Iron regulatory protein 1 is not an early target of cadmium toxicity in mice, but it is sensitive to cadmium stress in a human epithelial cell line.(Report)

Introduction

As a widespread pollutant and a metal with various technological uses, cadmium has long been studied for its deleterious effects on mammals and other living species (Deckert 2005; Satarug et al. 2003; Satoh et al. 2002). A striking conclusion that can be drawn from the large number of studies carried out over the years is the diversity of toxicity mechanisms that have been associated with cadmium intoxication (Martelli et al. 2006; Waalkes 2003). Perturbation of the homeostasis of essential metals underlies many of the involved processes (Goyer 1997; Martelli et al. 2006). The variety of disorders occurring after cadmium exposure is consequently large (Satoh et al. 2002; Waalkes 2003). Among them, iron deficiency anemia has been evidenced (Hays and Margaretten 1985; Schafer et al. 1990). Decreased production of erythropoietin from poisoned kidneys is a likely contributor to the symptom (Horiguchi et al. 2000; Horiguchi et al. 1994; Obara et al. 2003). Other important steps of iron homeostasis, such as iron absorption from the diet, contribute to cadmium toxicity. Enhanced production of divalent metal transporter 1 (DMT1), a cellular importer of both metals (Gunshin et al. 2001), indeed occurs at the plasma membrane of enterocytes in the case of iron deficiency (Bressler et al. 2004; Elisma and Jumarie 2001; Ryu et al. 2004). Therefore, further investigation appears useful for a better assessment of the correlation between cadmium exposure and dysregulation of iron homeostasis.

At the cellular level, iron homeostasis is maintained by iron regulatory proteins (IRPs). The two known members, IRP1 and IRP2, bind RNA motifs called iron responsive elements (IRE) in the noncoding sequences of several transcripts. Among the regulated proteins in mammals, DMT1 and ferroportin of enterocytes have been shown to be sensitive to cadmium exposure as a function of the iron status (Bressler et al. 2004; Elisma and Jumarie 2001; Kim et al. 2007; Ryu et al. 2004). The activity of the regulators is modulated in different ways. IRP1 is a bifunctional protein: when it does not regulate iron homeostasis, it contains a [4Fe-4S] cluster and functions as cytosolic aconitase, inter-converting citrate and isocitrate in iron-loaded cells. Unlike IRP1, IRP2 undergoes iron-dependent degradation in iron-replete cells (Guo et al. 1995b; Iwai et al. 1998). Under iron starvation, the [4Fe-4S] cluster of IRP1 is dismantled, whereas IRP2 is stabilized and both apoproteins bind IRE.

The influence of cadmium exposure on the IRP-centered regulating system does not seem to have been previously investigated. In the present study this question has been examined in two different settings. On the one hand, the iron regulatory system of a human epithelial cell line has been studied under cadmium stress, by following the concentration of the involved proteins and their activities. On the other hand, groups of mice have been intoxicated by adding cadmium to drinking water. The doses of cadmium were chosen so as to induce accumulation of the metal in all considered organs to concentrations largely exceeding those considered safe in various human populations (Satarug et al. 2003). The treatments were not continued for a long time to determine whether dysregulation of iron homeostasis was an early consequence of cadmium intoxication. Selected organs have been analyzed by measuring the IRP activity in relation to the metal concentration of these tissues. Our data show that exposure to cadmium does impact the IRP system, and the detailed action of the toxic element on these proteins has been investigated.

Materials and methods

Cell cultures, cadmium treatments, and extraction of proteins

Epithelial human cervix carcinoma (HeLa) cells were grown and exposed to cadmium as previously described (Rousselet et al. 2008b). To prepare protein extracts, the cell pellets were resuspended and left for 15 min at 4 [degrees] C in 200 mL of 10 mmol/L HEPES (pH 7.6), 3 mmol/L MgSO4, 40 mmol/L KCl, 10% glycerol, and 1% of a cocktail of protease inhibitors devoid of metal chelators (Sigma-Aldrich, St. Louis, Missouri) in the presence of 1% Triton X-100 (lysis buffer) or 30 mg/mL digitonin. After freezing and thawing 3 times, the mixture was centrifuged at 17 500g for 30 min at 4 [degrees] C, and the supernatant was used for activity measurements.

Animals and treatments

The experiments involved 3 groups of 4 female mice (strain C57BL/6) aged 4-8 weeks at the start of the experiment. All groups were fed a diet containing all nutrients in amounts that equaled the recommendations for laboratory mice (RM1, Special Diet Services, Witham, Essex, UK) including 114 mg of iron / kg. Cadmium intoxication of the animals was carried out by adding 100 or 200 mg/L (ppm) cadmium chloride (Sigma-Aldrich) to drinking water for 3 weeks, whereas nonexposed animals were given distilled deionized water. Mice had free access to drinking water. The animals used in the present study were the same as those used in other parallel experiments (Viau et al. 2007). The duration of the treatment is slightly larger than the half-life of red blood cells in mice (Tremml et al. 1999). The animals were sacrificed by CO2 inhalation and the organs of interest were removed, in accordance with the Atomic Energy Commission Care and Use Committee.

Blood parameter measurements

Blood samples were collected with S-Monovette systems (Sarstedt, Numbrecht, Germany). Hemoglobin concentrations, hematocrit, red blood cell counts, mean corpuscular volume, platelets, and white blood cell levels where determined in …