Abstract
Trichloroethylene (TCE) is a common organic solvent that has been widely used in industrial applications. Hundred cases of allergic reactions occurred after the workers were occupationally exposed to TCE in China in the past decade, but the underlying effector mechanisms of TCE remain unclear. The purpose of the present study is to examine the alteration of hepatic metabolic enzyme gene and apoptosis-related gene messenger RNA (mRNA) expression in L02 human hepatocytes (L02 cells) after treatment with TCE. L02 cells were cultured either with various doses of TCE (0.25, 0.5, 1.0 and 2.0 mmol/L) for 24 h or with a single dose of TCE (1.0 mmol/L) for different time intervals, whereas samples treated with dimethyl sulfoxide served as control. Quantitative real-time polymerase chain reaction analysis was performed to detect the mRNA expression of hepatic metabolic enzyme genes (CYP1A2, CYP3A4 and CYP2E1) and apoptosis-related genes (BAX and BAD). It was found that the transcript levels of hepatic metabolic enzyme genes and apoptosis genes including BAX and BAD were significantly increased after TCE treatment at various doses for 24 h when compared with controls. Additionally, when the cells were treated with a single dose of TCE (1.0 mmol/L) for different periods of time (3, 6, 12 and 24 h), the mRNA expression of these genes also increased significantly compared with control (p < 0.05 or p < 0.01). The conclusion of the study is that TCE could induce alteration of mRNA expression of hepatic metabolic enzyme genes and apoptosis genes, which might be implicated in the effector mechanisms of TCE cytotoxicity in vivo.
Introduction
Trichloroethylene (TCE), one of the common organic solvents, has been widely used in industrial applications, such as cleaning and degreasing of metal products as well as in computer-chip manufacturing. 1 –5 TCE is highly lipophilic and readily absorbed into the circulation following oral, dermal or inhalation exposure. Several human epidemiologic studies have focused on evaluating the health effects related to occupational and environmental TCE exposure. Consequently, an association between TCE exposure and the development of hypersensitivity dermatitis was noted. 2,6,7 Because of its widespread commercial use and improper disposal, TCE has also become a major occupational and environmental pollutant. It has been reported that TCE has various toxicities and can induce skin damage, liver damage and even heart or kidney impairment. 5,6,8 In recent years, we have found that some workers exposed to TCE suffered from allergic dermatitis, namely TCE-induced dermatitis or TCE-induced allergic disorder. Studies by others have described it as TCE-related generalized skin disorder, TCE-related hypersensitivities or TCE-induced medicamentosa-like dermatitis, which resembles drug hypersensitivities. 9 –11 Most patients with dermatitis or liver impairment usually demonstrate elevated enzyme activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). 7,12 –15 In China, the number of cases reported with these TCE-induced disorders or subsequent death has been increasing after the mid-1990s.
Although many studies have been conducted on TCE toxicity, the mechanism of TCE-induced allergic disorder remains unclear. Our previous work found that only a few TCE-exposed people suffered from hypersensitivity dermatitis with liver impairment, indicating that the individual susceptibility or TCE metabolism difference might be the critical factor determining the disease occurrence. It has been reported that TCE is mainly metabolized in liver by cytochrome P450 (CYP450). Therefore, the present study was undertaken to investigate the alteration of hepatic metabolism gene and apoptosis gene messenger RNA (mRNA) expression in L02 human hepatocytes (L02 cells) treated with various TCE concentrations for different durations. The objective of this study was to explore the possible association between TCE-induced allergic disorder and hepatic metabolism gene and apoptosis gene expression after TCE treatment.
Materials and methods
Reagents
Roswell Park Memorial Institute (RPMI) 1640 culture medium, fetal bovine serum (FBS), penicillin/streptomycin and trypsin were obtained from Gibco (Grand Island, New York, USA); TCE, dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma Co. (St Louis, Missouri, USA); Trizol reagent was from Invitrogen (Grand Island, New York, USA), ExScriptTMRT reagent kit and SYBR Premix Ex Taq were purchased from Takara (Daliang, China), the primers of target genes and β-actin were procured from Shanghai Biological Engineering Co., Ltd (Shanghai, China). L02 normal human-derived hepatocytes (L02 cells) were purchased from the American Type Culture Collection (ATCC, Shanghai Cytobiological Institute of China Academy of Science, Shanghai, China).
Cell culture and TCE treatment
L02 liver cells were cultured in 6-well plates with RPMI medium 1640 containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin under the condition of 37°C and 5% CO2. When the cells reached approximately 80% confluence, the medium was discarded and the cells were washed 3 times using phosphate-buffered saline, then the cells were cultured in 2 ml RPMI medium 1640 without FBS. TCE was dissolved in DMSO to make a proper solution for cell treatment. Four groups of cells were treated with TCE at the final concentration of 0.25, 0.5, 1.0 and 2.0 mmol/L, respectively, for 24 h. Group 5 was treated with DMSO only (used as solvent control) for 24 h, and group 6 was not treated with any reagents (used as negative control). In addition, a single TCE (1.0 mmol/L) concentration was incubated with groups of cells that were treated for different periods of time (3, 6, 12 or 24 h) to explore the time–effect relationship.
Cell viability assay
To evaluate the cytotoxicity of TCE on L02 cells, the cell viability was determined by MTT method as described by Mossmann (1983), with minor modifications. Briefly, L02 cells were seeded in 96-well plates, MTT solution was added to each well (final concentration of 0.5 mg/ml) and the cells were cultured for another 4 h, then 100 ml of 10% sodium dodecyl sulfate (SDS) 0.01 N HC1 (the SDS solution) were added to each well after mixing on a plate mixer. The culture plates were incubated overnight at 37°C in a 5% CO2 incubator. The absorbance was recorded at 590 nm using the automated microplate reader (BioTek ELX808IU, USA). Results were presented as percentage viability relative to the blank control values.
Real-time fluorescent quantitative PCR of target gene expression
The cells were harvested and total RNA from cells treated with TCE was extracted with Trizol kit according to the manufacturer’s specifications. Approximately 2 μg of RNA was used to synthesize the first-strand complementary DNA (cDNA) according to the protocol of reverse transcription kit. SYBR Green polymerase chain reaction (PCR) kit was used for real-time quantification of double-stranded DNA targets. The cDNA acquired was stored at −80°C. Real-time fluorescent PCR was performed with primers as mentioned in Table 1 and cDNA as template. Two microliters of 5-fold diluted cDNA were used for every PCR reaction in a final volume of 25 μl, containing 0.5 μl forward primer (10 mmol/L) and 0.5 μl reverse primer (10 mmol/L). A pair of conventional PCR primers for target genes was used for the amplification reaction on the real-time PCR amplifier (ABI 7900HT, USA). The reagent used for PCR reaction are as follows: 2× SYBR Green Q-PCR master mix 12.5 μl, forward primer (10 μmol/L) 0.5 μl, reverse primer (10 μmol/L) 0.5 μl, ROX reference dye (50×) 0.5 μl, cDNA 2 μl and double distilled water (ddH2O) 9 μl. The group with cDNA replaced by ddH2O was used as the no-template control. The following thermal cycling profile was used: 95°C for 10 s, followed by 40 cycles of 95°C for 5 s and annealing for 30 s. The annealing temperatures for each gene are as follow: CYP1A2 54°C, CYP3A4 54°C, CYP2E1 55°C, BAX 54°C and BAD 58°C. The relative expression levels of the target gene mRNA were determined with reference to the expression of the β-actin gene.
Real-time fluorescent PCR primer sequences and length of target genes
Statistical analysis
All data were expressed as means ± standard deviation. The means of the different groups were compared by one-way analysis of variance and post hoc analysis using Student–Newman–Keuls multiple range test. Differences between groups were considered significant when p value <0.05.
Results
Effect of TCE on cell viability
L02 cells were cultured in 96-well culture plates and were treated with TCE at various concentrations (0, 0.125, 0.25, 0.5, 1.0 or 2.0 mmol/L) for 12, 24 or 48 h, respectively. The effect of TCE treatment on L02 cell viability was determined by MTT test. As shown in Figure 1, the cell viability quantitatively decreased after 12 or 24 h of TCE treatment. However, there was a significant decrease in cell viability at higher TCE concentrations (1.0 or 2.0 mmol/L) after 48 h. These results indicated that TCE treatment decreased the cell viability at higher concentrations and for a period of time longer than 24 h.
The cell viability of L02 cells treated with trichloroethylene (TCE) for different periods of time. L02 cells were cultured in 96-well culture plates and were treated with TCE at various concentrations (0, 0.125, 0.25, 0.5, 1.0 and 2.0 mmol/L) for 12, 24 or 48 h, respectively, followed by the addition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) solution to each well and the cells were cultured for another 4 h, and the cell viability was determined by MTT test. Data are expressed as means ± SD of three experiments. *p < 0.05 and **p < 0.01 when compared with control.
Effect of TCE on hepatic metabolic enzyme gene expression
There was a 1.36- and 1.56-fold increase in the levels of CYP1A2 and CYP3A4 mRNA expression, respectively, when compared with negative controls at a dose of 0.25 mmol/L TCE; and these differences were statistically significant (p < 0.05). When TCE concentrations reached 0.5, 1.0 and 2.0 mmol/L, the levels of CYP1A2, CYP3A4 and CYP2E1 expression increased markedly. Notably, at doses of 0.5, 1.0 and 2.0 mmol/L TCE, CYP1A2 mRNA expression was 4.44, 6.56 and 8.26; CYP3A4 mRNA expression was 3.89, 4.87, 6.25 and CYP2E1 mRNA expression was 1.61, 1.78 and 1.94, respectively. As shown in Figure 2, these differences were statistically significant (p < 0.01). Moreover, different durations of TCE treatment also had notable effects on the expression of hepatic metabolic enzyme genes. When the cells were treated with TCE for 3 h, the levels of CYP1A2, CYP3A4 and CYP2E1 mRNA expression increased, and the differences were statistically significant (p < 0.05 or p < 0.01) compared with control (the expression levels of various genes in control were set as 1). When the cells were treated with TCE for 6–24 h, CYP1A2, CYP3A4 and CYP2E1 mRNA expression elevated further and reached the highest levels (3.38, 2.42 and 2.40, respectively) for each gene, and the differences were significant (p < 0.01; see Figure 3).
Messenger RNA (mRNA) expression of hepatic metabolic enzyme genes in L02 cells treated with different doses of trichloroethylene (TCE) for 24 h. L02 cells were cultured in 6-well plates and were treated with various concentrations of TCE (0, 0.25, 0.5, 1.0 and 2.0 mmol/L) for 24 h, respectively. Total RNA was extracted from L02 cells with Trizol kit and then reverse transcribed to complementary DNA (cDNA); real-time fluorescent polymerase chain reaction (PCR) was performed with primers and cDNA of the target genes. Data are presented as means ± SD of three experiments. *p < 0.05 and **p < 0.01 when compared with control.
Messenger RNA (mRNA) expression of hepatic metabolic enzyme genes in L02 cells treated with 1.0 mmol/L trichloroethylene (TCE) for different periods of time. L02 cells were cultured in 6-well plates and were treated with 1.0 mmol/L TCE at 0, 3, 6, 12 and 24 h, respectively. Total RNA was extracted from L02 cells with Trizol kit and then reverse transcribed to complementary DNA (cDNA), real-time fluorescent polymerase chain reaction (PCR) was performed with primers and cDNA of the target genes. Data are expressed as means ± SD of three experiments. *p < 0.05 and **p < 0.01 when compared with control.
Effect of TCE on apoptosis genes expression
As shown in Figure 4, at the dose of 0.25 mmol/L TCE, the levels of BAD mRNA expression was 1.29 when compared with control (mRNA expression level in control was set as 1), the differences are statistically significant (p < 0.05). When the doses reached 0.5, 1.0 and 2.0 mmol/L, the levels of BAX and BAD mRNA expression increased notably, the levels of BAX mRNA expression were 1.10, 1.32 and 1.43, respectively, while the levels of BAD mRNA expression were 1.42, 1.68 and 1.87, respectively, and the differences were significant except BAX at the dose of 0.5 mmol/L TCE.
Messenger RNA (mRNA) expression of apoptosis genes in L02 cells treated with different doses of trichloroethylene (TCE) for 24 h. L02 cells were cultured in 6-well plates and were treated with different doses of TCE (0, 0.25, 0.5, 1.0 and 2.0 mmol/L) for 24 h, respectively. Total RNA was extracted from L02 cells with Trizol kit and then reverse transcribed to complementary DNA (cDNA), real-time fluorescent polymerase chain reaction (PCR) was performed with primers and cDNA of the target genes. Data are expressed as means ± SD of three experiments. *p < 0.05 and **p < 0.01 when compared with control.
Furthermore, different durations of TCE treatment also had notable effects on apoptosis gene expression. According to the data shown in Figure 4, the levels of BAD significantly increased when the cells were treated with TCE for 3 h in comparison with control (expression level was set as 1). When the cells were treated with TCE for 6–24 h, the levels of BAX and BAD mRNA expression increased further and reached the highest levels (1.59 and 2.13, respectively) for each gene, and the differences were quite significant (p < 0.01; see Figures 4 and 5).
Messenger RNA (mRNA) expression of apoptosis genes in L02 cells treated with trichloroethylene (TCE) at 1.0 mmol/L for different periods of time. L02 cells were cultured in 6-well plates and were treated with TCE for 0, 3, 6, 12 and 24 h, respectively. Total RNA was extracted from L02 cells with Trizol kit and then reverse transcribed to complementary DNA (cDNA), real-time fluorescent polymerase chain reaction (PCR) was performed with primers and cDNA of the target genes. Data are presented as means ± SD of three experiments. *p < 0.05 and **p < 0.01 when compared with control.
Discussion
In recent years, much attention has been paid to occupational hazards and carcinogenesis of TCE in the world. International Agency for Research on Cancer has classified TCE as a probable human carcinogen (group 2A). Animal studies have shown that TCE is carcinogenic and could induce hepatic carcinoma and lung cancer in B6C3F1 and Swiss mice, and renal cells carcinoma in rats. 16,17 Ritz and his colleagues’ large cohort study showed that workers engaged in metal parts cleaning displayed higher hepatic cancer mortality. 18 Another case–control study illustrated that continuous exposure to large amount of TCE increased the incidence of renal cell carcinoma. 19,20 Besides, TCE has immunotoxicity, teratogenic and mutagenic effects. 21,22
We found that only a few TCE-exposed workers suffered from allergic disorder, and this kind of disease had some special features as follows: (1) there was no obvious dose–response relationship between TCE exposure and occurrence of allergic disorder, the latency ranged from a few days to a few months, sometimes the latency was 3–5 days, but at most circumstances it was no longer than 3 months; (2) almost all the patients appeared to have fever, skin damage including red maculopapule, skin blisters and epidermal necrosis, liver damage including liver dysfunction, elevated levels of serum ALT and AST activities; (3) no significant gender and age differences were found among the patients with TCE-induced allergic disorder; (4) the contents of urinary TCE metabolite trichloroacetic acid (TCA) appeared to increase, but there was no parallel relationship between TCA levels and the patient’s severity; (5) the patients could be sick again when they are exposed to TCE for the second time even if they have recovered; (6) skin patch tests of TCE showed positive results on the volunteers. 15 Because of these characteristics, we speculated that the pathological process of TCE-induced disorder might belong to allergic diseases, it could be related to some allergens formed during metabolism in some individuals or the expression level of some hepatic metabolic enzymes changed.
There are two main pathways to metabolize TCE in human body: (a) the CYP450-dependent oxidation pathway and (b) the glutathione (GSH)-dependent combination pathway. Through the CYP450 and GSH pathways, TCE metabolites are mainly toxic to kidney, liver and lung 21,23 ; therefore, in the present study we investigated the effects of TCE on expression of CYP450 genes by treating L02 cells with TCE so that we could understand the roles of CYP in TCE metabolism.
CYP1A2 is one of the primary members of the CYP superfamily, composing approximately 13% of the amount of liver CYP450. Our results showed that TCE treatment with L02 cells at the doses of 0.5, 1.0 and 2.0 mmol/L TCE for 24 h increases the levels of CYP1A2 expression to 444%, 656%, and 826%, respectively, in comparison with control. Treatment with TCE at 1.0 mmol/L for 6–24 h resulted in the CYP1A2 expression reaching the highest level at 3.38-fold as high as the control. These data illustrated that CYP1A2 expression elevated significantly after TCE treatment, the role of CYP1A2 was quite obvious in metabolizing TCE, so CYP1A2 is one of the major metabolic enzymes of TCE. It has been reported that CYP1A2 is involved in the metabolism of numerous drugs and exogenous chemicals.
CYP3A4 is a predominating oxidase in CYP superfamily, which takes up approximately 30% of the amount of liver CYP450, it is an important drug metabolizing enzyme, participating in the biotransformation of about 50% of clinical drugs. When L02 cells were treated with TCE (0.5, 1.0 and 2.0 mmol/L) for 24 h as mentioned above, the levels of CYP3A4 expression were 3.89-, 4.87-, 6.25-fold as high as control, respectively, indicating that CYP3A4 was involved in TCE metabolism, or TCE could induce CYP3A4 expression. Additionally, CYP2E1 is also an important hepatic metabolic enzyme, although its expression level increased by 61–94% after TCE (0.5, 1.0 and 2.0 mmol/L) treatment for 24 h in comparison with control, the elevation of CYP2E1 is not as obvious as CYP1A2 or CYP3A4, indicating TCE’s induction in CYP2E1 is not as strong as that in CYP1A2 or CYP3A4. Based on the previous study of TCE’s occupational hazards, we found only a few TCE-exposed workers suffered from allergic disorder with skin damage and liver damage, and we speculated that TCE was metabolized differently by CYP and produced different metabolites which might be the source of allergens in the TCE-exposed workers. Therefore, we assume that the liver could play an important role in TCE-induced pathological process, which is worthy of further study.
The apoptosis gene expression was also detected in this study. As we know, cell apoptosis is involved in numerous pathological processes or some diseases. Bcl-2 is a apoptosis-inhibiting gene, Bcl-2 functions in the form of Bcl-2 and Bax heterodimers, and the ratio of Bcl-2 and Bax determines whether apoptosis could happen, whereas the conjugate of apoptosis-promoting protein BAD and the survival-promoting member of Bcl-2 family Bcl-xl enhances apoptosis. 1,24,25 It was found that L02 cells treated with TCE at 0.5–2.0 mmol/L concentration increased the levels of BAX and BAD mRNA expression by 10–33% and 42–87%, respectively. These findings indicated that changes in expression of apoptosis gene might be one of the important mechanisms by which TCE could induce hepatotoxicity, which warrants further study on TCE’s hepatotoxicity.
Taken together, our data showed that TCE administration induced obvious change in mRNA expression of hepatic metabolic enzyme genes and apoptosis genes, which require further identification of protein levels of those transcript factors since gene expression is influenced by some elements; therefore, these findings are the preliminary data that could provide some clues for further study in this field. On the other hand, TCE-induced liver dysfunction is one of the serious occupational health events in China, so far only little is known about the mechanism of this disorder. The present study is aimed at exploring the possible relationship between TCE-induced hepatotoxicity and mRNA expression of hepatic metabolism genes and apoptosis genes, drawing much attention in exploration of this complex problem worldwide. Also we plan to conduct P4502E1 and apoptotic protein assays to confirm the current conclusion that mRNA expression of hepatic metabolic enzyme genes and apoptosis genes might be implicated in the effector mechanisms of TCE cytotoxicity in vivo.
Footnotes
Funding
This project was supported in part by the grants from Shenzhen Science and Technology Foundation to Dr Xinyun Xu (200802073 and 201101015).