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

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).

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.

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.

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.