ORGAN TOXICITY AND MECHANISMS
Yoshio Nakagawa Æ Sumiko Tayama
Metabolism and cytotoxicity of bisphenol A and other bisphenols
in isolated rat hepatocytes
Received: 25 October 1999 /Accepted: 31 January 2000
Abstract The relation between the metabolism and the
cytotoxic e€ects of bisphenol A (BPA, 2,2-bis(4-hy-
droxyphenyl)propane) has been studied in freshly iso-
lated rat hepatocytes and isolated hepatic mitochondria.
The incubation of hepatocytes with BPA (0.25–1.0 mM)
elicited a concentration- and time-dependent cell death,
accompanied by losses of intracellular ATP and total
adenine nucleotide pools. BPA at a low-toxic level (0.25
mM) in the hepatocyte suspensions was rapidly con-
verted to its major conjugate, BPA-glucuronide, and
other minor products without marked loss of cell via-
bility, although at a toxic level (0.5 mM), more than
65% of the compound presented in an unaltered form
2 h after the incubation. Addition of salicylamide (2
mM), non-toxic to hepatocytes during the incubation
period, enhanced BPA-induced cytotoxicity and reduced
the loss of BPA and the formation of BPA-glucuronide.
The addition of BPA to isolated hepatic mitochondria
caused a concentration (0–0.5 mM)-dependent increase
in the rate of state 4 oxygen consumption in the presence
of an FAD-linked substrate (succinate), indicating an
uncoupling e€ect, whereas the rate of state 3 oxygen
consumption was inhibited by BPA. Further, the addi-
tion of BPA (0.25 mM) reduced state 3 respiration with
NAD
+
-linked substrates (pyruvate plus malate) and/or
with the FAD-linked substrate, whereas state 3 respi-
ration with ascorbate plus tetramethyl-p-phenylenedi-
amine (cytochrome oxidase-linked respiration) was not
significantly a€ected by BPA. A comparative study of
the toxic e€ects of BPA and some bisphenols on cell
viability (at 1.0 mM) and mitochondrial respiration (at
0.25 mM) revealed that 4,4¢-(1,2-diethyl-1,2-ethe-
nediyl)bisphenol (diethylstilbestrol) was more toxic than
BPA, followed by 4,4¢-methylenediphenol and 4,4¢-bi-
phenol. These results indicate that the onset of cyto-
toxicity caused by BPA may depend on the intracellular
energy status and that mitochondria are important tar-
gets of the compound. The toxicity caused by the inhi-
bition of ATP synthesis may be related to the
concentration of unmetabolised free BPA remaining in
the cell suspensions. In addition, the toxic potency of
bisphenols to hepatocytes and mitochondria depends on
the relative elongation and/or molecular size of the hy-
drocarbon bridge between the phenolic groups.
Key words Bisphenol A Æ Mitochondrial
dysfunction Æ Metabolites Æ Cytotoxicity Æ Rat
hepatocytes
Introduction
Bisphenol A (BPA; 2,2-bis(4-hydroxyphenyl)propane) is
an important monomer used in the manufacture of a
multitude of chemical products, including epoxy resins
and polycarbonate. However, it is known that trace
amounts of BPA are eluted from polycarbonate plas-
ticware and from resins used for food packaging mate-
rials for industrial processing and consumer use.
Because of its widespread usage, the potential toxicity of
BPA has been investigated in vivo and in vitro to assess
various toxicological and xeno-oestrogenic properties. It
has reported that absorption of large amounts of BPA
through the skin causes extensive damage to the liver,
kidneys, and other organs in humans (Sax 1985). In rats
and mice, oral administration of BPA may be associated
with adverse reproductive e€ects (Bond et al. 1980;
Morrissey et al. 1987) or with an increased risk of cancer
of the haemopoietic system (National Toxicology Pro-
gram 1982). Furthermore, high incidences of multinu-
cleated hepatocellular giant cells, and degeneration and/
or regeneration of renal tubules were observed in male
and female mice treated with BPA (National Toxicology
Program 1982). However, the mechanisms by which
BPA elicits these adverse e€ects are not clear.
Arch Toxicol (2000) 74: 99–105 Ó Springer-Verlag 2000
Y. Nakagawa (&) Æ S. Tayama
Department of Toxicology, Tokyo Metropolitan Research
Laboratory of Public Health, 3-24-1, Hyakunin-cho,
Shinjuku-ku, Tokyo 169-0073, Japan
e-mail: yoshio@tokyo-eiken.go.jp; Fax: +81-3-33684060

In chemical structure, the bisphenolic compound
BPA is similar to the synthetic oestrogens diethylstil-
bestrol and hexestrol. Recently, considerable attention
has focused on BPA as well as other phenolic com-
pounds as endocrine disrupting chemicals having weak
oestrogenic activity in in vivo (Nagel 1997; vom Saal
et al. 1998) and in vitro (Gaido et al. 1997) bioassays.
The predominant metabolic pathway for BPA in rats is
conjugation of a phenolic hydroxyl group to glucuronide
(Knaak and Sullivan 1966). The major excretory con-
jugate is BPA-glucuronide rather than sulphate, because
the sulphate is undetectable in rat urine after the oral
administration of BPA (Knaak and Sullivan 1966). In
addition, however, some of the BPA added to rat he-
patic microsomal suspensions is converted to 5-hydroxy
bisphenol by cytochrome P-450-dependent mixed-func-
tion oxidases, and consequently the hydroxylated inter-
mediate is oxidised to bisphenol-o-quinone (Knaak and
Sullivan 1966; Atkinson and Roy 1995a). Although bi-
sphenol-o-quinone reacts with DNA and induces the
formation of DNA adducts in vivo and in vitro (At-
kinson and Roy 1995b), several in vivo and in vitro
genotoxicity studies have obtained negative results
(Ashby and Tennant 1988; British Industrial Biological
Research Association 1989; Ivett et al.1989).
Despite the fact that much is known about the met-
abolic pathway of and toxicity of BPA, no extensive
study has been conducted on the relation between the
cytotoxicity and metabolism of BPA. The isolated he-
patocyte system is rich in various drug-metabolising
enzymes and cofactors associated with Phase I and
Phase II (Molde´ us et al. 1978) and is a useful system
with which to study the intracellular target sites and
temporal sequences leading to cell damage caused by
chemicals and their metabolites. In the present study, we
investigated the action of BPA and other bisphenols on
freshly isolated rat hepatocytes and isolated hepatic
mitochondria, and discuss the mechanisms of the toxic
e€ects of BPA and its metabolism.
Materials and methods
Materials
The chemical compounds used were obtained from the following
companies: BPA and 4,4¢-biphenol (4,4¢-dihydroxybiphenyl, puri-
ties of >97%) from Tokyo Kasei Co., Tokyo, Japan; 4,4¢-methy-
lenediphenol (purity >98%) from Aldrich Chemical Co. Inc.,
Milwaukee, Wis.; 4,4¢-(1,2-diethyl-1,2-ethenediyl)bisphenol (dieth-
ylstilbestrol, purity >99%), adenine nucleotides, b-glucuronidase,
sulphatase and bovine serum albumin from Sigma Chemical Co.,
St. Louis, Mo.; collagenase and salicylamide from Wako Pure
Chemicals, Osaka, Japan. All other chemicals were of the highest
purity commercially available. The chemical structures of bisphe-
nols used in this study are shown in Fig. 1.
Isolation and incubation of hepatocytes
Male F344/DuCrj (240–260 g) rats were obtained from Charles
River Japan Inc., Yokohama, Japan and were housed in wire-
bottom cages. The rats were allowed food (CE-2, Clea Japan Inc.,
Tokyo, Japan) and water ad libitum before hepatocytes were pre-
pared. The hepatocytes were isolated by collagenase perfusion of
the liver, as described previously (Molde
´
us et al. 1978). Hepatocyte
viability was assessed by Trypan blue exclusion and initial cell vi-
ability in each experiment was more than 85%. Hepatocytes (10
6
cells/ml) were suspended in Krebs-Henseleit bu€er, pH 7.4, con-
taining 12.5 mM HEPES and 0.1% albumin. All incubations were
performed in rotating, round-bottomed flasks at 37 °C, under a
constant flow of humidified carbogen (95% O
2
/5% CO
2
). Reac-
tions were started by the addition of BPA or other bisphenols
dissolved in DMSO (final concentration, <1%). Corresponding
control groups received an equivalent volume of DMSO. In some
experiments using salicylamide as a inhibitor of glucuronide and/or
sulphate conjugation (Burke et al. 1977; Orrenius et al. 1978), the
compound (2 mM) dissolved in DMSO was added to the hepato-
cyte suspension 30 min before the addition of BPA. Aliquots of
incubation mixture were taken at intervals to monitor cell death
and the concentrations of intracellular adenine nucleotides, and
BPA and its metabolites.
Preparation of liver mitochondria and measurement
of respiration rates of mitochondria
Liver mitochondria were isolated from male F344/DuCrj rats by
di€erential centrifugation in medium containing 0.25 M sucrose, 5
mM Tris-HCl, pH 7.4, and 1 mM EDTA (Cain and Skilleter 1987).
EDTA was omitted for the final wash and resuspension. The rate of
oxygen consumption was measured polarographically with a
Clark-type oxygen electrode (model 5300; Yellow Springs Instru-
ments) at 30 °C in the presence (state 3) and after exhaustion (state
4) of 50 lM ADP (Cain and Skilleter 1987). Respiration bu€er (3
ml, pH 7.4) contained 0.2 M sucrose, 20 mM KCl, 3 mM MgCl
2
,
and 5 mM potassium phosphate. The respiration substrates were
5 mM pyruvate plus 0.5 mM malate, 5 mM succinate containing
1 lM rotenone for inhibition of NAD
+
-linked oxidation, or 1 mM
ascorbate plus 50 M tetramethyl-p-phenylenediamine (TMPD)
containing 1 lM rotenone/50 nM antimycin A for inhibition of
electron transport at complex IV (cytochrome c reductase), and the
amount of mitochondria was 1 mg of protein/ml. The respiratory
control index (RCI) was calculated as the ratio of state 3/state 4
respiration.
Biochemical assays
Adenine nucleotides (ATP, ADP, and AMP) in hepatocytes were
measured using HPLC according to Jones (1981). Protein was de-
termined by the method of Lowry et al. (1951) using bovine serum
albumin as standard. Cell death of hepatocytes was assessed by
Trypan blue (0.16%, w/v) uptake under light microscopy (Molde
´
us
Fig. 1A–D Chemical structures of BPA and other bisphenols utilized
in this study. (A) 4,4¢-biphenol, (B) 4,4¢-methylenediphenol, (C) BPA
and (D) diethylstilbestrol
100