Short-term inhalation exposure of B6C3F1 mice to
styrene causes
necrosis of centrilobular (CL) hepatocytes. However, in spite of continued exposure, the necrotic parenchyma is rapidly regenerated, indicating resistance by regenerated cells to
styrene toxicity. These studies were conducted to test the hypothesis that resistance to repeated
styrene exposure is due to sustained cell proliferation, with production of hepatocytes that have reduced metabolic capacity. Male mice were exposed to air or 500 ppm
styrene (6 h/day); hepatotoxicity was evaluated by microscopic examination, serum liver
enzyme levels, and
bromodeoxyuridine (
BrdU)-labeling index (LI). Metabolism was assessed by measurement of blood
styrene and
styrene oxide. Both single and repeated exposures to
styrene resulted in mortality by Day 2; in mice that survived, there was CL
necrosis with elevated
BrdU LI at Day 6, and complete restoration of the necrotic parenchyma by Day 15. The
BrdU LI in mice given a single exposure had returned to control levels by Day 15. Re-exposure of these mice on Day 15 resulted in additional mortality and hepatocellular
necrosis, indicating that regenerated CL cells were again susceptible to the cytolethal effect of
styrene following a 14-day recovery. However, in mice repeatedly exposed to
styrene for 14 days, the
BrdU LI remained significantly increased on Day 15, with preferential labeling of CL hepatocytes with enlarged nuclei (karyomegaly). If repeated exposures were followed by a 10-day recovery period, CL karyomegaly persisted, but the
BrdU LI returned to control level and CL hepatocytes became susceptible again to
styrene toxicity as demonstrated by additional mortality and acute
necrosis after a challenge exposure. These findings indicated a requirement for continued
styrene exposure and
DNA synthesis in order to maintain this resistant phenotype. Analyses of
proliferating-cell nuclear-antigen (
PCNA) labeling were conducted to further characterize the cell cycle kinetics of these hepatocytes. The proportion of cells in S-phase was increased by repeated exposure. However,
PCNA analysis also revealed an even larger increase in the G1 cell compartment with repeated exposures, without a concurrent increase in G2 phase or in mitotic cell numbers. These data indicate that resistance to
styrene-induced
necrosis under conditions of repeated exposure is not due to sustained cell turnover and production of new, metabolically inactive cells, but rather is due to some other, as yet unknown, protective phenotype of the regenerated cells.