[Optimization of primary hepatocytes model and study on the cytotoxicity of styrene and styrene oxide].

OBJECTIVE: To establish a model in vitro for primary cultured mouse hepatocytes with high viability and function, and evaluate the acute toxicity of the primary hepatocytes exposed to the chemicals such as styrene and styrene oxide (SO). METHODS: Based on the classical method, the two-step collagenase digestion method was optimized by reverse and intermittent perfusion, restriction of digestion time as well as purification of percoll liquid. Hepatocytes were isolated from BALB/C mouse by an improved isolated method and then cultured in monolayer and sandwich configuration. The primary cultured hepatocytes model was assessed by various indexes including cell morphology, cell viability, intracellular glycogen granules, as well as albumin (ALB), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and blood urine nitrogen (BUN) levels in the supernatant. In addition, the primary cultured hepatocytes were treated with various concentrations from 0.2 to 25 micromol/L of styrene and styrene oxide during different time from 3 to 48 hours. The cytotoxicity induced by the two toxicants was assessed by CCK-8 and LDH assays. RESULTS: On average, the isolation using this improved method resulted in the cell viability of (90.3 +/- 5.2) %, the cell purity of (95.3 +/- 4.2)% and the yield of (2.4 +/- 0.9) x 10(7) viable cells. More than 90% cells showed a typical morphological feature of hepatocytes in sandwich configuration within 7 days, and contained a large number of glycogen granules on the third day. The ALB secretion, ALT and LDH leakage and BUN synthesis as well as cell viability fluctuated during 8 days, and they stayed at stable levels between 3 to 7 days in sandwich configuration. But they fluctuated during 6 days in monolayer configuration. In comparison with the monolayer configuration, the levels of ALB and BUN were distinctly increased and the levels of LDH and ALT were significantly decreased in sandwich configuration. The levels of ALB [ (1.42 +/- 0.20) g/L ] and BUN [(1.97 +/- 0.22) mmol/L] as well as cell viability were the highest, while the levels of LDH [ (7.30 +/- 2.33) U/L] and ALT [ (6.51 +/- 1.86) U/L] were the lowest in sandwich configuration on the third day. The relative low cytotoxicity and high cell survival rate ( more than 90%) were shown in treated hepatocytes with styrene and styrene oxide within 6 hours by CCK-8 and LDH measurements, and there was no distinct difference in the determination of cytotoxicity between the two methods. With the prolonged exposure time, the cell survival rate was lower by CCK-8 assay (less than 85%) than the one by LDH assay. The relative obvious cytotoxicity and low cell survival rate (about 85%) by CCK-8 method were revealed in treated cells with 5 micromol/L of styrene and styrene oxide for 24 hours, but there was no significant difference between CCK-8 and LDH assays. With the increase of the concentrations, the cell survival rate was lower by CCK-8 assay (less than 80%) compared with LDH assay. CONCLUSION: The improved two-step collagenase digestion method combination with sandwich culture method might maintain the morphology and function of primary cultured mouse hepatocytes for seven days. The cytotoxic effects of styrene and styrene oxide might be accurately evaluated by means of primary cultured hepatocyte model from 3 to 7 days. The chemicals might have major adverse effects on the functions of the organelles in hepatocytes such as mitochondria, but little influence to the cell membrane damage.

[Effect of occupational exposure to toluene diisocyanate on workers' health].

OBJECTIVE: To investigate the effect of occupational exposure to toluene diisocyanate (TDI) on the workers' health. METHODS: A total of 76 workers exposed to TDI (exposure group) and 64 management staff members (control group) were selected from a factory as the study subjects. Area sampling was performed for the place with exposure to TDI according to the method in GBZ 159-2004 Specifications of air sampling for hazardous substances monitoring in the workplace, and gas chromatography was applied to measure the concentration of TDI in workplace air. The workers' personal information was collected with questionnaire, pulmonary ventilation function was determined with a portable spirometer, hematological parameters were analyzed by automatic blood analyzer and blood chemistry analyzer, and the indicators of oxidative damage and energy metabolism were measured by the reagent kit provided by Nanjing Jiancheng Bioengineering Institute. SPSS 17 software was applied for statistical analysis. RESULTS: The exposure group had significantly lower forced vital capacity (FVC), forced expiratory volume in 1 second(FEV1.0), and FEV1.0/FVC ratio than the control group (P <0.05). Compared with the control group, the exposure group had significantly higher red blood cell count, platelet distribution width, mean platelet volume, lymphocyte count, and neutrophil count(P<0.01), and significantly lower activities of lactate dehydrogenase(LDH), superoxide dismutase, and succinodehydrogenase (SDH)(P <0.01). In the exposure group, the length of exposure was negatively correlated with the activities of SDH and LDH in the serum (r=-0.319, P <0.05; r=-0.239, P <0.05), and the length of exposure was not found to be correlated with the activity of SOD and pulmonary function indices. CONCLUSION: TDI can induce inflammatory response and lung ventilation function impairment in workers exposed to TDI, as well as oxidative stress and imbalance of energy metabolism. Therefore, it can cause damage to workers' health, and protective measures should be enhanced.

The Distribution Pattern and Species Richness of Scorpionflies (Mecoptera: Panorpidae).

The uneven distribution of species diversity on earth, with mountainous regions housing half of the high species diversity areas, makes mountain ecosystems vital to biodiversity conservation. The Panorpidae are ecological indicators, ideal for studying the impact of climate change on potential insect distribution. This study examines the impact of environmental factors on the distribution of the Panorpidae and analyzes how their distribution has changed over three historical periods, the Last Interglacial (LIG), the Last Glacial Maximum (LGM), and Current. The MaxEnt model is used to predict the potential distribution area of Panorpidae based on global distribution data. The results show that precipitation and elevation are the primary factors affecting species richness, and the suitable areas for Panorpidae are distributed in southeastern North America, Europe, and southeastern Asia. Throughout the three historical periods, there was an initial increase followed by a decrease in the area of suitable habitats. During the LGM period, there was a maximum range of suitable habitats for cool-adapted insects, such as scorpionflies. Under the scenarios of global warming, the suitable habitats for Panorpidae would shrink, posing a challenge to the conservation of biodiversity. The study provides insights into the potential geographic range of Panorpidae and helps understand the impact of climate change on their distribution.