Studies On The Molecular Mechanisms Of Lung Cancer Induced By Nickel Compounds

Nickel is a kind of essential trace element for animal and it is widely distributed in environment and occupational exposed to human beings. Accumulated epidemiological evidence and animal experiments confirm that exposure to nickel compounds are associated with tumor, especially lung cancer and nasal cancer. The International Agency for Research on Cancer (IARC) classified nickel compounds as group I carcinogen to human in 1990. The crystalline Ni₃S₂ is of the highest carcinogenic activity among the nickel compounds, and the Ni₂O₃ is second. The high consumption of nickel-containing products inevitably leads to environmental pollution, utilization, and disposal. Nickel can accumulated in human bodies after exposure to it.Although the molecular mechanisms by which nickel compounds cause cancer are still under intense investigation, the carcinogenic actions of nickel compounds are thought to involve four aspects. The first is genomic DNA mutation induced by nickel compounds, which leads to change the tumor related gene expression pattern and result in carcinogenesis. For example, transversion of base G→T in p53 gene is produced during lung cancer induced by nickel. Although many studies have revealed that nickel compounds have a potential to cause DNA damage in cells and tissues in vitro, it is quite clear that nickel compounds have low mutagenic activity in most of the mutational systems that have been examined. The second is generation of reactive oxygen species (ROS) induced by nickel exposure, which leads to DNA and chromatin damage. And then tumor related gene expression is changed to cause cancer. The third one is that nickel exposure activates the signaling pathway leading to transcription factor transactivation, by which nickel regulate the expression of specific genes related to tumor development. For example, nickel exposure to 3T3 cell to causetranscriptional factor NFkB up-regulation and induce cell transformation. The fourth, nickel exposure has been shown to have significant effect on epigenetics, e.g, on DNA methylation and histone acetylation in various cell lines.Recently, numerous epidemiologic and experimental investigations about carcinogenesis and acute lunge injury induced by nickel compounds have been reported and some hypotheses have been proposed. However, the exact mechanisms of nickel-induced carcinogenesis are not known due to its complex metabolic mechanism in human body. In the present work, the molecular mechanism of lung cancer induced by nickel compounds has been studied on cellular transformation, aberrant expressions of antioxidant enzymes and related gene as well as the epigenetic changes in the process of carcinogenesis in order to obtain more understanding on chemoprevention and treatment of occupational lung cancer.MethodsI Human lung fibroblasts ( HLF )cells were treated by Ni2O3 of different concentrations(0.5².0mg/L) four times in vitro. The identification of malignancy in transformation of HLF cells was carried out by the tests of ConA and semisolid agar culture. The DNA lesions of HLF cells treated with different concentrations of Ni3S2, Ni2O3 and NiSO4 were observed with the method of single cell gel electrophoresis (SCGE) or Comet Assay. II The alveolar macrophages (AM) were cultured with exposure to Ni2O3 (5|Jg/ml) in vitro. The cell activity, nitric oxide (NO) , reactive oxygen species (ROS), malondialdehyde (MDA) and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT )and nitric oxide synthase (NOS) were detected. Inducible nitric oxide synthase (iNOS) mRNA expression was measured with RT-PCR. Human lung fibroblasts (WI-38 )cells were also treated with Ni2O3 ( 5|Jg/mk lOjJg/ml and 15[Jg/ml ) and the expression changesof gene hypoxia-inducible transcription factor-1 (HIF-1), vascular endothelial growth factor(VEGF), thioredoxin(TRX), and heme oxygenase-l(HO-l) were tested by RT-PCR and Real-time PCR. 20 male SD rats receives three times intramuscular injection of 10 mg of Ni3S2or Ni2O3 suspended in 0.2 ml of Chloromycetin solution and 10 control rats were given Chloromycetin solution only, as a solvent, in the right hind legs (the triceps surae muscle). Sixteen month later, the detection of SOD, GSH-Px, MDA, NO and NOS concentration in serum, liver and cardiac muscle were carried out. Meanwhile, pulmonary alveolar macrophages of all experimental group rats were collected. Apoptosis of AM was measured by propidium staining and flow cytometry (FCM). Fluo-3 AM staining and FCM were used to detect the intracellular free calcium. Mitochondrial membrane electric potential was measured by rhodamine 123 staining with FCM. Ill DNA methylation of CG island in promoter regions of P16 and RASSF1 in nickel-treated WI-38 cells and P16, RASSF1, MGMT and DAPK1 in two rats lung cancer tissue induced by nickel were analyzed using methylation-specific-PCR (MSP) method.ResultsI The malignancy transformation of HLF cells was induced by Ni2O3. The transformed cell proliferated rapidly. The transformed colonies exhibited in extensively random orientation and the cells were crossed -over. The frequency of transformation showed dose-response manner in the experiment. The transformed cells could be agglutinated by lowered concentration of ConA and could grow in semisolid agar. The DNA lesions of HLF cells treated with different concentrations of Ni3S2, Ni2O3 and NiSO4 were greater than those of control cells by SCGE ( P<0.01 ). The average tail moment of control cells was 10.06±0.32|Jm;but in Ni3S2 groups ( 2.5|Jg/ml, 5pg/ml and lOpg/ml) they were 65.50± 0.44pm , 114.08±1.08|Jm and 189.23±0.62pm ( compared with control group P<0.01 ) , respectively. In Ni2O3 groups ( 2.5pg/ml, 5pg/ml and 10Mg/ml ) they were 75.86±1.86|Jm , 87.89±2.07|Jm and 133.09±1.17jJm ( compared with control group P<0.01 ) , respectively, and in NiSO4 groups ( 155Mg/ml , 309|Jg/ml and 464|Jg/ml ) they were 62.20±0.42Mm, 111.40±1.73|Jm and 412.49±2.46Mm ( compared with control group P<0.01 ) , respectively. II Ni2O3 could increase mortality and decrease survival activity of AM in vitro. It could also increase MDA, NO and NOS activity, and decrease SOD, GSH-Px, CAT activities and up-regulate the expression of iNOS mRNA. VEGF and HO-1 were over-expressed and TRX was down-regulated in nickel treated WI-38 cells compared with control cells. HIF-1 expression of 5Mg/ml and lOMg/ml nickel group increased at 48 hours and decreased after 72 hours. However 15Mg/ml nickel group dropped dramatically at 48 hours. SOD concentration in serum and cardiac muscle, GSH-Px in cardiac muscle were decreased in nickel treated rats. MDA, NOS and NO in serum, liver and cardiac muscle were all increased compared with control group. FCM revealed that the apoptosis rate of AM in Ni3S2 treated rats was higher than control group while the intracellular calcium increased and mitochondrial membrane electric potential decreased. Ill P16 methylation rate detected in nickel-treated WI-38 cells was 27.8% and RASSF1 38.9%, while non-methylation occurred in control cells. DNA methylation in promoter regions of P16, RASSF1, MGMT and DAPK1 gene were observed in two rats lung cancer tissue induced by nickel, but normal tissues were not found.ConclusionsI Ni2O3 can strongly induce the malignant transformation of HLF cells and nickel compounds (Ni2O3 , Ni3S2 and NiSO4 ) can induce DNA damage and are considered to be carcinogenic chemicals. II The lipid peroxidation of AM, antioxidant system damage in rats and expression changes in genes associated with oxidative stress such as VEGF, HIF-1, TRX and HO-1 induced by nickel implicated that nickel-mediatedoxidative processes may participate in the molecular pathways accounting for its carcinogenicity. Changes of intracellular calcium and mitochondrial membrane electric potential may play pivotal roles in apoptosis of AM induced by nickel. Because of the apoptosis of AM, the ability to prevent against pathogenic microbe and lung injury and antigen presenting ability decreased, which could assist in development of cancer. Ill The methylation of CG island in the promoters of PI6, RASSF1, MGMT and DAPK1 genes is an important alternative mechanism of gene inactivation and linked with the initiation of lung cancer induced by nickel compounds.To sum up, oxidative stress, changes of gene expression and epigenetics are three important pathways in the process of carcinogenesis induced by nickel compounds.