<i>In vitro</i> measurement of carcinogen‐resistant liver cells during hepatocarcinogenesis

Abstract

Abstract Following the transfer of rat liver cells from 2‐acetylaminofluorene‐induced, grey‐white hyper‐plastic nodules to a primary, monolayer cell culture system, the proportion of the nodule cells which were resistant to the cytotoxic effects of aflatoxin B1 was determined The proportion of the resistant nodule cells was expressed as a percentage of the number of attached, viable nodule cells remaining after aflatoxin B1 treatment divided by the number of attached, viable nodule cells remaining without aflatoxin B1 treatment. In each experiment, the results were compared with the proportion of resistant liver cells derived from normal, untreated rats. Following a 3‐h attachment period, nodule and normal cell cultures were exposed to aflatoxin B1 concentrations of 10 −7 mol/l to 10 −5 mol/l for either 24 h or 48 h. Trypan blue was added directly to the culture flasks and viability was scored as the number of attached cells that would not stain with trypan blue. The resistant nodule cell populations demonstrated a remarkable insensitivity to increasing concentrations of aflatoxin B1 compared with resistant normal cell populations. At all doses of aflatoxin B1 tested, the size of the resistant nodule population remained comparatively constant for periods of treatment varying from 24 to 48 h, whereas the size of the resistant normal population diminished greatly over periods from 24 h to 48 h. Morphologic differences between normal and nodule hepatocytes were great following aflatoxin B1 treatment. Aflatoxin B1 treatment inhibited the spreading of normal hepatocytes, whereas the highly resistant nodule hepatocytes continued to spread out on the plastic substratum. Supplementation of the culture medium with dexamethasone routinely increased the proportion of resistant normal cells exposed to some concentrations of aflatoxin B1 but did not significantly increase the size of the resistant nodule cell population.