Toxic Effects Of Cr(Ⅵ) On Mice By Oral Administration: A Study At Molecular And Cellular Level

Chromium is an ubiquitous element in nature that exists in primarily two valences states, trivalent (Cr(III)) and hexavalent (Cr(VI)). The two valence states can be transformed mutually under the influence of physical, chemical and biological factors in the environment. However, the effects of the two states on human health are quite different. Cr(III) is an essential micronutrient for human. It is a part of the glucose tolerance factor, which plays an important role in regulating glucose metabolism for the maintenance of normal glucose levels. In contrast, Cr(VI) is regarded as one of the most toxic chemical substances, as well as an international recognized heavy metal carcinogen.With wide use of the chromium compounds in industries, the waste water, exhaust gas and waste residue emitted from these industries resulted in severe chromium pollution. Great attention has been payed to this increasing severe pollution by the world. Cr(VI) scattered in the environment cycles with the water in ion state. It can not be decomposed by microorganism, but can be absorbed by animals and plantsand then affect the balance of the whole ecosystem. Cr(VI) is toxic to humans due to the effect of biological amplification of Cr(VI) along food chain. Cr(VI) may enter into the human body via dermal contact, inhalation and ingestion. Dermal contact and inhalation are the two major routes of occupational Cr(VI) exposure. Studies have demonstrated that the workers exposed to Cr(VI) chronically were vulnerable to dermatitis and eczema, as well as perforation, ulcer, chronic rhinitis and bronchitis. Cr(VI) may also result in emphysema, and consequently affect lung function. The most toxic effect on humans via inhalation is that it increases the risk of suffering from respiratory tract cancer. Water and food intake is a common route of Cr(VI) exposure for nonoccupational people. Several cases about which ingestion of food contaminated by Cr(VI) caused adverse effects on humans have been reported by researchers at home and abroad.A plenty of in vitro experiments have demonstrated that Cr(VI) could induce toxic effects on many types of cells including hepatocytes, renal cells, fibroblasts and lymphocytes. Animal experiments also confirmed that Cr(VI) exposed by subcutaneous or intraperitoneal injection could cause several organs damage, especially for liver and kidney. However, due to the special toxic mechanisms of Cr(VI), animal experiments established by subcutaneous or intraperitoneal injection could not properly reflected the toxicity induced by oral administration of Cr(VI) in vivo. Up to now, only a few animal experiments administrated with Cr(VI) by gavage or drinking at liberty have been reported, moreover, conclusions on toxicity induced by oral administration of Cr(VI) remains uncertain.To illustrate the toxicity of Cr(VI) exposed by oral administration in vivo, it is necessary to study the potential toxic mechanisms at molecular and cellular level. Overview on a series of in vitro and in vivo researches, it can be concluded that an important aspect of Cr(VI) toxicity is the formation of plentiful reactive oxygenspecies (ROS) and the consequent oxidative stress. Recently, a large number of studies have showed a close relationship between ROS and apoptosis because ROS could disturb normal apoptosis through many cell signaling pathways. Therefore, investigating the apoptosis induced by Cr(VI) may contribute to illustrating the toxic mechanisms of Cr(VI) in vivo.The present study was designed to give Cr(VI) to the experimental animals via oral administration (gavage). It was planed to study the toxic effects of Cr(VI) in vivo by comparing the damage in different organs. Furthermore, evaluating the changes of apoptotic related proteins in the process of cell apoptosis was also planed to investigate the toxic mechanisms of Cr(VI) in vivo.According to the experimental design, the animals were randomly distributed into four groups and were orally administrated (gavage) with potassium dichromate (K₂Cr₂O₇) at a dose of 0, 25, 50, 100 mg/kg body weight once daily for 1 day or consecutive 5 days. The mice were continued breeding for 24 h after the last Cr(VI) treatment. The blood was collected by extirpating the eyeballs, and the blood lymphocytes were separated out for comet assay immediately. The tissues of liver and kidney were quickly dissected off after the mice were sacrificed. One part of the tissues were used for TUNEL assay and transmission electron microscope, the remains were frozen in liquid nitrogen for p53, Bcl-2, Bax, cytochrome c protein level and caspase-3 activation determined by Western blot, and also for ROS level, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity and catalase (CAT) activity evaluation.Main results:1. The comet assay of peripheral blood lymphocytes showed that the tail length in treatment groups significantly increased in a dose-dependent manner after 1 day or 5days of K₂Cr₂O₇ exposure. Except for the lowest dose group at 1 day of exposure, the tail moment at the other treatment groups were obviously higher than that of the control groups. However, comparing with 1 day of exposure, the increasing tendency of the value on the tail length and the tail moment declined after the mice exposed to K₂Cr₂O₇ for 5 days.2. After the mice were exposed to K₂Cr₂O₇ for 1 day or 5 days, ROS levels in mice liver in treatment groups were all higher than those in control groups. Except for the lowest dose group at 1 day of exposure, there was a significant difference between the control group and the other treatment groups.3. SOD activity in mice liver was not modified by the administration of K₂Cr₂O₇ at 50 and 100 mg/kg dose for 1 day. However, the value showed a non-significant increase at 25 mg/kg dose compared with the controls. SOD activity declined significantly at the three doses after 5 days of exposure as compared to the controls.4. CAT activity in mice liver showed a dose-dependent decrease in all K₂Cr₂O₇ treated groups and the difference was significant at 50 and 100 mg/kg doses as compared to the controls.5. The MDA content in mice liver increased in a dose-dependent manner after administration of K₂Cr₂O₇ to mice for 1 day or 5 days, but no statistical differences were observed between the control and treatment groups.6. No significant changes were observed on ROS, MDA, SOD and CAT in kidney after administration of K₂Cr₂O₇ to mice between the control and treatment groups.7. Transmission electron microscopy revealed the ultrastructural alterations of hepatocytes undergoing apoptosis in mouse liver treated with K₂Cr₂O₇ for 1 and 5 days. The typical apoptosis alterations were described as follows: the nucleus of mouse hepatocytes become shrunken. The chromatin was condensed to form dense compact masses and arranged along the crinkly nuclear membrane. In addition, themitochondria became a compact and vacuolar structure with loss of cristae.8. TUNEL assay was used to evaluate DNA degradation in liver after the mice were treated with K₂Cr₂O₇ . The number of cells stained with brown significantly increased in K₂Cr₂O₇ -treated mice compared with the control animals after 1 day of exposure. Such phenomenon also occurred in liver cells after administration of K₂Cr₂O₇ for 5 days. Dose- and time-dependent increase was observed after 1 and 5 days of K₂Cr₂O₇ exposure to mice and the increase was significant between the control and treatment groups.9. After the mice were exposed to K₂Cr₂O₇ at 0, 25, 50, 100 mg/kg doses for 1 day or 5 days, it could be seen that the protein band of caspase-3 active segment (17 kD) hydrolyzed from caspase-3 zymogen in liver cells gradually became thick. Caspase-3 activation enhanced in accordance with the increase of K₂Cr₂O₇ , and the difference compared with the control animals was significant at the dose of 100 mg/kg.10. p53 protein level in liver showed that the administration of K₂Cr₂O₇ to mice for 1 or 5 days enhanced the p53 protein level, and the difference compared with the control animals was significant at the dose of 100 mg/kg.11. Bcl-2 protein level in liver showed a declining tendency with the increase of K₂Cr₂O₇ at 1 or 5 days. Furthermore, the Bcl-2 protein level at 100 mg/kg dose for 1 day as well as at 50 mg/kg and 100 mg/kg doses for 5 days decreased significantly compared with the control groups. Bax protein level increased in a dose-dependent manner, and the difference was significant only at 100 mg/kg dose for 5 days.12. Cytochrome c protein level in hepatocytes cytoplasm enhanced in accordance with the increase of K₂Cr₂O₇ after 1 day or 5 days of exposure, and the values at 50 mg/kg and 100 mg/kg doses for 1 day as well as at 100 mg/kg dose for 5 days showed a statistical significance compared with the controls.Main conclusions:1. ROS level increased and SOD and CAT activity decreased in mice liver after the animals were exposed with oral administration of Cr(VI). The results indicated that one important reason for the toxicity induced by Cr(VI) is that Cr(VI) exposed by oral administration could destroy the balance of antioxidative system and cause the following oxidative stress in vivo.2. Compared with liver, no significant oxidative stress was observed in kidney after oral administration of Cr(VI) to mice, indicating that the toxic effects on different organs may be different after Cr(VI) underwent redox metabolism in vivo.3. Oral administration of Cr(VI) induced DNA damage in mice lymphocytes, indicating that the DNA damage induced by ROS and/or Cr intermediates which formed in the Cr(VI) metabolism was one of the Cr(VI) toxic effects.4. Cr(VI) could induce hepatocytes apoptosis and alter the p53, Bcle-2, Bax, cytochrome c protein levels and caspase-3 activation, indicating p53, Bcle-2, Bax, cytochrome c and caspase-3 take part in the cell apoptosis induced by Cr(VI). These results suggests that an important mechanism on the Cr(VI) toxicity is to disturbe the normal process of cell apoptosis.