Spleen injury and apoptotic pathway in mice caused by titanium dioxide nanoparticules

Abstract

Nanoparticulate titanium dioxide (TiO₂) has been demonstrated to decrease immunity of mice, but very little is known about the injury of spleen involved immunomodulation and its molecular mechanism. In order to understand the spleen injury induced by intraperitoneal injection of TiO₂ nanoparticules (NPs) for consecutive 45 days, the spleen pathological changes, apoptosis, the expression levels of the apoptotic genes and their proteins, and oxidative stress in the mouse spleen were investigated. The results demonstrated that TiO₂ NPs had obvious accumulation in the mouse spleen, leading to congestion and lymph nodule proliferation of spleen tissue, and splenocyte apoptosis. TiO₂ NPs effectively activated caspase-3 and -9, decreased the Bcl-2 the levels of gene and protein, and increase the levels of Bax, and cytochrome c genes and their protein expression, promoted ROS accumulation. Taken together, this study indicated that TiO₂ NPs-induced apoptosis in the mouse splenocyte via mitochondrial-mediated pathway. These findings provide strong evidence that the TiO₂ NPs can induce the spleen pathological changes, apoptosis, leading to the reduction of immunity of mice.

Introduction

Titanium (Ti) either pure or in alloys is extensively used for a wide range of implanted medical devices, such as dental implants, joint replacements, cardiovascular stents, and spinal fixation devices, due to its advantageous combination of physico-chemical and biological properties. However, under mechanical stress or altered physiological conditions such as low pH, Ti-based implants can release large amounts of particle debris, both in the micrometer and nanometer size range (Brien et al., 1992, Buly et al., 1992, Arys et al., 1998, Cunningham et al., 2002). As new types of photo-catalyst, anti-ultraviolet light agents, and photoelectric effect agents, TiO₂ nanoparticules (NPs) are used in a variety of consumer products, such as toothpastes, sunscreens, cosmetics, food products (Gurr et al., 2005), paints and surface coatings (Fisher and Egerton, 2001), and in the environmental decontamination of air, soil, and water (Esterkin et al., 2005, Choi et al., 2006). The toxicological concern is due to the distinct properties of nanoparticles, such as small size, high number per given mass, large specific surface area, and generation of free radicals (Lynch et al., 2006).

It has been suggested that the biological responses to nanoparticles may exceed those elicited by micron-sized particles (Borm et al., 2006, Ne et al., 2006). Recent studies indicate that TiO₂ NPs are toxic to lung, liver, and gill of animals. Previous studies had shown that TiO₂ NPs produced pulmonary inflammation, cytotoxicity and adverse rat lung tissue effects by intratracheally instillation (Afaq et al., 1998, Warheit et al., 2007a, Warheit et al., 2007b), and interstitial thickening in the rat lung (Liu et al., 2009a). TiO₂ NPs were demonstrated to cause the oxidative stress in the gill of rainbow trout (Federici et al., 2007), and accumulated in the kidneys but had minimal effect on kidney functions of rainbow trout (Scown et al., 2009). TiO₂ NPs with oral gavage increased the ratio of alanine aminotransferase to aspartate aminotransferase, the activity of lactate dehydrogenase and the liver weight, and caused the hepatocyte necrosis and damaged the liver functions of mice (Wang et al., 2007), and with intraperitoneal injection could also increase the activities of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, leucine acid peptide, pseudocholinesterase, lactate dehydrogenase, and the levels of triglycerides, total protein, and albumin level, damaging the liver function (Liu et al., 2009b), and caused hepatocyte apoptosis and inflammatory cascade, which was closely related to significant alteration of both mRNA and protein expression levels of several inflammatory cytokines (Ma et al., 2009). The enhancement of lipid peroxides in the mouse brain and liver caused by TiO₂ NPs implicated an oxidative attack that was activated by a reduction of the antioxidative defense mechanism (Ma et al., 2010, Liu et al., 2010), and caused DNA cleavage of the mouse liver (Li et al., 2010). The liver function damage in mice caused by intragastric administration with TiO₂ NPs was demonstrated to be closely associated with the damage of blood system haemostasis and the reduction of immunity (Duan et al., 2010). In addition, this last may be due to the spleen damage. The spleen is the largest immune organ in animals, participating in immune response, generating lymphocytes, eliminating aging erythrocytes and storing blood. The immune system is affected by the toxicological activity of many pollutants, including heavy metals (Bernier et al., 1995, Burns et al., 1995). Heavy metals can cause dose dependent immunomodulation by disturbing the fine balanced mechanisms of immune cell regulation. Depending on the particular metal, its concentration, biologic availability and a range of other factors, the outcome of this modulation may be either immunosuppression or immunoenhancement (Burns et al., 1995, Khangarot et al., 1999, Krocova et al., 2000, Lawrence and McCabe, 2002). However, to our best knowledge, so far, the researches on splenic toxicity of nanomaterials are rarely reported. Is the bio-toxicity of nanomaterials on the spleen also related to apoptosis, leading to the decrease of immunity and the damage of liver function? And the mechanisms of nanomaterials-induced toxicity in animals need investigation.

In the present paper, the accumulation of TiO₂ NPs, the spleen pathological changes, the splenocyte ultrastructure, the expression levels of the apoptotic genes and their proteins, and oxidative stress in the mouse spleen were investigated to understand mechanism of the splenic injury in mice caused by this compound.