Inorganic arsenic induces MDM2, p53, and their phosphorylation and affects the MDM2/p53 complex in vitro.

Arsenic, as a human carcinogen, has posed a certain threat to environmental health globally. However, the underlying mechanism of the arsenic carcinogenic effect remains largely undetermined. The up-regulation of MDM2 seems to play a crucial part in tumors in especial carcinomas of the diffuse type. The interaction of MDM2 and p53 is closely relevant to the pathogenesis of tumors. In this study, we aimed to investigate the effect on MDM2, p53, and their phosphorylation after As(III). In the epidemiological study, we investigated that MDM2 expression was up-regulation and was positively linked to methylated metabolites (monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)) after As(III)-exposure. In vitro studies employing A549 and 16HBE cells confirmed the epidemiological data. Studies on MDM2 phosphorylation sites consisting of Ser166, Ser260, and Ser394 in response to arsenic exposure, which have not been studied presently, indicated that As(III) could induce the expression of MDM2 phosphorylation. Moreover, we studied the alterations of p53 and its N-terminus phosphorylation sites of Ser9, Ser15, and Ser33, which demonstrated that p53 and its phosphorylation were highly expressed after As(III) exposure. Subsequently, Co-immunoprecipitation assays validated our hypothesis that the bonding of MDM2 and p53 was altered by arsenic exposure. What's more, outcomes coming from different cell types of A549, 16HBE, and 60 T-16HBE revealed that MDM2 and its phosphorylation expression existed a significant difference. The study provides evidence that As(III) and its methylated metabolites modulate the expression of MDM2, p53, and their phosphorylation and then affect the interaction between MDM2 and p53.

Arsenic induces gender difference of estrogen receptor in AECII cells from ICR fetal mice.

Arsenic is a confirmed human lung carcinogen with estrogenic activity. There are gender differences in the incidence of lung cancer. Estrogen receptors (ER) play an important role in the process of the development of lung cancer. In order to understand the gender difference effects of ER during carcinogenesis of lung induced by arsenic, the effects of arsenic and estrogen receptor antagonist (ICI182780) on expression levels of estrogen receptor beta (ERß), extracellular regulated protein kinase (ERK1/2) and nuclear factor κB (NF-κB/P65) in type II alveolar epithelial cells (AECII) from different sex ICR fetal mice lung were detected. Results showed that arsenic increased the expression levels of mRNA and protein of ERß, ERK1/2 and NF-κB/P65, and ICI182780 inhibited the increase. Furthermore, there remains a gender difference in these changes. To summarize, the observations here strongly suggested that estrogen receptor and its mediated signal pathway molecules might have critical roles of the gender difference of incidence of lung cancer in arsenic induced.

[Gender-dependent Expression of ERß in AEC â ¡ of Fetal Mice Exposed to Arsenic and Estrogen Receptor Antagonist].

OBJECTIVE: To determine the effects of arsenic and estrogen receptor antagonist (ICI182, 780) on the expression of estrogen receptor beta (ERß) in alveolar Ⅱ epithelial cells (AECⅡ) of female and male mice. METHODS: Nineteen or twenty day fetus mice were obtained through caesarean section of ICR mice. Purified AECⅡ cells were separated from the female and male fetus,respectively,and confirmed using immunofluorescence staining. The cells were exposed to sodium arsenite (NaAsO2) at a low,medium,or high dosage determined by MTT and cultured for 24 h. The NaAsO2 (5 µmol/L) exposed cells were compared with those treated (for 24 h) with dimethyl sulfoxide (DMSO) or ICI182, 780 (1×10-4 mol/L). Apoptosis rates of the cells were measured by flow cytometry. Real-time fluorescence quantitative PCR method and Western blot technique were used to detect the expression ofERßmRNA and protein in AECⅡ. RESULTS: Purity of AECⅡ cells reached (87.0±2.5)%. NaAsO2 exposure was set at a concentration of 0.5 (low),1.25 (medium),and 5 (high) µmol/L. The cells exposed to medium and high dosage of NaAsO2 had higher apoptosis rates than the blank controls (P<0.05),without sex differences. Female cells exposed to medium and high dosage of NaAsO2 had higher levels of expressions ofERßmRNA and protein than the blank controls (P<0.05) and male cells exposed to the same dosage of NaAsO2 (P<0.05). No significant differences were found in the expressions ofERßmRNA and protein between the exposed male cells and the blank controls. ICI182, 780 lowered the expression levels ofERßmRNA and protein in the female exposed cells (P<0.01). CONCLUSION: Arsenic exposure increases expressions of AECⅡ's ERß,more so in female cells than in male cells. This can be blocked by estrogen receptor antagonists.

[Gender-dependent expression of ERalpha in arsenic exposed mice offspring's lung tissue].

OBJECTIVE: To observe theexpression variations of estrogen receptor alpha (ERalpha) in different periods of mice offspring and the gender-dependent differences in the lung tissue with parental arsenic exposure. METHODS: Parental female mice were exposed to arsenic by gavage from gestational day 8th to offspring infancy, and offspring were exposed to arsenic by drinking water after infancy. The expression level of ERalpha mRNA and protein in lung tissue of the male and female offspring in different developmental periods and different doses (low, middle, high) of sodium arsenite exposure were detected by real-time PCR and Western blot. RESULTS: ERalpha mRNA expression in female lung tissue was lower than male in embryonic period (P<0.05); ERalpha mRNA expression in female lung tissue was higher than that of male in infant and adult periods (middle dose of infancy P<0.05, middle and high doses of adulthood P<0.05); No statistical significances were observed in embryo, infancy and adulthood control groups. ERalpha mRNA expression in female lung tissue of infancy and adulthood was higher than that in embryonic period (low, middle and high dose groups P<0.05). ERa protein expression in arsenic exposed female lung tissue was higher than that of male in infant and adult periods, it was also increased by compared with corresponding control groups (P<0.05). The expression level of ERalpha protein in exposed adult female and male offspring were higher than that of infancy. CONCLUSION: Arsenic infected during pregnancy can increase the lung tissue's ERalpha expression level of female offspring in infancy and adulthood. This result is significant to elucidate the role of environment pollutants in gender difference of lung cancer's occurrence.

Metabolites of arsenic and increased DNA damage of p53 gene in arsenic plant workers.

Recent studies have shown that monomethylarsonous acid is more cytotoxic and genotoxic than arsenate and arsenite, which may attribute to the increased levels of reactive oxygen species. In this study, we used hydride generation-atomic absorption spectrometry to determine three arsenic species in urine of workers who had been working in arsenic plants,and calculated primary and secondary methylation indexes. The damages of exon 5, 6, 8 of p53 gene were determined by the method developed by Sikorsky, et al. Results show that the concentrations of each urinary arsenic species,and damage indexes of exon 5 and 8 of p53 gene in the exposed population were significantly higher, but SMI was significantly lower than in the control group. The closely positive correlation between the damage index of exon 5 and PMI,MMA, DMA were found, but there was closely negative correlation between the damage index of exon 5 and SMI. Those findings suggested that DNA damage of exon 5 and 8 of p53 gene existed in the population occupationally exposed to arsenic. For exon 5, the important factors may include the model of arsenic metabolic transformation, the concentrations of MMA and DMA, and the MMA may be of great importance.

Comparison of immunotoxic effects induced by the extracts from methanol and gasoline engine exhausts in vitro.

Gasoline engine exhaust has been considered as a major source of air pollution in China. Due to lower cyto- and geno-toxicity effects of methanol engine exhaust, methanol is regarded as a potential substitute for gasoline. We have previously compared cyto- and geno-toxicities of gasoline engine exhaust with that of methanol engine exhaust in A549 cells (Zhang et al., 2007).To characterize the immunotoxic effects for gasoline and methanol engine exhausts in immune cell, in this study, we further compared effects of gasoline and methanol engine exhausts on immune function in RAW264.7 cell and rabbit alveolar macrophages. Results showed that both gasoline and methanol engine exhaust could evidently inhibit RAW264.7 cell proliferation, promote RAW264.7 cell apoptosis, decrease E-rosette formation rate and inhibit anti-tumor effects of alveolar macrophages, at the same time, these effects of gasoline engine exhaust were far stronger than those of methanol engine exhaust. In addition, gasoline engine exhaust could significantly inhibit activities of ADCC of alveolar macrophages, but methanol engine exhaust could not. These results suggested that both gasoline and methanol engine exhausts might be immunotoxic atmospheric pollutants, but some effects of gasoline engine exhaust on immunotoxicities may be far stronger than that of methanol engine exhaust.

Oxidative damage of the extracts of condensate, particulate and semivolatile organic compounds from gasoline engine exhausts on testicles of rats.

Oxidative damage induced by extracts of condensate, particulate matters and semivolatile organic compounds from gasoline engine exhausts were investigated in testicles of adult Sprague-Dawley rats. The results showed that gasoline engine exhaust could increase the contents of malondialdehyde and carbonyl protein, decrease activities of superoxide dismutase and glutathione peroxidase, and induce DNA damage in testicle of rat. Taking together, the gasoline engine exhaust could promote oxidative damage of bio-macromolecular in testicles of rat and oxidative stress might be an alternative mechanism for male reproductive function of male mammals.

[Oxidative damage of gasoline engine exhausts to rat lung tissues].

OBJECTIVE: To study the effects of extracts of condensate, particulates and semivolatile organic compounds from gasoline engine exhaust on DNA damage, 8-oxoguanine DNA glycosylase-1 (OGG1) expression, and changes of ultra-structures in lungs of rats. METHODS: Organic extracts of gasoline engine exhaust (GEE) was intratrachealy instilled into rat lungs at 0, 5.6, 16.7, and 50.0 L/kg body weight, respectively, once a week for a month. The single DNA strand break was measured by comet assay. The OGG1 was determined using immunohistochemistry method. The ultrastructure of lung cells was observed with electronic microscope. RESULTS: The rates of tailed cells detected by the comet assay increased significantly when the rats were exposed to 16.7 and 50.0 L/kg of GEE compared with those exposed to solvent only (P < 0.05). However, the tail length did not differ significantly between the groups. Similarly, exposure to 16.7 and 50.0 L/kg of GEE led to increased OGG1 significantly. Significant changes of mitochondria in type I and II alveolar cells as well as respiratory bronchiole epithelial cells were observed, which included decrease of numbers, pyknosis and swelling. CONCLUSION: Gasoline engine exhausts induce single DNA strand break, increase OGG1 expression, decrease numbers of mitochondria, and destroy ultrastructures of mitochondria in various lung cells of rats.

[Cytotoxicity induced by gasoline engine exhausts associated with oxidative stress].

OBJECTIVE: To evaluate the relationship between cytotoxic effects of the extracts of condensate, particulates and semivolatile organic compounds from gasoline engine exhausts (EGE) and oxidative stress. METHODS: After A549 cells were treated with various concentrations of EGE for 2h, and cell viabilities were detected induced by EGE were examined by MTT assay. Meanwhile, the reactive oxygen species (ROS) in A549 cells induced by EGE were examined, 2',7'-dichlorodihy-drofluorescin diacetate (DCFH-DA) was used to catch ROS and its level measured by value of pixel fluorescence intensity. Furthermore, A549 cells pretreated with different concentrations of glutathione (GSH) were exposed to various concentrations of EGE for 2h, and then cell viabilities were examined. RESULTS: Viabilities of A549 cells significantly decreased in comparison to the solvent group when the concentrations of EGE were more than 3.9 ml/ml (P < 0.05). There were a dose-response relationships between the viabilities and the concentration of EGE (r = -0.81, P < 0.01). At the concentrations of 31.3 ml/ml and 62.5 ml/ml, the values of pixel fluorescence intensity were (125.0 +/- 19.2) and (168.9 +/- 16.9), which were significantly higher than those of control (8.5 +/- 1.4). In addition, the viabilities of cells pretreated with GSH gradually increased with the increases of the concentrations of GSH. There were also a significant difference between the pretreated and non-pretreated group at the concentrations of 0.5 mmol/L and 1.0 mmol/L. CONCLUSION: Oxidative stress could be one of the mechanisms of cytotoxic effects of EGE.

[Effects of the extracts of condensate, particulates and semivolatile organic compounds from gasoline exhaust on oxidative damage and genotoxicity in testicles of rats].

OBJECTIVE: To determine the effects of the extracts of condensate, particulates and semivolatile organic compounds from gasoline engine exhaust (EGE) on oxidative damage and DNA single strand break in testicles of rats. METHODS: Rats were exposed to the organic extracts of EGE at the concentrations of 0, 5.6, 16.7, and 50.0 L/kg body weight via intratracheal instillation, once a week for a month. The body weight and testicle weight were measured, and the contents of malondialdehyde (MDA) and carbonyl protein (CP), and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in testicles tissue were determined. In addition, the DNA single strand break was examined by comet assay. RESULTS: The body weight increment and the absolute and relative weight of the testicles in the group rats treated with various concentrations of EGE were not significant in comparison with those of control group rats. In comparison with the solvent group, the contents of MDA and CP significantly increased at the concentrations of 16.7 and 50.0 L/kg EGE (P < 0.05). On the contrary, the activities of SOD decreased at tested groups (P < 0.05). But for GSH-Px, the activity reductions were significant only at the concentration of 50.0 L/kg EGE (P < 0.05). In comet assay, at doses of 16.7 and 50.0 L/kg EGE, the rates of tailed cells were (50.06% +/- 7.62%) and (83.25% +/- 8.97%) respectively, the differences were significant in comparison with the solvent group (P < 0.05). However, statistical differences in tail lengths were observed only at the dose of 50.0 L/kg EGE in comparison with the controls (P < 0.05). CONCLUSION: Gasoline engine exhaust could enhance oxidative damage and induce DNA single strand break in testicles of rats.

Increased damage of exon 5 of p53 gene in workers from an arsenic plant.

Mutagenesis is a multistage process. Substitution mutations can be induced by base modified through alteration of pairing property. Mutations of exon 5 and 8 of p53 gene have been found in most arsenicosis patients with precarcinomas and carcinomas, but never in arsenicosis individuals without precarcinomas and carcinomas. This study investigates whether base modification exists in exon 5 and 8 of p53 gene, and explores the dose-effect relationship between damage of exon 5 of p53 gene and urinary arsenic. Concentrations of urinary 8-hydroxydeoxyguanine (8-OHdG) are analyzed to identify the occurrence of DNA damage. The real-time PCR developed by Sikorsky et al. is applied to detect base modification in exon 5 and 8 of p53 gene for apparently healthy participants. Our results show that the mean total arsenic concentrations of two exposed groups from an arsenic plant are significantly elevated compared with the control group, and the damage level of exon 5 of the high-exposed group is significantly higher than that of the control group, but which does not happen in exon 8. The closely correlation between the damage index of exon 5 and urinary organic arsenic concentration are found. Concentration of 8-OHdG of the high-exposed group is significantly higher than that of the control group. These results imply that base modification in exon 5 of p53 gene can be induced by arsenic. In addition, our study suggests that the damage level of exon 5 is a useful biomarker to assess adverse health effect levels caused by chronic exposure to arsenic.

Enhanced sensitivity to DNA damage induced by cooking oil fumes in human OGG1 deficient cells.

Cooking oil fumes (COFs) have been implicated as an important nonsmoking risk factor of lung cancer in Chinese women. However, the molecular mechanism of COFs-induced carcinogenicity remains unknown. To understand the molecular basis underlying COFs-induced cytotoxicity and genotoxicity as well as the roles of hOGG1 in the repair of COFs-induced DNA damage, a human lung cancer cell line with hOGG1 deficiency, A549-R was established by using a ribozyme gene targeting technique that specifically knockdowned hOGG1 in A549 lung adenocarcinoma cells. MTT and comet assays were employed to examine cell viability and DNA damage/repair, respectively, in A549-R and A549 cell lines treated with COF condensate (COFC). RT-PCR and Western blot results showed that the expression of hOGG1 in A549-R cell line was significantly decreased compared with that in A549 cell line. The concentration of COFC that inhibited cell growth by 50% (the IC50) in the A549-R cell line was much lower than that in the A549 cell line, and more COFC-induced DNA damage was detected in the A549-R cell line. The time course study of DNA repair demonstrated delayed repair kinetics in the A549-R cell line, suggesting a decreased cellular damage repair capacity. Our results showed that hOGG1 deficiency enhanced cellular sensitivity to DNA damage caused by COFC. The results further indicate that hOGG1 plays an important role in repairing COF-induced DNA damage. Our study suggests that COFs may lead to DNA damage that is subjected to hOGG1-mediated repair pathways, and oxidative DNA damage may be involved in COF-induced carcinogenesis.

Suppression of a DNA base excision repair gene, hOGG1, increases bleomycin sensitivity of human lung cancer cell line.

Bleomycin (BLM) has been found to induce 8-oxoguanine and DNA strand breaks through producing oxidative free radicals, thereby leading to cell cycle arrest, apoptosis and cell death. Cellular DNA damage repair mechanisms such as single strand DNA break repair/base excision repair (BER) are responsible for removing bleomycin-induced DNA damage, therefore confer chemotherapeutic resistance to bleomycin. In this study, we have investigated if down-regulation of human 8-oxoguanine DNA glycosylase (hOGG1), an important BER enzyme, could alter cellular sensitivity to bleomycin, thereby reducing chemotherapeutic resistance in human tumor cell. A human lung cancer cell line with hOGG1 deficiency (A549-R) was created by ribozyme gene knockdown technique. Bleomycin cellular sensitivity and DNA/chromosomal damages were examined using MTT, colony forming assay, comet assay as well as micronucleus assay. We demonstrated that hOGG1 gene knockdown enhanced bleomycin cytotoxicity and reduced the ability of colony formation of the lung cancer cell lines. We further demonstrated that bleomycin-induced DNA strand breaks resulted in an increase of micronucleus rate. hOGG1 deficiency significantly reduced DNA damage repair capacity of the lung cancer cell lines. Our results indicated that hOGG1 deficiency allowed the accumulation of bleomycin-induced DNA damage and chromosomal breaks by compromising DNA damage repair capacity, thereby increasing cellular sensitivity to bleomycin.

Comparison of cytotoxicity and genotoxicity induced by the extracts of methanol and gasoline engine exhausts.

Gasoline engine exhaust has been considered a major source of air pollution in China, and methanol is considered as a potential substitute for gasoline fuel. In this study, the genotoxicity and cytotoxicity of organic extracts of condensate, particulate matters (PM) and semivolatile organic compounds (SVOC) of gasoline and absolute methanol engine exhaust were examined by using MTT assay, micronucleus assay, comet assay and Ames test. The results have showed that gasoline engine exhaust exhibited stronger cytotoxicity to human lung carcinoma cell lines (A549 cell) than methanol engine exhaust. Furthermore, gasoline engine exhaust increased micronucleus formation, induced DNA damage in A549 cells and increased TA98 revertants in the presence of metabolic activating enzymes in a concentration-dependent manner. In contrast, methanol engine exhaust failed to exhibit these adverse effects. The results suggest methanol may be used as a cleaner fuel for automobile.

Compositions and oxidative damage of condensate, particulate and semivolatile organic compounds from gasoline exhausts.

The effects of extracts of condensate, particulate and semivolatile organic compounds from absolute gasoline exhausts on DNA single strand break, levels of malondialdehyde (MDA), carbonyl protein and activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were investigated in lungs and brains of adult Sprague-Dawley rats of both sexes. In addition, the non-conventional components of the extracts and concentrations of 13 polycyclic aromatic hydrocarbon (PAHs) in gasoline exhaust were measured by GS/MS. Extract of gasoline exhaust at different doses (5.6, 16.7 and 50.0L/kg) were given to administered animals by intratracheal instillation once a week for 4 weeks, while blank control and solvent groups were given with physiological saline and dimethyl sulfoxide (DMSO). Our results showed that gasoline exhaust increased DNA single strand break, promoted lipid peroxidation and oxidative protein damage and decreased activities of SOD in lungs and brains. While, it decreased the activities of GPx in lungs but not in brains. The present data suggested that gasoline exhaust exposure could cause oxidative damage to lung and brain of rats. That was to say that gasoline is a toxin to brain of mammals, not only to lung.