| Over the last decade, air pollution has becoming increasingly prominent. Air pollutants, either in gas or particle phase, are found to have direct impact on human health. Exposure to air pollutants has been suggested to be associated with the respiratory and cardiovascular diseases. In recent years, toxicity assessment of air pollutants has become an effective tool to evaluate the health effects of air pollution. To evaluate the toxicity, a number of in vitro tests have been developed to examine cell viability, oxidative stress, and genotoxicity. However, these commonly used assays are time-consuming, and only can be used to evaluate specific toxicological endpoints. Hence, a rapid and cost-effective toxicity assessment bioassay is needed to determine the overall toxicity of single chemical compounds and complex chemical mixtures present in the air. Bioassays using reporter genes regulated by cellular stress-responsive gene promoters have been suggested as promising tools for toxicity assessment.Among the various cellular stress response pathways, the heat shock response (HSR) is one of the major pathways characterized by up-regulation of a family of proteins called heat shock protein (HSP). HSPA1A (Hsp70-1), as one of the most important HSPs, is a highly conserved molecular chaperone that plays essential roles in nascent protein synthesizing, folding, and misfolded protein refolding or degradation. Under stressful conditions (heat, heavy metals, and toxic chemicals), the heat shock factor (HSF) will assemble into a trimer and accumulate within the nucleus. Activation and binding of HSF to the heat shock element (HSE) in the HSPA1A promoter region will initate the gene transcription. Because of its stress responsiveness, HSPA1A expression is becoming a sensitive indicator of the early cellular damage induced by environmental stressors. Transgenic technology, using reporter genes under the control of HSPA1A gene promoter, provides a novel method for toxicity assessment of environmental pollutants. In fact, human cells carrying an HSPA1A promoter-driven reporter gene have been used to assess the toxicity of water pollutants and soil contaminants such as heavy metals, organochlorine compounds, and eluates of industrial wastes. However, whether the HSPA1A promoter-driven reporter can be used in the toxicity assessment of single chemical compounds and complex chemical mixtures such as polycyclic aromatic hydrocarbons (PAHs), formaldehyde, urban traffic emissions and coke oven emissions from the polluted air is an open question.The HepG2cells, derived from a human liver hepatoma, are reported to retain many properties of primary cells such as containing many functional phase-1and-II enzymes which are lost in most cultured cell lines. In the present study, the HepG2cells were stably transfected with a human HSPA1A promoter-driven luciferase reporter (HepG2/HSPA1A cells). The HepG2/HSPA1A cells were treated with different concentrations of pyrene, benzo[a]pyrene, formaldehyde, urban traffic emissions, and coke oven emissions (COEs) collected from the top, side, and bottom of a coke oven battery for24h. After the treatments, luciferase activity, cell viability, malondialdehyde (MDA) concentration, Olive TM value, and micronuclei frequency were determined, respectively. We also assessed the toxicological sensitivity of HepG2/HSPA1A cells by comparison with a battery of established in vitro toxicity tests, namely, cell viability assay, MDA assay, comet assay, and micronucleus test. Our hypothesis was that transcriptional activation of the HSPA1A promoter-driven luciferase reporter is a sensitive indicator of the overall toxicity of environmental pollutants.Part I Establishment of stable HSPA1A promoter-driven luciferase reporter HepG2cells (HepG2/HSPA1A cells)The HSPA1A promoter-driven luciferase reporter HepG2cells (HepG2/HSPA1A cells) were developed by using the lipofectamine transfection technology. Firstly, the human HSPA1A promoter was amplified and inserted into the pGL4.17plasmid. Then, HepG2cells were stably transfected with the validated plasmid, and positive clones were selected with culture medium containing0.1mg/ml G418for14days. Finally, the stable HepG2/HSPA1A cells were validated by heat shock treatment. The HepG2/HSPA1A cells were heated at42℃for1h and then recovered at37℃for different time points, and then levels of HSPA1A and relative luciferase activity were determined by Western blotting analysis and luciferase reporter system, respectively. Compared with the control group, heat shock induced a progressive increase in levels of HSPA1A peaking at4h of recovery. And the results of three independent experiments showed that the relative level of HSPA1A at4h of recovery was over3times the control level. The relative luciferase activities in the recovery groups were also significantly increased after heat shock treatment. While, at4h of the recovery, the relative luciferase activity was greater than9times the control level. The Spearman rank test showed that the relative luciferase activity was positively correlated with the level of HSPA1A. Heat shock treatment is the classic inducer of heat shock reponse. In the present study, both the levels of HSPA1A and relative luciferase activity were obvious increased after heat shock treatment. Our data validated the sensitivity of the newly developed HepG2/HSPA1A cells, and the cells can be used in the detection of the toxic effects induced by environmental stressors.Part II Using HepG2/HSPA1A cells to assess the toxicity of single chemical compounds present in the airIn order to evaluate the potential of HepG2/HSPA1A cells in assessing the toxicity of single chemical compounds, we treated the cells with different concentrations of three single chemical compounds (noncarcinogenic pyrene, carcinogenic benzo[a]pyrene, and mutagenic formaldehyde) that are ubiquitous in the air for24h. After the treatments, luciferase activity, cell viability, oxidative damage indicated by MDA concentration, and genotoxic damage indicated by both Olive TM value and micronuclei frequency were determined, respectively. We also assess the toxicological sensitivity of HepG2/HSPA1A cells by comparison with four established in vitro toxicity tests including cell viability assay, MDA assay, comet assay, and micronucleus test. Pyrene induced a dose-dependent increase in relative luciferase activity up to2.4times the control level (P<0.01) at100μM. Compared with the control level, the increases in relative luciferase activity were statistically significant (P<0.05). Benzo[a]pyrene led to a2.7times induction of relative luciferase activity at20μM. The low dose of formaldehyde (1μM) led to a significant increase in relative luciferase activity that progressively increased to1.6times the control level at40μM. In HepG2/HSPA1A cells exposed to pyrene, relative luciferase activity was only positively correlated with MDA concentration (P<0.01). In cells treated with benzo[a]pyrene and formaldehyde, relative luciferase activity was positively correlated with Olive TM value and micronuclei frequency (P<0.01). Significant increases in relative luciferase activity were observed at concentrations that were as low, or lower than the concentrations that the tested chemical compounds decreased cell viability, and increased MDA concentration, Olive TM value and micronuclei frequency. This suggests that the relative luciferase activity in HepG2/HSPA1A cells is a sensitive indicator of the cellular damage induced by these individual chemical compounds. These findings validate the relative luciferase activity in HepG2/HSPA1A cells as a sensitive and responsive indicator of the overall toxicity of single chemical compounds. The HepG2/HSPA1A cells can be used to assess the overall toxicity of single chemical compounds present in the air.Part III Using HepG2/HSPA1A cells to assess the overall toxicity of complex chemical mixtures present in the airAfter validating the application of HepG2/HSPA1A cells in toxicity assessment of single chemical compounds, in this part we will evaluate the sensitivity of the stable metabolically competent HepG2/HSPA1A cells for assessing the overall toxicity of complex chemical mixtures present in the air. Urban traffic is a major source of air pollution, and everyday many people are exposed to the toxic compounds from urban traffic. Coke oven emissions (COEs) represent the coal-burning pollution in the air. The COEs contain a variety of genotoxic agents including mutagens and carcinogens. Exposure to COEs has been shown to result in a dose-dependent risk of cancer in coke oven workers. In the present study, the urban vehicle emissions were collected at a heavily traveled road in Wuhan, while the coke oven emissions in ambient air were collected from different sites where the coke oven workers worked in a state-run coke oven plant located in Central China. Then, we evaluated the toxicity of urban vehicle emissions and coke oven emissions in HepG2/HSPA1A cells. The urban traffic emissions induced significant increases in relative luciferase activity up to1.4times the control level at0.6ng/1. Compared with the control level, significant increase (P<0.01) in relative luciferase activity was also observed at the lowest tested concentration (6pg/1). The bottom COEs induced significant increases (P<0.01) in relative luciferase activity up to1.4times the control level at0.15μg/1. The low dose of side COEs (0.02μg/1) led to a significant increase (P<0.01) in relative luciferase activity that progressively increased to2.1times the control level at65.4pg/1. The top COEs produced a strong dose-dependent induction of relative luciferase activity up to over5times the control level at the highest concentration tested (202μg/1). In HepG2/HSPAlA cells treated with traffic EOM, relative luciferase activity was positively correlated with both Olive TM values and micronuclei frequency (P=0.001and P=0.007, respectively). InHepG2/HSPA1A cells treated with the bottom COEs, relative luciferase activity was positively correlated with MDA concentration (r=0.404, P<0.05). For the three COEs samples, positive correlations were observed between relative luciferase activity and Olive TM values and micronuclei frequency. Significant increases in relative luciferase activity were observed at concentrations that were as low, or lower than the concentrations that the urban traffic emissions and coke oven emissions decreased cell viability, and increased MDA concentration, Olive TM value and micronuclei frequency. The relative luciferase activity in HepG2/HSPA1A cells can sensitively reflect the toxicity of urban traffic emissions and coke oven emissions. When we used HepG2/HSPA1A cells to assess the toxicity of complex chemical mixtures, the mixtures were considered as a whole. The response of relative luciferase activity in HepG2/HSPA1A cells may reflect an integrated stress response summarizing the total cellular damage induced by the complex chemical mixtures. We conclude that the HepG2/HSPA1A cells are a valuable tool for rapid screening of the overall toxicity of complex chemical mixtures present in the air.In summary, the main results of our study are:(1) The relative luciferase activity was increased by all the tested single chemical compounds and complex chemical mixtures present in the air including the non-carcinogenic pyrene and low concentrations of urban traffic emissions (pg/1).(2) Significant increases in relative luciferase activity were observed at concentrations that are generally as low, or lower than those doses at which the tested air pollutants decreased cell viability, increased oxidative and genotoxic damage. These findings indicate that the relative luciferase activity in HepG2/HSPA1A cells can serve as an early indicator of toxicant exposure.(3) Although a limited number of air pollutants were screened, linear relationships between relative luciferase activities and levels of cellular damage were detected. This suggests that relative luciferase activity in HepG2/HSPA1A cells can reflect the overall toxic effects induced by air pollutants.(4) The relative luciferase activity in HepG2/HSPA1A cells is generally as sensitive, or more sensitive to the environmental pollutants tested, compared with the cell viability assay, MDA assay, comet assay, and micronucleus test.(5) Furthermore, compared with those in vitro toxicity tests that are developed to evaluate the specific toxicological endpoints, the HepG2/HSPA1A cells can be used to detect the overall toxicity of environmental pollutants present in the air.There are some innovations and advantages in the present study. Firstly, the metabolic competent HepG2cells were used to construct the toxicity test system. Secondly, three single chemical compounds (noncarcinogenic pyrene, carcinogenic benzo[a]pyrene, and mutagenic formaldehyde) and two complex chemical mixtures (urban traffic emissions and coke oven emissions) that are ubiquitous in the air were tested to validate the application of HepG2/HSPA1A cells in toxicity assessment of environmental pollutants. Finally, the toxicological sensitivity of HepG2/HSPA1A cells was evaluated by comparing the reporter gene assay with other toxicity tests including cell viability assay, MDA assay, comet assay, and micronucleus test.However, there are some limitations needed to be addressed. Firstly, data with other single chemical compounds and complex chemical mixtures present in the air are necessary to confirm our findings and further validate the application of the HepG2/HSPA1A cells in toxicity assessment. Secondly, more toxicity tests should be included in the evaluation of the toxicological sensitivity of HepG2/HSPA1A cells. Finally, further study is also needed to be conducted to assess the toxicity of environmental pollutants in vivo. |
PDF Full Text Request