastoma nasus_, (CN) and _Squalius cephalus_ (SC); ***\(p<0.001\) in the case of gills: SC compared to BB and CN; ***\(p<0.001\) in the case of kidneys: BB compared to CN and SC; ***\(p<0.001\) in the case of liver: SC compared to BB and CN; (**C**) Correlations of the histopathological lesion grade (range 1-3) in the gills, kidneys, and liver of _Barbus barbus_ (BB), _Squalius cephalus_ (SC), and _Chondrastoma nasus_ (CN) with PCNA level determined in the same tissues of all three analyzed species. Statistical results mark the calculated significance between the PCNA concentration and the lesion grade in the respective organ per each species: *** significant difference at \(p<0.001\). It is probably that the immunoreactivity for PCNA could be correlated with the severity of DNA injury, mainly for the gills and kidney, which might suggest that the activation of repair pathways depends on the level of alterations induced by exposure to heavy metals. Although the severity of tissue damage was recorded at the highest level for carnivorous species (BB), the repair mechanisms were unable to keep up with the damage (Figure 6B,C). According to our data, the analyzed CN organs presented the best capacity of proliferation, meaning that PCNA expression could be correlated with liver [41], kidney [42], and gill [43] tissue regeneration. The omnivorous SC presented a similar capacity for kidney regeneration with CN, but a lower one for the gills and liver. The correlation between PCNA expression in the three organs and food chain position of the three species revealed a significant inverse relationship, i.e., the herbivorous species likely activated the repair mechanisms at the highest level (Figure 6C). More studies are necessary to explain this observation.