Abstract
Accidental inhalation of selenium (Se) derivatives, such as dimethyl selenide (DMSe), has been associated with damage of respiratory tissues. However, systemic effects of inhaled Se have not been thoroughly established. We have investigated whether mouse kidney and liver show cellular pathology as a result of a single intratracheal instillation of two different doses of DMSe (0.05 and 0.1 mg Se/kg BW). The animals were sacrificed 1, 7, 14, and 28 days after either 1 of the 2 DMSe treatments; samples were studied by light microscopy. Instillation of the low DMSe dose resulted in acute and transient tubular disease of the kidney expressed by swelling and vacuolation of epithelial cells of proximal tubules; in some mice, tubular necrosis was observed. After 14 days of the DMSe treatment, these lesions were ameliorated and, by day 28, the kidney tubular epithelium depicted a normal morphology. The same low dose of DMSe caused sustained damage to centrilobular hepatocytes characterized by swollen and vacuolized liver cells. After the instillation of the high DMSe dose, the mice presented sustained liver and kidney focal necrosis. Our data suggest that inhalation of DMSe results in: (i) acute tubular injury of the kidney and damage to centrilobular liver cells and (ii) this systemic pathology induced by DMSe is a dose-dependent phenomenon.
Introduction
Selenium (Se) derivatives are among the toxic airborne substances that workers may be accidentally exposed to in a number of industries, such as smelter, refinery, and glass bangle industries (Brune et al., 1980; Wester et al., 1981; Gerhardsson et al., 1985, 1986, 1988; Holness et al., 1989; Rastogi et al., 1991; Gerhardsson and Nordberg, 1993). When neutron activation analysis and atomic absorption spectrophotometry was used to analyze the concentration of elemental Se in samples of human lung tissue from deceased smelter workers, it was found that they showed significantly higher levels of Se in their lungs than what was observed in the lungs of rural controls (Gerhardsson et al., 1988; Gerhardsson and Nordberg, 1993). The levels of 23 elements, including Se, in the lungs, liver, and kidneys of autopsy specimens from Se-exposed workers in Northern Sweden were 2–16 times higher than those of a control group (Brune et al., 1980). In addition, Diskin et al. (1979) reported on the autopsy of a man who had been employed in a Se refinery and had been exposed to the red elemental Se. This patient died from congestive heart failure and the autopsy revealed that the lungs showed numerous perivascular granulomas and fibrosis. Also, high concentrations of Se were detected in the peribronchial lymph nodes, lung, thyroid, and kidney of the patient.
We have reported recently that intratracheal instillation of dimethyl selenide (DMSe) in mice causes lung inflammation and injury (Cherdwongcharoensuk et al., 2003, 2004). We have looked for early systemic effects of DMSe on the pathology of kidney and liver of mice subjected to a single intratracheal instillation of this Se derivative.
Materials and METHODS
Dimethyl Selenide (DMSe)
DMSe (C2H6Se) in liquid form, at analytical grade, was purchased from Fluka Chemical Co (ref. no. 41572) with a purity greater than 99%.
Study Design
We have used 64 individual one-month-old CD-1 mice (Charles River strain) of both sexes (32 males and 32 females), purchased from a commercial breeder (Charles River Laboratories, SA, Spain). Each experimental group of mice was made up of 6 animals (Table 1). Additional mice were employed to determine mortality rates. The mice weighed about 20 g, were kept under standard housing conditions of the animal house that follows the standard requirements of EC law, and had unrestricted access to commercial rodent chow (Rodent Chow, Panlab S.I., Barcelona, Spain) and water. They were treated in accordance with the European Union law on animal protection (directive 86/609/EC). Mice were exposed to intratracheal instillation of DMSe at either 1 of 2 different doses: 0.05 or 0.1 mg Se/kg BW. An additional 16 mice were used as controls.
Intratracheal Instillation of DMSe
The CD-1 mice were anesthetized by intramuscular injection of 4.0–8.0 mg/kg BW of ketamine (Ketalar, Parke-Davis Co., Barcelona, Spain), and of 0.8–1.6 mg/kg BW of xilazine (Rompun, Bayer Co., Amadora, Portugal). The anesthesia was 15–20 minutes long. Anesthetized mice were placed on a surgical board, and their neck exposed by surgical dissection until reaching the tracheal wall. DMSe was delivered to the lungs through the tracheal wall with the use of a Hamilton microliter syringe. The total volume of pure DMSe instilled in trachea of the mice was 2 μl. In control mice the same volume of (0.2 μl) 0.9% saline was injected in the tracheal airway. After the instillation, mice were held up for 1 minute, the surgical wound was closed, and the animals returned to their cages. The mice were observed twice daily for signs of toxicity. The 6-mouse groups were sacrificed at 1, 7, 14, and 28 days after the DMSe treatment. Controls were also sacrificed 1, 7, 14, and 28 days after the intratracheal injection of saline. Kidneys and livers of the sacrificed animals were removed rapidly and fixed by immersing in Bouin’s solution. The tissue samples were processed for paraffin embedding by standard methods, and sections of the tissues were cut in a Leica RM 2125 RT microtome. They were stained with haematoxylin and eosin and Mallory’s trichrome and examined by light microscopy.
Results
Mortality
We found that the DMSe-induced acute mortality of mice (i.e., death of animals up to 1 hour after treatment) depended on the dose instilled in the tracheal airway. Of the 31 mice treated with the low dose (0.05 mg Se/kg BW) only 1 died, whereas 9 out of 60 mice (i.e., 15%) treated with the high dose (0.1 mg Se/kg BW) died within 1 hour (Table 2). Total mortality (i.e., death of animals during the overall 28 days period of this investigation) of mice submitted to the low dose of DMSe was lower (3/31, i.e., 9.7%) than that of mice treated with the high dose (23/60, i.e., 38.3%).
Clinical Symptoms
The earliest clinical change observed in mice after the DMSe intratracheal instillation was tachypnea (i.e., increase frequency of respiratory movements) that started within 2–3 minutes of the treatment and continued for at least 10–15 minutes. In most of the mice treated with the high DMSe dose, there was evidence of dyspnea throughout the first 2 weeks of the study. In addition, some of these mice showed tremor, loss of mobility, and decreased food intake. Necropsies of mice that died from the DMSe treatments were not conclusive regarding the cause of death.
Pathology Induced by Low Dose of DMSe (Table 3)
Kidney
Swelling and vacuolation of epithelial cells of proximal tubules were found in mice that had been treated 1 and 7 days before with DMSe (compare Figure 1A, control, with Figure 1B, treated). The epithelial cells were enlarged and showed light cytoplasm staining due to the presence of vacuoles of different size. The severity of this tubular damage decreased later: just 1 of the 6 animals showed injury 14 days after the DMSe instillation. The kidney morphology of mice sacrificed 28 days after the DMSe treatment revealed complete recovery of the early lesions that had been caused by the DMSe instillation.
Liver
The presence of swollen hepatocytes located around the central lobular vein was the major morphological alteration observed in liver samples of mice treated with DMSe. In contrast with the kidney lesions, this alteration of liver cells was found throughout the whole course of this investigation, i.e., since day 1 and up to day 28 after the administration of DMSe to the animals. Focal necrosis of the liver was also observed in 1 out of the 6 mice, both 1 and 14 days after the DMSe instillation.
Pathology Induced by High Dose of DMSe (Table 3)
Kidney
Tubular injury of the kidney was seen in samples of all of the mice submitted to this treatment. It was expressed by swelling and vacuolation of kidney cells from day 1 to day 28 after the tracheal instillation. At day 7, one-third of the mice also showed tubular necrosis (Figure 1C).
Liver
Hepatocyte swelling was again the major injury detected in samples from the treated animals (compare Figure 2A, control, with Figure 2B, treated). In addition, the high DMSe dose caused condensation of the nuclear chromatin and focal necrosis that was still present in the liver of mice sacrificed 28 days after the DMSe administration. Focal areas of necrosis were detected on a background of general swelling of liver cells (Figure 2C). This was observed throughout the whole extension of the study. In addition, some liver cells showed condensation of the nuclear chromatin; the nuclear membrane appeared to be intact with a clear area surrounding the condensed nucleoplasm (Figure 2D).
Discussion
The present investigation documents that intratracheal instillation of mice with DMSe results in systemic pathology of both kidney and liver. The use of a low dose of DMSe allowed the identification of the domains that appear to be the most vulnerable to the toxic effect of DMSe: the epithelium of the proximal tubules of the kidney, and the centrilobular parenchyma of the liver. The kidney tubular cells showed damage in response to DMSe whereas the centrilobular hepatocytes presented sustained cytoplasmic vacuolization. These lesions evolved focal liver or kidney necrosis if a higher dose of DMSe was given to the mice.
Intratracheal Instillation vs. Inhalation
The respiratory airways are presumably the most important exposure route for Se present in the atmosphere. Experiments have been done concerning the effect of inhaled toxic agents on the respiratory system, by either aerosol inhalation or intratracheal instillation. The latter has considered to be sufficiently close to inhalation exposure; it has been used to treat animals with both soluble and insoluble particles (Henderson et al., 1995; Nonavinakere et al., 1999; Bell et al., 2000). This method allows the definition of the exact amount of the agent and also decreases the chance of contamination from other the external agents (Bell et al., 2000). In addition, it must be stated that particle deposition and distribution patterns are slightly different from that of the aerosol method (Pritchard et al., 1985). Experiments of instillation of Se by both routes have shown that the respiratory lesion is similar.
Inhalation and intratracheal instillation were electively compared by Brain et al. (1976) using technetium-sufur colloid administered to the lung of rats and hamsters. They concluded that the advantages of the intratracheal instillation technique are: (i) the actual dose delivered to each animal can be measured more accurately and administered more uniformly; (ii) the procedure is simple and minimizes hazards to laboratory personnel when the materials are highly toxic; (iii) this technique permits the instillation of large and therefore effective doses of material in a short time; (iv) this technique decreases skin exposure to the material; (v) it allows high local exposures in the lung. Oberdorster et al. (1997) have also administered 85Sr-labeled particles (3μ) the lower respiratory tract of rats either by inhalation or by intratracheal instillation, and they compared the 2 techniques regarding their suitability for measuring normal and impaired particle clearance rates. They documented that the 2 methods showed no significant differences.
Kidney and Liver Lesions Induced by Airborne DMSe
Previous studies with inhaled Se have shown that it is rapidly absorbed into blood from the lung and translocated to liver, kidney, spleen, and heart; in these organs Se as biological half-time of 30–40 days (Medinsky et al., 1981; Weissman et al., 1983).
In spite of kidney and liver being the major detoxification organs of the body, few studies have addressed the histopathology of kidney and liver after Se inhalation. Early work by Dudley and Miller (1937) revealed that exposure of guinea pigs to acute inhaled hydrogen selenide in amounts of 0.002 to 0.57 mg/L (10, 30, and 60 minutes) resulted in slight thickening of the alveolar wall and fatty metamorphosis of liver and kidney. One-hour long inhalation of rats with DMSe vapor at 1607, 4499, or 8034 ppm produced increase in RNA and reduction of DNA contents of the lung, in absence of morphological changes of lung, kidney, and liver (Al-Bayati et al., 1992). Mild congestion and mild atrophy of the liver was observed in rats exposed to 30 mg Se (dust)/m3 for 8 hours (Hall et al., 1951).
The herein found elective susceptibility of proximal tubules of the kidney to DMSe may be due to the high concentration that Se reaches in this portion of the nephron before being eliminated in the urine (Kobayashi et al., 2002; Zhu et al., 2002; Meotti et al., 2003). Regarding the susceptibility of centrilobular liver to the toxicity of systemic Se, it is pertinent to recall that centrilobular hepatocytes are nourished by oxygen-poor blood, these cells are often the first liver cells to be damaged by circulating toxic molecules (Farina et al., 2003; Meotti et al., 2003).
Thus our findings are consistent with the observations of Medinsky et al. (1981) and Weissman et al. (1983) describing that Se or selenious acid can be absorbed through the lung and transferred to kidney and liver via blood circulation. Also, there are reports of high concentrations of Se in the kidney and liver of smeltry and refinery workers (Diskin et al., 1979; Brune et al., 1980). Our data are in contrast with those of Al-Bayati et al. (1992) who found no changes in kidney and liver of rats after being submitted for 1 and 7 days to DMSe inhalation at 1607, 4499, or 8034 ppm. The difference between the 2 investigations may be related from our study having used a higher dose of DMSe than it was employed in the work of Al-Bayati et al. (1992).
In conclusion, both kidney tubular cells and centrilobular hepatocytes are early targets for the dose-dependent toxic effects of systemic DMSe reaching blood circulation after being absorbed through the respiratory tissues.
Footnotes
Acknowledgments
The authors are very grateful to Mr. Antonio Costa e Silva, Mr. Emanuel Monteiro, Ms. Madalena Costa, and Mrs. Alexandrina Ribeiro for technical assistance. This work was supported in part by grants from Fundação Oriente and FCT (Pocti/Feder), Portugal.