The present study shows that long-term Cr(pic)3 treatment does not influence abnormal glycemic status or indices of growth of OZR. Further, the treatment is not associated with exacerbated oxidative DNA damage. Rather, the treatment exerts mild-moderate beneficial effects on several markers of oxidative stress and inflammation in OZR.
The interest in chromium supplementation stems from earlier studies that suggested an essential role for trivalent chromium in carbohydrate and lipid metabolism [10
]. These studies showed that rats consuming a diet lacking chromium developed an inability to efficiently dispose of blood glucose [17
]. This defect was reversed by addition of chromium-enriched food or by supplementation with synthetic trivalent chromium. Subsequent observation that patients on total parenteral nutrition also develop a deficit in carbohydrate metabolism, which can be alleviated with trivalent chromium, established the essential role of trivalent chromium in human diet as well [17
]. Although the exact molecular events subserving the effect of chromium on glucose metabolism remain to be established, a number of mechanisms have been proposed that collectively lead to amplification of insulin signaling (e.g., increased insulin receptor binding but inhibition of insulin receptor tyrosine phosphatase) [28
]. In light of these reports, we had expected that long-term Cr(pic)3 treatment of OZR would improve insulin resistance. Surprisingly, however, the treatment did not beneficially influence several indices of glycemic control or the marked histopathological features of pancreatic islets in OZR. Nonetheless, the results are consistent with a recent report indicating that Cr(pic)3, at a dose of 1000 μg/day, does not improve insulin sensitivity or several other features of metabolic syndrome in obese adults [30
]. Human studies have typically used chromium at a dose of 200-1000 μg/kg [29
]. Assuming an average body weight of 70 kg, this corresponds to a range of about 3-14 μg/kg of chromium supplementation. Animal studies, on the other hand, have used dosages similar to (and even far higher than) the ones used in this study (about 190 to 410 μg/kg/day for OZR; 5 Cr and OZR; 10 Cr groups, respectively) [22
]. Thus, it is likely that far higher doses of the formulation would be required to unmask a beneficial effect on glycemic control in the setting of marked insulin resistance as it occurs in OZR. Nonetheless, the results raise question about the efficacy of Cr(pic)3, alone, in doses that are commonly consumed by human subjects although the possibility that such doses may exert additive or synergistic effects with physical exercise and/or other interventional modalities remains to be explored. This notion is consistent with a report indicating that Cr(pic)3 supplementation increases insulin sensitivity and improves glycemic status of type 2 diabetic patients who are on sulfonylurea agents [32
It is well-established that abnormal glycemic state of obesity and diabetes mellitus are associated with increased oxidative stress and subsequent oxidative DNA damage [3
]; indeed, generation of 8-OHdG is considered as a surrogate marker of enhanced oxidative stress and associated oxidative DNA damage [33
]. Thus, we conjectured that Cr(pic)3-induced improvement in glycemic control should reduce 8-OHdG production. On the other hand, if indeed Cr(pic)3 treatment increases oxidative DNA damage, then the treatment should further increase 8-OHdG generation in the setting of increased oxidative and nitrosative stress that are features of OZR [7
]. Consistent with this notion, urinary excretion of 8-OHdG was significantly higher in the OZR than LZR, suggesting an increase in whole body oxidative stress. On the other hand, renal tissue nitrotyrosine level was increased in kidneys of OZR than LZR reflecting an increase in local oxidative/nitrosative stress. Interestingly, however, Cr(pic)3 treatment did not increase either urinary excretion of 8-OHdG or renal tissue nitrotyrosine content. Rather, the treatment was associated with mild reductions in both parameters that were sufficient enough to abrogate statistical significance compared to the LZR group. Interestingly, the reductions in 8-OHdG excretion and tissue nitrotyrosine level were not associated with any improvement in glycemic status of OZR. In this context, Chander and colleagues [7
] have shown that treatment of OZR with ebselen (an antioxidant and peroxynitrite scavenger) reduces lipid peroxidative products and 3-nitrotyrosine-modified proteins without affecting blood glucose.
Since urinary excretion of 8-OHdG is a surrogate biomarker of whole body oxidative DNA damage, we also carried out immunohistochemical examination of the kidney for 8-OHdG. The rationale for this relates to the fact that trivalent chromium is known to accumulate in a number of organs although the kidney accumulates it to a greater extent [17
]. Indeed, as shown in Figure , Cr(pic)3-enriched diets caused significant increases in kidney chromium content of the OZR. Thus, we conjectured that any adverse effect of Cr(pic)3 treatment on oxidative DNA damage should be more readily detected in the kidney. Interestingly, however, immunostaining of renal tissue did not reveal differential patterns or intensity among the experimental groups; similar findings were noted for the pancreas. These observations coupled with the demonstration that urinary excretion of 8-OHdG increases in conditions associated with oxidative stress suggest that the modified nucleoside is largely released rather than being accumulated in the tissue(s). Nonetheless, immunostaining findings and urinary excretion profile of 8-OHdG do not support the notion that Cr(pic)3 enhances oxidative DNA damage in OZR. As indicated earlier, a major concern regarding Cr(pic)3 relates to reports of increased risk of genotoxicity associated with its use [13
]. Genotoxicity of trivalent chromium has been shown in acellular systems where direct interaction occurs with the genetic material [34
]. However, trivalent chromium compounds, including Cr(pic)3, have produced conflicting results in ex vivo and in vivo studies likely due to multiple reasons including low cellular uptake of trivalent chromium and the demonstration that it does not avidly accumulate in DNA containing organelles such as the nucleus or the mitochondria [18
]. The results of this study are in agreement with this notion and the more recent reports indicating lack of a significant DNA toxicity of Cr(pic)3 [20
Consistent with other reports, the OZR displayed a number of renal abnormalities compared to their lean counterparts including greater albuminuria and histopathological changes indicative of extracellular matrix deposits, inflammatory infiltrates and tubulointerestitial injury. Nonetheless, renal tubular dilatations were noted for both LZR and OZR thereby suggesting lack of a correlation of this abnormality with metabolic derangements associated with obesity and (or) type 2 diabetes. It is noteworthy that there were numerous lipid droplets in renal tissue of OZR than LZR; ectopic lipid accumulation is believed to contribute importantly to pathophysiology of organ dysfunction in obesity/type 2 diabetes (i.e., lipotoxicity) [36
]. More importantly, however, it is suggested that release of autocrine and paracrine factors from periorgan fat deposits contributes importantly to the pathogenesis of cardiac abnormalities in animals with dietary fat-induced obesity [37
]. Indeed, OZR display marked deposits of fat around the kidney, effectively encasing the organ. Nonetheless, the contribution of autocrine and paracrine release of factors from adipose tissue surrounding the kidney, an encapsulated organ, to eventual manifestation of renal pathology in OZR remains to be established. Importantly, however, Cr(pic)3 treatment did not result in significant beneficial effects on renal function (e.g., albuminuria) or histopathological findings of OZR.
The lack of significant effects of Cr(pic)3 on renal function and structure are seemingly inconsistent with its mild-moderate ameliorating effects on indices of oxidative stress (e.g., tissue nitrotyrosine) and inflammation (e.g., urinary MCP-1 excretion and renal tissue COX-2 level). However, it is important to note that multiple mechanisms contribute to genesis of proteinuria and renal dysfunction associated with obesity and type 2 diabetes including metabolic, hemodynamic (both systemic and intrarenal), oxidative stress and a variety of inflammatory cytokines and chemokines [3
]. Ultimately, the net effect of these changes results in loss of glomerular membrane permeability barrier and albuminuria. It is noteworthy that systemic hemodynamics and metabolic status of the 3 Cr(pic)3-treated OZR groups were generally similar thereby suggestive of similar contribution of these determinants to albuminuria. On the other hand, although the parameters that were measured in this study are known to contribute to genesis of proteinuria, it is likely that the mild Cr(pic)3-induced changes in their levels are not sufficient to beneficially influence albuminuria. Alternatively, Cr(pic)3 in doses that were used in this study may not be sufficient enough to alter the course and/or extent of proteinuria.
In conclusion, long-term Cr(pic)3 intake in doses that exceed those consumed by human subjects does not exert beneficial effects on glycemic control or adversely affect growth of OZR. In addition, despite renal accumulation of chromium, kidney function and structure were generally similar among untreated and treated OZR. Importantly, the treatment did not increase either urinary excretion of 8-OHdG or its immunohistochemical pattern and intensity in the kidney and the pancreas. Rather, Cr(pic)3 treatment of OZR was associated with attenuations in indices of oxidative stress and inflammation thereby diminishing the differentials with LZR.