Trichloroethylene-induced Mitochondrial Damage In Keratinocytes And Protective Effect Of Ginkgo Biloba Extract

Background Trichloroethylene (TCE) is an organic solvent mainly used as degreaser in industry. Occupational exposure to TCE had been causing severe dermatitis in workers, with clinical manifestation of exfoliative dermatitis, erythema polymorphe, even Stevens-Johnson syndrome (SJS) or acantholysis bullosa. However, the toxicity mechanism is still unknown. Available data suggest that cutaneous cells especially keratinocytes (KC) death at least partially take part in the clinical manifestation of dermatitis. In vitro study also demonstrated that TCE can damage keratinocytes and induce apoptosis. It is known that, mitochondria are vital in regulation of cell's fate, and ginkgo biloba extract (GBE) is regarded as an effective mitochondrial protective agent in many published literatures.Objective Purpose of current study is to observe mitochondrial changes in KC after stimulation with TCE and the influence of GBE on these changes. Not only with intention to elucidate dermal toxicity of TCE but also throw light on prevention and cure of related occupational dermatitis.Methods Normal human KC were isolated from foreskins of healthy donors undergoing circumcision by two-step trypsin digestion and cultured in serum-free medium. Cells were treated with 0.125, 0.5 or 2.0 mmol/L TCE for different time (4, 8, 12 or 24 hours), medium and 1% acetone (volume fraction) were served as blank control and vehicle control respectively. While observe GBE's protective effect, cells were pre-incubated different concentrations of GBE (10, 50, 100 or150 mg/L) for 2 hours, then stimulated with 2.0 mmol/L TCE. After treating, MTT assay and ATPase activity detection were conducted, cellular viability and inhibition ratio of mitochondrial enzyme were calculated according to optical density (OD) value of MTT assay. Change of mitochondrial membrane potential (?Ψm) was detected by flow cytometer (FCM) after double-stained with rhodamine123 (Rh123) and propidium iodide (PI). Morphological changes were observed by transmission electron microscope (TEM).Results①Cell viability: Both 0.5 and 2.0 mmol/L TCE stimulate KC for 4h or 8h can decrease cell viability (compare with control group, P<0.01), but 0.125 mmol/L TCE seemed don't work like that with 4 h or 8 h incubation (P>0.05). Cell viability in all three dose group decreased while been stimulated for 12 h even more longer (24 h), and displayed an tendency of decline accompany with increase of dose of TCE. Cell viability in any GBE-pretreating group was higher than non-pretreating group (treated only with TCE) (P<0.01), especially in 100 and 150 mg/L GBE-pretreating group, cell viability were as same as vehicle control (P>0.05).②Inhibition ratio of mitochondrial enzyme: While treating for 4 h or 8 h, 0.125 mmol/L TCE didn't cause change of inhibition ratio of mitochondrial enzyme, differences had statistical significance only between 0.5 and 2.0 mmol/L TCE group with vehicle group(P<0.01). While treating for 12 and 24h, inhibition ratio of mitochondrial enzyme of all three TCE-treating group had statistical significance compare with vehicle control (P<0.01). The inhibition lightened while pre-treating with GBE, 100 and 150 mg/L GBE pre-treatment fully protected mitochondrial enzyme from being inhibited (compared with medium, P>0.05).③ATPase activity: cells treated with 0.125, 0.5, or 2.0 mmol/L TCE for 4 h, 0.125 or 0.5 mmol/L TCE for 8 h, didn't have changes of ATPase activity compared with vehicle control (P>0.05). 2.0 mmol/L TCE×8 h treatment decreased ATPase activity(P<0.05). 0.125, 0.5, or 2.0 mmol/L TCE incubated with TCE for 12 h and 24 h, caused a dose-dependently decrease of ATPase activity. 10 mg/L GBE didn't have protective effect. ATPase activity in 50 mg/L GBE-pretreating group was higher than TCE-treating group. In 100 and 150 mg/L GBE pretreating group, ATPase activities were as the same level as vehicle control (P>0.05).④Change of ?Ψm: FCM results showed that after treating with 2.0 mmol/L TCE, fluorescences density of Rh123 in 4, 8, 12 and 24 h group were lower than control group (P<0.01). But after 8 h, Rh123 maintained at the level equal to that of 8 h. The same phenomena also emerged in Rh123-/PI- cell proportion. The results also showed that Rh123-/PI- cell proportion and fluorescence density of Rh123 changed with dose of TCE. On the other hand, GBE pretreatment elevated fluorescence intensity of Rh123 and decreased Rh123-/PI- cell proportion, especially in 100 and 150 mg/L GBE-pretreating group (compared with 2.0 mmol/L TCE group, P<0.01; with control group, P>0.05).⑤Mitochondrial morphology changes: Under TEM, mitochondria in TCE-treated group appeared extensive swelling and vacuolar degeneration with less matrix and obscure or vanished mitochondria cristae; but in control group and GBE-pretreating group, mitochondrial structure was integrity, with uniform matrix and visible mitochondria cristae.Conclusion Current in vitro study showed that trichloroethylene can decrease keratinocytes'viability, ATPase activity and inhibit mitochondrial enzyme, as a result, dysfunction of cellular and mitochondrial energy metabolism, as well as caused diminish of mitochondrial membrane potential, mitochondria swelling and damage. Results also demonstrated that ginkgo biloba extract can protect mitochondria and keratinocyte from damage caused by trichloroethylene. We concluded that mitochondria damage contribute to cytotoxicity of trichloroethylene and may play an important role in its dermal toxicity, ginkgo biloba extract can be an valid candidate for development of effective protect agent aim to related occupational dermatitis.