The molecular mechanism(s) underlying induction of the hepatic microso
mal cytochrome P4502E1 (2E1) by xenobiotics (e.g. ethanol and acetone)
is controversial. Proposed mechanisms include increased rates of enzy
me synthesis due to elevated 2E1 mRNA levels, enhanced translation of
pre-existing mRNA, or stabilization of 2E1 protein. To further assess
which, if any, of these events predominates during the initial stages
of 2E1 protein induction, we investigated the effects of acetone treat
ment on 2E1 content in cultured rabbit hepatocytes, an in vitro system
that allows for precise control of the cellular mileau. Hepatocytes h
arvested from female rabbits and plated on plastic dishes with serum-s
upplemented medium were 90-100% viable for at least 48 hr in culture.
Analysis of immunoreactive 2E1 content and aniline hydroxylase activit
y in microsomes isolated from hepatocytes cultured for up to 24 hr rev
ealed that 2E1 expression was equal to that of microsomes from unplate
d cells and by 48 hr of culture, 2E1 levels decreased by only 35%. Mor
eover, microsomes isolated from cells exposed to 17 mM acetone for 24
hr exhibited a 53 and 62% increase in aniline hydroxylase activity and
2E1 content, respectively, compared to untreated cells. To explain th
ese increases, the rate of 2E1 protein synthesis was determined in unt
reated cells or in cells treated with 17 mM acetone by first exposing
hepatocytes to medium supplemented with S-35-labeled methionine and cy
steine ([S-35]Met/Cys) and subsequently assessing radiolabel incorpora
tion into 2E1 protein. While no difference was found between untreated
and acetone-treated cells in the incorporation of [S-35]Met/Cys into
trichloroacetic acid-precipitable microsomal proteins, immunoaffinity
purification of 2E1 revealed that incorporation of S-35-labeled amino
acids specifically into 2E1 was elevated by acetone to 200% of control
values. Treatment of hepatocytes with the transcriptional inhibitor,
alpha-amanitin, markedly inhibited this acetone-mediated increase in [
S-35]Met/Cys incorporation into 2E1. Analysis of hepatocyte RNA reveal
ed that acetone increased 2E1 mRNA to 130 and 160% of control levels a
t 6 and 24 hr, respectively, and that these increases were prevented b
y pretreatment with alpha-amanitin. Our results indicate that acetone
increases 2E1 protein levels in cultured rabbit hepatocytes by stimula
ting its rate of de novo synthesis. Since this increase in 2E1 synthes
is stems, at least in part, from the acetone-mediated enhancement of h
epatocyte 2E1 mRNA content and is inhibitable by alpha-amanitin, trans
criptional activation of the rabbit CYP2E1 gene is apparently involved
in the induction of 2E1 protein by acetone.