Despite the many benefits of nanotechnology, some studies indicate that certain nanoparticles may cause adverse effects because of their small size and unique properties (Service, 2003
; Hoet et al., 2004
). Indeed, their size makes them highly mobile in both the human body and the environment. Nanomaterials can enter human tissues through several ports via the lungs after inhalation (Oberdorster, 2001
), through the digestive system (Jani et al., 1990
), and possibly through the skin (Kreilgaard, 2002
; Lademann et al., 1999
). Systemic distribution of nanoparticles has been demonstrated after inhalation and oral uptake (Jani et al., 1990
; Oberdorster et al., 2002
), and nanoparticles have been found to cross the blood–brain barrier, reaching the olfactory bulb and the cerebellum (Borm and Kreyling, 2004
; Oberdorster et al., 2004
). Chen and colleagues also reported that nanoparticles can penetrate the blood-testis barrier (Chen et al., 2003
). Although organ- or cell-specific drug delivery through nanoparticles is a promising area of medicine, and nanoparticles might be used some day as sensors for intracellular mechanisms, few toxicology studies are available. Many of the artificially manufactured nanoparticles are made of nonbiodegradable pollutants, such as carbon black and metals, and the long-term behavior of such substances is not known.
Toxicants that impair normal reproductive functions are an important public health issue. A decrease in semen quality of approximately 2% per year over the preceding 50 years has been reported for industrialized countries (Carlsen et al., 1992
). It has been hypothesized that exposure to toxic chemicals is an important cause of the decline, although great regional differences exist for the same level of environmental contamination. Nevertheless, studies have shown that high exposure of men to various chemicals in certain occupational settings resulted in lower semen quality. For example, dibromochloropropane (DBCP), a nemotocide, resulted in lower sperm counts because of the destruction of undifferentiated spermatogonia (Potashnik et al., 1978
). Therefore, it has now become critical to understand the molecular mechanisms leading to reproductive toxicity. Several in vivo
animal models have been used to assess the testicular toxicity of many compounds. However, these models necessitate the sacrifice of animals at the end of experimentation, and they do not allow the manipulation or dissection of intracellular pathways to elucidate mechanisms of toxicity at the molecular level. In vitro
model alternatives have been established, and some of them have tried to reproduce in the petri dish the complex cell–cell interactions that take place between the different germ cells and Sertoli cells (Hadley et al., 1985
; Yu et al., 2005
). However, these models are limited by the poor viability of the freshly isolated germ cells.
In this study, we used a cell line with spermatogonial stem cell characteristics to evaluate the toxicity of different types of nanoparticles on the germline. We used three parameters widely used in toxicological studies, such as the ability of mitochondria to reduce MTS, the integrity of the plasma membrane, and the activation of apoptotic pathways.
Our results indicate that the C18–4 cells provide a suitable test system for cytotoxicity in the germline. The MTS and LDH assays can be used for rank ordering of chemical and nanoparticle toxicity on mitochondrial function and plasma membrane integrity. Cadmium is a recognized toxicant that has been classified as a probable human carcinogen. It is a heavy metal that has the potential to cause lysosomal damage and DNA breakage in mammalian hepatocytes (Fotakis et al., 2005
) and many other cells and tissues (Satoh et al., 2002
). Cadmium also disrupts mitochondrial function both in vivo
(Belyaeva et al., 2002
) and in vitro
(Pourahmad and O'Brien, 2000
), and promotes apoptosis (Pulido and Parrish, 2003
). In the testis, cadmium induces lysosomal damage in testicular Sertoli cells (Boscolo et al., 1985
), but its main toxic effects appear in germ cells. In male rats subcutaneously injected with 0.6 mg cadmium chloride/kg body weight, histological examination of the testes revealed an accumulation of cadmium only in spermatogonia and spermatocytes, but not in somatic cells (Aoyagi et al., 2002
). Subsequently, a decrease in the number of spermatogonia in relation to the time of exposure was observed, followed by a decrease in the number of spermatocytes and, ultimately, sperm cells. In humans, male infertility is strongly linked to cadmium exposure, but is rather due to a failure of the acrosomal reaction in sperm cells (Benoff et al., 2000
). In our spermatogonial stem cell line, exposure of the cells to cadmium chloride induced a significant decrease in their metabolic activity. However, like hepatocytes (Fotakis et al., 2005
), cadmium chloride had no effect on the integrity of the plasma membrane, as monitored by the LDH release assay. Importantly, the deleterious effects of cadmium were enhanced for cells exposed to the particulate, insoluble form of cadmium, such as cadmium oxide. These effects mimic well-documented data obtained on macrophages and other cell lines (Goering et al., 2000
). In this case, not only mitochondrial function decreased drastically, but LDH was released in the cell environment as a function of cadmium oxide concentration. Because oxidative stress and lipid peroxidation have been reported after exposure to both cadmium chloride and cadmium oxide, the higher toxicity of cadmium oxide might be related to the size of the particles entering into contact with the plasma membrane. Furthermore, our study shows that the sensitivity of the C18–4 cells to cadmium oxide is comparable to the sensitivity of the BRL 3A liver cells regarding their metabolic activity (Hussain et al. in press
) (). The membrane integrity of the C18–4 cells is less affected. This is corroborated by the fact that at lower concentrations cadmium oxide promotes apoptosis rather than necrosis in the C18–4 cells, thus leaving the plasma membrane intact.
Calculated EC50 Values of CdO and Ag Nanoparticles for C18–4 Spermatogonial Stem Cells Compared to BRL 3A Liver Cells
Because the C18–4 cells showed an increased sensitivity to the particulate form of cadmium rather than the soluble form, we next studied the effects of metal nanoparticles on these germline stem cells. As predicted, silver carbonate, which was used as a control, was not toxic as shown by the MTS and LDH leakage assays. In contrast, silver nanoparticles (15 nm) reduced mitochondrial function drastically and increased membrane leakage. Our data show that the C18–4 cells are more sensitive than the BRL 3A cells in that respect (). There are studies showing that silver nanoparticles could be used in bone cement or other implantable devices as antimicrobial agents (Alt et al., 2004
), but our study shows that silver in nanoparticulate form could be toxic for the bone-lining cells and other tissues.
Although molybdenum in soluble form is considered to be a mildly toxic substance, it did not significantly affect the metabolic activity or the membrane integrity of the C18–4 cells. Molybdenum as a nanoparticulate did not affect metabolic activity either, at least up to a concentration of 40 μg/ml (EC50 = 90 μg/ml). At higher concentrations (over 50 μg/ml), the molybdenum nanoparticles become significantly toxic. Interestingly, whereas the effect on mitochondrial function is mild, molybdenum nanoparticles seem to promote some plasma membrane leakage at very low concentrations (5 μg/ml and 10 μg/ml). The same pattern of toxicity is shown for aluminum nanoparticles; however, the morphology of the cells did not change, indicating that at these concentrations apoptosis still occurs. This finding was corroborated by the Vybrant assay. It is known that extreme and visible membrane damage occurs only in the late stages of apoptosis.
In conclusion, we have demonstrated that the C18–4 germline stem cells are a valuable tool with which to study in vitro toxicity in the germline. The sensitivity of these cells to Ag nanoparticles is greater than that of BRL 3A liver cells, which are widely used in toxicity studies. However, in the case of cadmium and the other nanoparticles tested, the sensitivities of the two cell lines are comparable. The molecular mechanisms of nanoparticles toxicity are still poorly understood, and the availability of a cell line with which to gain an understanding of these processes in the germline is of paramount importance.