| Hydroquinone (HQ) is a widely used solvent in industry and daily life. Its exposure is mainly to those industrial workers or people who use HQ containing cosmetic or medicine, also HQ is one product of benzene metabolizations, and it plays important roles in benzene leukemia toxicity. HQ could be absorbed from skin, alimentary canal and respiratory system, mainly metabolized in liver, then distributed all over the body. It combines with glucuronate or sulfate and be expeled from urine, but partly could cumulate in bone marrow where it is further oxidated to p-benzoquinone. Human epidemiology and animal experiments both find HQ induces toxic effects to body. It could do harms to liver, kidney, bone marrow and hematopoiesis. HQ also induces injury of conjunctiva and cornea, eyesight falling, and skin stimulation such as erythema, pruritus, and permanent trauma, etc.The mechanism of HQ toxicity has been studied a lot, and mainly focus on the gene level, such as DNA break, chromosome aberration, DNA adduction. The deficiency is they all confined to one or a few targets, and couldn't give a fully description of mechanism. Recently, as science and technology develops, the analysis of cellular proteins expressed by genome, in a cell or a tissue, termed proteomics becomes a hot spot. High-throughput screening techniques (HTSTs) of transcription and proteomics have been proposed as an ideal approach to non-selectively gather information about the effects of environmental stimuli on cell gene transcription and protein expression., and it also represents a powerful analytic technology to enhance the study of the diagnosis, treatment and prevention of human disease. Combination of techniques including two-dimensional gel electrophoresis (2-DE), mass spectrometry (MS) and bioinformatics. Proteomics can be expected to show the changes in the protein expression profile during disease development and progression, thus leading to the identification of new molecular markers and potential therapeutic targets.So in the present study, we employed 2-DE to determine whether HQ stimulation can change protein expression profile in L-02 cells and to screen HQ responsive proteins. Firstly, subcultured L-02 cells were treated by HQ for 24 h at a 1×10~4 mol/L concentration, while the control group was done nothing. Immediately after treatment, total cellular proteins were extracted and stored in -80℃ freezer after determining protein concentration by Bradford assay. 150 μg protein was subjected to isoelectric focusing on an IPG strip (17 cm, pH 5-8 linear), including rehydration for 12 h. The second separation was carried out on 12% SDS slab gels at 200 volt for 6.5 h when the BPB line went to the bottom of the gel. Gels were silver stained and scanned by GS-800 densitometer, then analyzed for differential expression with control in PDQuest 7.1 software package. The experiment was totally repeated 3 times with 3 repetitions for each group every time. The well-repeated differential expressed protein spots were cut from the gels and subjected to in-gel digestion with trypsin. Peptidemixtures were analyzed with a voyager-DE STR MALDI-TOF mass spectrometer using a delayed ion extraction and ion mirror reflector mass spectrometer and proteins were identified using the MASCOT software. The database (NCBInr) searches were performed using the following parameters: homo sapiens (human), trypsin digest (allowed up to 1 missed cleavage), cysteine as carbamidomethylated, methionine as oxidized, mass tolerance of 100 ppm using internal calibration.On average, about 1000 protein spots were detected in each gel. Compared with control group, 17> 18n 24 spots were significantly different in 3 times experiments, going upward or downward for at least 2 folds. The 4 well-repeated spots were identified by MALDI-TOF MS as Rho GDP dissociation inhibitor GDI alpha, 6-phosphogluconolactonase, erbB3 binding protein EBP1 and lamin A/C, isoform 1 precursor. They are involved in cell skeleton, signal transduction and energy metabolization in functional classification.2-DE of L-02 cells exposed to HQ revealed that protein SSP6305 was down-regulated and MS analysis identified it was EBP1. To further confirm the results, a western blot was employed with specific antibody, and showed the same.In conclusion, HQ could change the L-02 cells protein expression profile, and the 2-DE coupled with MS is an effective technique to screen the differentially expressed proteins in liver cells between HQ-treated and control. These proteins may offer new insights into the mechanism of HQ toxicity. But their expression in cells of hematological system need further studies.
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