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Cytotoxicity and modulation of gene expression in adult rat hepatocytes by benzo(a)pyrene and related aromatic hydrocarbons
The goal of the present study was to define the cellular and
molecular targets of BaP and related AHs in liver
parenchymal cells, and to define the relative contribution of
AhR-dependent events and/or free radical mechanisms in the
responses elicited by these chemicals. The cytotoxic
responses of rat hepatocytes to selected AHs were assessed
to determine if class-specific differences in toxicity
potential exist among these chemicals. Hepatocytes were
exposed to various concentrations of BaP, TCDD, TCDF, NAPH,
2-MNAPH, or PCP for designated times. 3H-Thymidine
incorporation into DNA, cellular levels of GSH, mitochondrial
membrane fragility, and LDH leakage were monitored as indices
of cytotoxicity. The data indicated that BaP, TCDD and TCDF
induced a concentration-dependent inhibition of DNA
synthesis, while NAPH, 2-MNAPH, and PCP were without effect.
Significant increases in GSH levels were induced by BaP at
all concentrations, while only modest increases occurred in
cultures treated with TCDD or TCDF. Upregulation of cellular
GSH levels in treated cultures was preceded by depletion at 6
to 18 hr. All other hydrocarbons failed to modulate cellular
GSH levels significantly, Only BaP and PCP induced
significant increases of mitochondrial membrane fragility
after exposure for 4 hr, while LDH leakage was only observed
in hepatocytes treated with PCP at 1000 []m. To study
molecular targets of AHs in hepatocytes, we investigated the
modulation of protooncogene expression induced by BaP or
TCDD. Hepatocytes were exposed to 3 []M BaP or 1 nM TCDD for
4 hr and patterns of c-fos, c-jun, c-myc, and c-Ha-ras
expression monitored by Northern analysis. BaP and TCDD
increased c-ras and c-myc gene expression, but decreased c-
fos and c-jun mRNA levels. Inhibition of AP1-TRE binding was
observed using a gel shift assay when hepatocytes were
exposed to 3 []M BaP, but not 1 nM TCDD for 2 hr. To
investigate the contribution of AhR-dependent events to the
responses elicited by BaP and related AHs, the effects of AhR
antagonists ([]-naphthoflavone) or of inhibitors of CYPlAl-
catalyzed metabolism of AHs (ellipticine), as well as AH
analogs of low affinity for the Ah receptor (e.g. BeP and
perylene) on selected endpoints were examined. The results
of these studies implicated the AhR in the inhibition of DNA
synthesis and cellular GSH levels, as well as in the
modulation of protooncogene expression. In studies to assess
the role of free radical mechanisms in the hepatotoxic
effects of BaP and related AHs, we found that treatment of
cells with diamide to induce oxidative stress and
downregulate antioxidant capacity increased mitochondrial
membrane fragility and protooncogene expression in a manner
consistent with the pattern induced by BaP. Collectively,
these data suggest that the hepatotoxic effects of BaP and
related AHs involve AhR-, as well as oxidative stress-
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Includes bibliographical references: pages 95-128.