0022-3565/02/3002-273–281$3.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 300:273–281, 2002
Vol. 300, No. 2
4213/952624
Printed in U.S.A.
Mechanisms of Circadian Rhythmicity of Carbon Tetrachloride
Hepatotoxicity
JAMES V. BRUCKNER, RAGHUPATHY RAMANATHAN,1 K. MONICA LEE,2 and SRINIVASA MURALIDHARA
Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, the University of Georgia, Athens, Georgia
Received June 7, 2001; accepted September 21, 2001
This paper is available online at http://jpet.aspetjournals.org
Many physiological and biochemical processes in laboratory animals and humans have been found to vary rhythmically over a 24-h period (i.e., to exhibit a circadian or diurnal
rhythm). Rhythms are considered as sequences of biological
events, repeating themselves in the same order and at the
same intervals. Temporal differences in drug action and toxicity have been recognized for over 50 years in humans and in
laboratory animals. A number of cyclic physiological processes can affect the absorption, distribution, metabolism,
and elimination of drugs and other chemicals (Labrecque and
Belanger, 1991).
This work was supported by U.S. Environmental Protection Agency (EPA)
Cooperative Agreement CR-811215 (to J.V.B). As the work described in the
paper has not been peer reviewed or policy reviewed by the EPA and does not
necessarily reflect the views of the EPA, no official endorsement should be
inferred. This article was presented in part at the 8th International Conference on Chronopharmacology and Chronotherapeutics, Williamsburg, VA, August 24 –27, 1999 and at the Society of Toxicology Meeting, San Francisco, CA,
March 26 –29, 2001.
1
Present address: Wyle Laboratories, Life Sciences, 1290 Hercules Drive
120, Houston, TX 77058.
2
Present address: Toxicology Northwest MS K4-10, Battelle PO Box 999,
Richland, WA 99352.
dosed with CCl4 near the beginning of their dark/active cycle.
Blood acetone, hepatic CYP2E1 activity, and covalent binding
of 14CCl4/metabolites to hepatic microsomal proteins in untreated rats fed ad libitum followed circadian rhythms similar to
that of susceptibility to CCl4. Parallel fluctuations of greater
amplitude were seen in rats fasted for 24 h. Hepatic glutathione
levels were lowest at the time of greatest susceptibility to CCl4.
Acetone dose-response experiments showed high correlations
between blood acetone levels, CYP2E1 induction, and CCl4induced liver injury. Pretreatment with diallyl sulfide suppressed
CYP2E1 and abolished the circadian rhythmicity of susceptibility to CCl4. These findings provide additional support for
acetone’s physiological role in CYP2E1 induction and for
CYP2E1’s role in modulating CCl4 chronotoxicity in rats.
The chronotoxicity of solvents and other industrial chemicals has received relatively little attention. Volatile organic
compounds (VOCs) are a class of solvents to which many
people are exposed occupationally and environmentally.
Early studies of chloroform (Lavigne et al., 1983), carbon
tetrachloride (CCl4) (Harris and Anders, 1980; Bruckner et
al., 1984), and 1,1-dichloroethylene (Jaeger et al., 1973) revealed circadian rhythms in susceptibility of rats to liver
damage by the chemicals. Some of the investigators speculated that periods of enhanced susceptibility might be due to
cyclic increases in metabolic activation of the VOCs. Temporal variations in P450-catalyzed metabolism of a variety of
substrates by rat liver microsomes have been reported, but
correlations of substrate metabolism with P450 levels have
generally been poor (Labrecque and Belanger, 1991).
Miyazaki et al. (1990) described the time dependence of several P450 isozymes active in catalyzing testosterone hydroxylation in rat liver in a preliminary report. Furukawa et al.
(1999) observed high alkoxycoumarin O-dealkylase activities
in the liver of rats sacrificed during the animals’ dark cycle
but relatively stable total P450 levels. It is possible that
ABBREVIATIONS: VOCs, volatile organic compounds; P450, cytochrome P450; CYP2E1, cytochrome P450IIE1; GSH, glutathione; S-D, SpragueDawley; DAS, diallyl sulfide; ALT, alanine aminotransferase; SDH, sorbitol dehydrogenase; ICD, isocitrate dehydrogenase; AUC, area under the
curve; GC, gas chromatograph; NPSH, nonprotein sulfhydryl; GSHPx, glutathione peroxidase; PNP, p-nitrophenol; 4-NC, 4-nitrocatechol; ␤HB,
␤-hydroxybutyrate; AA, acetoacetate.
273
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ABSTRACT
The toxicity of carbon tetrachloride (CCl4) and certain other
chemicals varies over a 24-h period. Because the metabolism
of some drugs follows a diurnal rhythm, it was decided to
investigate whether the hepatic metabolic activation of CCl4
was rhythmic and coincided in time with maximum susceptibility to CCl4 hepatotoxicity. A related objective was to test the
hypothesis that abstinence from food during the sleep cycle
results in lipolysis and formation of acetone, which participates
in induction of liver microsomal cytochrome P450IIE1
(CYP2E1), resulting in a diurnal increase in CCl4 metabolic
activation and acute liver injury. Groups of fed and fasted male
Sprague-Dawley rats were given a single oral dose of 800 mg of
CCl4/kg at 2- to 4-h intervals over a 24-h period. Serum enzyme
activities, measured 24 h post dosing as indices of acute liver
injury, exhibited distinct maxima in both fed and fasted animals
274
Bruckner et al.
Materials and Methods
Chemicals. Analytical-grade CCl4 was purchased from J. T.
Baker Chemical Co. (Philadelphia, PA). 14CCl4 with specific activities of 4 and 61 mCi/mmol was supplied by PerkinElmer Life Sciences (Boston, MA) and Amersham Pharmacia Biotech (Arlington
Heights, IN), respectively. Carbasorb was purchased from Packard
Instrument Co. (Downers Grove, IL). All other chemicals and biochemicals were obtained from Sigma Chemical Co. (St. Louis, MO).
Animals. Male Sprague-Dawley (S-D) rats (250 –265 g) were purchased from Harlan Sprague Dawley Inc. (Indianapolis, IN). The
animals were housed in their own room in an American Association
for the Accreditation of Laboratory Animal Care-approved animal
care facility. The experimental protocol was reviewed and approved
by an institutional animal care committee. The rats were acclimated
to a 12-h light/dark cycle (light 6:00 AM– 6:00 PM, i.e., 0600 –1800 h)
in a temperature (25°C)- and humidity (40%)-controlled room for at
least 2 weeks prior to use. Animals were housed in groups of six in
stainless steel cages in a ventilated rack. Tap water and food (Purina
Rat Chow 5001; Purina, St. Louis, MO) were available ad libitum to
some animals. Other groups of rats were fasted for 24 h prior to
blood/tissue sampling or CCl4 dosing in experiments requiring fasting. Food was withheld from all animals for 3 h post dosing, but
water was provided at all times.
CCl4 Dosing and Blood Sampling. The body weight of animals
at the time of experimentation ranged from 320 to 360 g. Appropriate
amounts of CCl4 were diluted in corn oil, such that an oral dose of
800 mg of CCl4/kg b.wt. could be given in a total volume of 1.5 ml/kg.
Randomized groups of rats were administered a single dose of 800
mg of CCl4/kg by gavage, at 2- to 4-h intervals over a 24-h period. A
curved, ball-tipped intubation needle was used to give the oral bolus
dose. Controls similarly received 1.5 ml/kg of corn oil. The rats were
sacrificed 24 h after CCl4 dosing. Blood was drawn by cardiac puncture, and serum was obtained for assays of enzyme activities.
In a follow-up experiment, nonfasted rats received 20 mg of diallyl
sulfide (DAS)/kg p.o. in corn oil 12 h before CCl4. The choice of the 20
mg/kg p.o. dose of DAS was based upon the results of pilot time
course and dose-response experiments, in which maximal inhibition
of hepatic CYP2E1 activity (65–70% decrease from control) was
manifest in rats 12 to 24 h after oral administration of 20 to 40 mg
of DAS/kg (data not shown). Groups of the DAS-pretreated rats were
gavaged with 800 mg of CCl4/kg in corn oil at 4-h intervals over a
24-h period. Blood was withdrawn by cardiac puncture for serum
enzyme measurements 24 h after CCl4 administration.
Serum Enzyme Assays. The activities of three enzymes in serum
were monitored as measures of hepatocellular damage by CCl4.
Alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH), and
isocitrate dehydrogenase (ICD) activities were measured by standard spectrophotometric techniques. ALT, SDH, and ICD are each
expressed as milliunits per milliliter of serum, in which 1 unit of
activity is defined as 1 ␮mol of NAD(H) converted/min/ml of serum.
Acetone Dose-Response Study. The first of a set of experiments
was conducted to determine the area under the blood concentration
versus time curve (AUC) for each of a series of doses of acetone. An
indwelling cannula was surgically implanted into the right carotid
artery of rats of 350 to 375 g. The cannula was passed under the skin
and exteriorized at the nape of the neck. Food and water were
supplied ad libitum during a 24-h recovery period. Each group of the
unanesthetized, freely moving animals was given one of the following doses of acetone by corn oil gavage between 9:00 and 10:00 AM:
100, 250, 500, 1000, or 2000 mg/kg. Serial blood samples of 5 to 50 ␮l
were taken at intervals of 2 to 120 min for 8 h post dosing and
analyzed for their acetone content as described below.
The dose dependence of hepatic CYP2E1 induction by acetone was
evaluated in a second experiment. Groups of six nonfasted rats of
⬃280 g were given 0, 50, 100, 250, 500, 1000, or 2000 mg of acetone/kg by corn oil gavage. The rats were dosed between 9:00 and
10:00 AM, a time when we found that hepatic CYP2E1 activity was
relatively low. The animals were sacrificed 24 h later, so their liver
could be processed and microsomal CYP2E1 activity measured as
described below.
The objective of the third experiment was to assess the dose
dependence of potentiation of CCl4 acute hepatotoxicity by acetone.
Groups of nonfasted rats of ⬃280 g were administered 0, 50, 100,
250, 500, 1000, or 2000 mg of acetone/kg by corn oil gavage between
9:00 and 10:00 AM. After 24 h they were given 800 mg of CCl4/kg by
corn oil gavage. The animals were sacrificed 24 h after CCl4 and
blood was taken by cardiac puncture for assay of SDH activity, as
detailed above.
Acetone Analysis. Blood samples of 5 to 50 ␮l, taken from an
indwelling carotid arterial cannula, were transferred to a 20-ml
headspace vial. The vial was immediately capped with a polytetrafluoroethylene-lined septum and tightly crimped. A PerkinElmer model
8500 gas chromatograph (GC) fitted with a flame ionization detector
and an HS-101 headspace autosampler was used. Analyses were
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diurnal rhythms in certain P450 isozymes account for the
periodicity in xenobiotic metabolism. Unfortunately, very little information relevant to this postulate is available. The
P450 substrates used by Furukawa et al. (1999) had low
isoform specificity.
Abstinence from food during the sleep cycle may be linked
to circadian rhythmicity in CCl4 metabolic activation and
acute toxicity. Fasting for 16 to 48 h is known to enhance the
metabolism and hepatotoxicity of CCl4 (Harris and Anders,
1980; Nakajima et al., 1982). Fasting has also been found to
induce hepatic microsomal cytochrome P450IIE1 (CYP2E1)
(Brown et al., 1995). CYP2E1 plays a major role in metabolism of CCl4 and other low molecular weight VOCs
(Guengerich et al., 1991; Zangar et al., 2000). It is possible
that levels of CYP2E1 vary rhythmically over a 24-h period
and that the isozyme’s activity at any given clock time will
determine the extent of CCl4 metabolic activation and hepatotoxicity. Fasting can result in significant lowering of hepatic glutathione (GSH) levels. The GSH redox cycle is
thought to play a protective role against CCl4-induced oxidative stress by metabolism of hydroperoxides and lipid hydroperoxides (Nishida et al., 1996). Fasting is also known to
result in increased circulating levels of ketone bodies, including acetone. CYP2E1 plays a major role in acetone catabolism, as demonstrated by a marked elevation in blood acetone
in diallyl sulfide-treated rats (Chen et al., 1994). Conversely,
acetone is an inducer of CYP2E1 and certain other P450s
(Johansson et al., 1988). Thus, it is possible that lack of food
intake during animals’ sleep cycle may trigger the formation
of acetone, which can contribute to CYP2E1 induction sufficiently to increase the production of cytotoxic CCl4 metabolites.
Objectives of this investigation were to 1) determine
whether acute CCl4 hepatotoxicity and hepatic microsomal
CYP2E1 exhibit predictable circadian rhythms in rats maintained on a constant light/dark cycle; 2) test the hypothesis
that abstinence from food during sleep results in lipolysis
and formation of acetone, which participates in induction of
hepatic CYP2E1, resulting in a diurnal rhythm in CCl4 metabolic activation and acute hepatotoxicity; and 3) assess the
potential contribution of circadian rhythms in glutathione
and glutathione peroxidase activity to CCl4 chronotoxicity.
Information gained from this study may be helpful in understanding the synchronization of metabolism of CCl4 and
other CYP2E1 substrates.
Mechanisms of Circadian Rhythmicity of CCl4 Hepatotoxicity
Results
Toxicity. Rats dosed with 800 mg/kg CCl4 exhibited a
pronounced circadian rhythm in susceptibility to the chemical’s hepatotoxic action. The three enzymes used as indices of
hepatocellular damage rose and fell in parallel over the 24-h
monitoring period (Fig. 1). CCl4 was most hepatotoxic to fed
rats when given at 6:00 PM, the beginning of the animals’
active/dark cycle. The chemical was least hepatotoxic during
the middle (i.e., 10:00 AM–2:00 PM) of their inactive/light
cycle (Fig. 1A). Serum enzyme levels in these subjects rose
very rapidly from a minimum at 10:00 AM to 2:00 PM to a
maximum at 6:00 PM. These increases were about 3- to
5-fold. SDH, ALT, and ICD levels did not vary significantly
during 24 h in undosed rats (data not shown). It is evident
when comparing Fig. 1A with 1B that fasted rats were much
more sensitive to CCl4 than fed rats. The diurnal rhythm of
cytotoxicity in the fasted animals had a substantially greater
amplitude. Maximal hepatocellular injury, as reflected by the
serum enzyme levels, however, occurred some 2 h sooner
than in the group fed ad libitum. The serum enzyme levels
declined more rapidly thereafter in these animals than in
their fed counterparts.
As shown in Fig. 1A, DAS pretreatment effectively blocked
acute CCl4 hepatotoxicity and abolished the diurnal rhythm
in susceptibility to the injury. Serum SDH and ALT activities
were similarly affected. The diurnal rhythm in hepatic
CYP2E1 activity, which is described below, was also suppressed (data not shown).
Metabolism. A circadian rhythm in hepatic microsomal
total P450 was not apparent in fed or in fasted rats. The P450
levels were comparable in both groups over the 24-h monitoring period (data not shown). Hepatic microsomal CYP2E1
activity, in contrast to total P450 levels, exhibited a definite
circadian rhythm (Fig. 2). CYP2E1 activity in fed rats progressively diminished during the animals’ inactive cycle,
reaching its lowest level at 2:00 PM. There was an increase in
CYP2E1 between 2:00 and 6:00 PM of ⬃35% in the fed
animals. This interval was followed by stable CYP2E1 activity for the remainder of the active cycle. In contrast, CYP2E1
diminished rapidly during the active cycle, but rose steadily
throughout the inactive cycle in the fasted rats, attaining its
maximal level at 6:00 PM, the beginning of the active cycle.
There was an increase of ⬃110% during this period (i.e., 6:00
AM– 6:00 PM). The isozyme’s activity was significantly
higher in the fasted than in the fed rats at all times except
6:00 AM.
The magnitude of covalent binding of 14CCl4/metabolites to
hepatic microsomal proteins exhibited a diurnal rhythm (Fig.
3). Binding was lowest during the inactive cycle. An ⬃80%
increase in binding occurred between 6:00 and 8:00 PM in
rats fed ad libitum. Thereafter, the level of bound radiolabel
remained elevated for the duration of the active period. Covalent binding of 14CCl4 to microsomal proteins of fasted rats
also showed a diurnal rhythm, but binding was substantially
greater than in the nonfasted animals. There was an abrupt
diurnal increase of ⬃80% in the fasted rats, with the rise
occurring some 4 h earlier than in the nonfasted subjects.
Binding remained high during the active cycle of the fasted
rats.
Acetone, CYP2E1, and Potentiation of CCl4 Toxicity.
Levels of acetone in the blood exhibited a unique circadian
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carried out on a 6⬘ ⫻ 1/8⬙ stainless steel column packed with 3%
OV-17 that was supplied by Alltech Associates (Deerfield, IL). The
temperatures for analysis were as follows: column run isothermal at
75°C, injector at 110°C, and detector at 200°C. Nitrogen was used as
the carrier gas at 5 psi. The headspace vials were heated at 110°C in
the thermostat-controlled autosampler chamber for 30 min before
being vented into the GC. Acetone was well resolved without interfering peaks. Standard solutions of acetone in distilled water were
analyzed each day as described above to derive a standard curve.
Liver Assays. The liver nonprotein sulfhydryl (NPSH) content of
freshly prepared liver homogenates from undosed rats was measured
colorimetrically. Other portions of liver were homogenized in cold
0.02 M Tris-KCl buffer at pH 7.4. Microsomes were prepared by
differential centrifugation and stored at ⫺80°C. Glutathione peroxidase (GSHPx) activity in the 105,000g supernatant was assayed
according to Lawrence and Burke (1976). The protein concentration
and total cytochrome P450 content of hepatic microsomes were determined by conventional spectrophotometric procedures. CYP2E1
activity was estimated by measuring p-nitrophenol (PNP)-hydroxylation. The hydroxylation of PNP to 4-nitrocatechol (4-NC) was determined by the procedure of Koop (1990). A substrate concentration
of 100 ␮M PNP was used in this assay. CYP2E1 activity was expressed as nmol of 4-NC/min/mg of protein.
Covalent Binding Study. Groups of undosed rats served as
liver donors for a study of covalent binding of 14CCl4 as a function
of clock time. Liver samples were taken every 2 to 4 h over a 24-h
period. Microsomes were prepared as described previously and
diluted with 0.05 M Tris-0.15 M KCl/EDTA buffer (pH 7.4) to a
microsomal protein concentration of 2 mg/ml. This preparation
was incubated with NADPH (1 mM), MgCl2 (5 mM), nicotinamide
(2.5 mM), and 400 nmol/ml 14CCl4 (0.25 ␮Ci/ml). The flasks were
capped with latex rubber septa immediately after addition of the
14
CCl4 and incubated for 20 min at 37°C. The reaction was stopped
with 2 ml of ice-cold ethanol, and the samples were centrifuged in
the cold at 1800 g for 15 min. The pellet was washed twice with
cold ethanol, dissolved in 0.5 N NaOH, neutralized with 1 N HCl,
and counted in a Beckman LS-7500 scintillation counter (Beckman Coulter, Inc., Fullerton, CA) after addition of an appropriate
scintillation cocktail. Blanks were prepared using boiled microsomes. The results are expressed as pmol of 14C bound/mg of
microsomal protein.
Safety Precautions. Studies were conducted in a biohazard facility to avoid CCl4 contamination of laboratory personnel and surrounding areas. Preparation of test solutions, dosing, sacrifices, and
blood and tissue sampling were performed under a 100% exhaust
hood. The rats were maintained in vented cage racks to avoid exposure of each other and personnel. Animal remains and other CCl4contaminated materials were sealed in double plastic bags, placed
into biohazard barrels, and disposed of by a licensed disposal firm.
14
CCl4 was used, monitored for, and disposed of in accordance with
university policy.
Statistical Analyses. Data were analyzed by one-way analysis of
variance. The significance of difference between fed and fasted
groups was determined by use of Tukey’s least significant differences
test. Area under the blood acetone concentration versus time curve
from 0 to 8 h (AUC80) values were calculated according to the linear
trapezoidal rule. The significance of apparent acetone dose-dependent differences in blood acetone AUC80, hepatic CYP2E1 activity,
and serum SDH activity were assessed by the Student-NewmanKeuls test using Modstat software by Statistics.Com (Bellingham,
WA). The minimum level of statistical significance for these tests
was p ⱕ 0.05. Correlation coefficients between AUC80 and hepatic
CYP2E1 activity and between AUC80 and serum SDH activity were
determined using Prism Version 3.02 software by GraphPad Software (San Diego, CA).
275
276
Bruckner et al.
rhythm (Fig. 4). There was a pronounced peak in the acetone
concentration at the beginning of the active cycle in both fed
and fasted rats. The blood levels rose abruptly between 2:00
and 6:00 PM and then dropped rapidly during the next 4 h.
Blood acetone concentrations were significantly higher
throughout the 24-h monitoring period in the fasted animals,
particularly at 6:00 PM.
Administration of a series of doses of acetone to rats by
corn oil gavage produced dose-dependent increases in blood
acetone concentrations, hepatic microsomal CYP2E1 activity, and serum SDH levels. The blood acetone time courses
exhibited plateaus rather than true peaks, so AUC80 was
calculated as the internal dosimeter. The AUC80 values were
directly proportional to the administered dose of acetone
(r2 ⫽ 0.99). The lowest acetone dosage, 50 mg/kg, induced
CYP2E1 and potentiated CCl4 acute hepatotoxicity (Table 1).
There were not significant increases in CYP2E1 or SDH
activities between 100 and 250 mg/kg, but there were statistically significant dose-dependent elevations in each index in
response to 500, 1000, and 2000 mg/kg. There were high
degrees of correlation between blood acetone AUC80 and
CYP2E1 activity (r2 ⫽ 0.96) and between AUC80 and serum
SDH activity (r2 ⫽ 0.95) (Fig. 5, A and B).
Nonprotein Sulfhydryls. A prominent diurnal rhythm
was seen in the NPSH content of the liver of rats fed ad
libitum (Fig. 6). Fasted rats displayed a similar rhythm of
lower amplitude. NPSH levels were significantly lower in
the fasted animals at each measurement period. Hepatic
NPSH in the fed rats diminished throughout the 12-h light
period and steadily increased during the last 10 h of the
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Fig. 1. Circadian rhythmicity of susceptibility of rats to CCl4 hepatotoxicity. Groups of rats were dosed at 2to 4-h intervals over a 24-h period
with 800 mg of CCl4/kg b.wt. SDH,
ALT, and ICD activities in serum
were measured 24 h after dosing.
Each point represents the mean ⫾
S.E. for six animals. For some
groups, S.E. bars are too small to be
visible. The rats were fed ab libitum
(A) or fasted for 24 h (B) before CCl4.
ICD activity is not included (in A)
for the sake of clarity. SDH levels in
rats pretreated with 20 mg of
DAS/kg p.o. 12 h before CCl4 are
shown instead. As illustrated by the
bar parallel to the x-axis, all animals were maintained on a 12-h
light/dark cycle with light from
6:00 AM to 6:00 PM (0600 –1800 h)
and dark from 6:00 PM to 6:00 AM
(1800 – 0600 h).
Mechanisms of Circadian Rhythmicity of CCl4 Hepatotoxicity
277
Fig. 2. Diurnal variation in CYP2E1
activity in hepatic microsomes of
fasted and nonfasted rats. Six rats
in each group were sacrificed every
4 h over a 24-h period. CYP2E1 activity was assessed by measuring
hydroxylation of p-nitrophenol to
4-NC. Points represent mean ⫾ S.E.
All values for the fasted groups are
significantly higher than corresponding values for the nonfasted
groups at p ⱕ 0.05, except at 6:00
AM.
dark period. There was a lag time of 2 to 4 h in the diurnal
rhythm of the fasted rats. Hepatic GSHPx activity did
not exhibit a circadian rhythm nor was there a significant difference between fed and fasted animals (data not
shown).
Discussion
Findings in the current investigation are consistent with the
hypothesis that abstinence from food during the inactive cycle
results in lipolysis and formation of acetone, which participates
in CCl4 metabolic activation and hepatotoxicity. The susceptibility of male S-D rats to CCl4 hepatotoxicity varies significantly during a 24-h period. The extent of liver damage upon
CCl4 exposure during the initial part of the animals’ active cycle
is substantially greater than that caused by the same dose of
chemical given during the inactive cycle. The abrupt increase
from minimal to maximal susceptibility consistently occurs just
prior to and during the first hours of feeding and other nocturnal activities.
Circadian rhythmicity in metabolic activation of CCl4 is
apparently important in the chemical’s chronotoxicity in
male S-D rats. CCl4 is metabolized by P450-dependent reductive dehalogenation to a trichloromethyl radical. This
moiety can covalently bind to lipids and proteins or react
further to form organic free radicals, peroxides, and other
reactive cytotoxic metabolites. We found covalent binding of
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Fig. 3. Diurnal variation in extent of
covalent binding of 14CCl4 metabolites
in vitro to hepatic microsomal proteins of fasted and nonfasted rats.
14
CCl4 was incubated with microsomes harvested every 2 to 4 h over a
24-h period from untreated rats. Each
point represents the mean ⫾ S.E. for
six animals. All values in the fasted
group are significantly different from
corresponding values in the nonfasted
group at p ⱕ 0.05.
278
Bruckner et al.
Fig. 4. Circadian rhythm of blood
acetone levels in fasted and nonfasted rats. Six rats from each group
were sacrificed every 4 h over a 24-h
period. Blood acetone concentrations
were measured by headspace gas
chromatography. Points represent
mean ⫾ S.E. S.E. bars are too small
to be visible for some groups. All values for the fasted groups are significantly higher than corresponding
values for the nonfasted groups at
p ⱕ 0.05.
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TABLE 1
Dose-dependency of acetone induction of CYP2E1 and potentiation of
CCl4 hepatotoxicity
Groups of six rats received 0, 50, 100, 250, 500, 1000, or 2000 mg of acetone/kg by
gavage. Serial blood samples were taken via an indwelling cannula for 8 h from one
set of rats and AUC80 values determined. A second set of animals was killed 24 h
post-acetone, and hepatic microsomal CYP2E1 activity was assessed by measuring
hydroxylation of p-nitrophenol to 4-NC. Members of the third set were given 800 mg
of CCl4/kg p.o. 24 h after acetone and killed 24 h post-CCl4 for measurement of serum
SDH activity. Values for the three indices are expressed as mean ⫾ S.E.
Acetone
Dosage
AUC80
CYP2E1
mg/kg
mg 䡠 min/ml
nmol 4-NC/min/mg protein
mU/ml
0
50
100
250
500
1000
2000
N.D.
N.D.
64 ⫾ 3a
108 ⫾ 9b
227 ⫾ 10c
456 ⫾ 32d
983 ⫾ 78e
0.9 ⫾ 0.04a
1.2 ⫾ 0.1b
2.1 ⫾ 0.1c
2.2 ⫾ 0.1c
3.1 ⫾ 0.3d
4.5 ⫾ 0.2e
5.5 ⫾ 0.1f
17 ⫾ 7a
64 ⫾ 14b
162 ⫾ 25c
196 ⫾ 42c
406 ⫾ 72d
1257 ⫾ 124e
1887 ⫾ 107f
SDH Activity
N.D., not determined.
a–f
Values for each index that are significantly different from one another at p ⱕ
0.05 are designated by a different letter.
14
CCl4 metabolites to hepatic microsomal proteins to be relatively high in rats during their active cycle. Total P450
levels were relatively constant over a 24-h period in the
current study. In contrast, CYP2E1 activity exhibited a diurnal rhythm, increasing significantly (by 6:00 PM), the time
of maximum 14CCl4 covalent binding and toxicity. CYP2E1 is
the principal P450 isozyme that catalyzes the metabolism of
CCl4 and other VOCs in rodents and humans (Guengerich et
al., 1991; Wong et al., 1998; Zangar et al., 2000). In the
current study, pretreatment of rats with DAS, a CYP2E1
inhibitor, negated the rhythmicity of CYP2E1 activity and
CCl4 cytotoxicity, demonstrating the central role of CYP2E1
in this phenomenon.
Circadian variations in CCl4 metabolism and toxicity appear to be related to restricted food intake during inactive/
sleep periods, because fasting is known to potentiate CCl4
hepatotoxicity. Nakajima et al. (1982), for example, observed
a 3.3-fold increase in CCl4 metabolism and a marked elevation of serum enzymes in male Wistar rats fasted for 24 h
before CCl4 exposure. Brown et al. (1995) reported time-
Fig. 5. A, correlation between area under the blood acetone concentration
versus time curves from 0 to 8 h post-acetone dosing (AUC80) and hepatic
microsomal CYP2E1 activity. Best linear regression (solid line): y ⫽
0.0074X ⫾ 1.27, r2 ⫽ 0.96, p ⫽ 0.004, n ⫽ 6. B, correlation between AUC80
and serum SDH activity. Best linear regression (solid line): y ⫽ 2.696X ⫾
(⫺47.63), r2 ⫽ 0.95, p ⫽ 0.004, n ⫽ 6.
dependent CYP2E1 increases in male F-344 rats fasted for 8,
16, and 24 h. We observed an increase in CYP2E1 activity of
⬃35% between 2:00 and 6:00 PM and ⬃50% between 2:00 PM
Mechanisms of Circadian Rhythmicity of CCl4 Hepatotoxicity
279
Fig. 6. Diurnal variation in hepatic
NPSH levels in fasted and nonfasted
rats. Six rats from each group were
sacrificed every 2 to 4 h over a 24-h
period for measurement of liver
NPSH content. Points represent
mean ⫾ S.E. For some groups, S.E.
bars are too small to be visible. All
values for the fasted groups are significantly different from the corresponding values for the nonfasted
groups at p ⱕ 0.05.
found that a 1-day fast enhanced the metabolism of CCl4 and
seven other VOCs in male Wistar rats. If acetone is a mediator of fasting-induced CYP2E1 increase and ensuing CCl4
metabolic activation/toxicity, the magnitude of each process
should vary directly with the duration of fasting. Miller and
Yang (1984), indeed, reported reasonably good correlation
between the duration of fasting and blood acetone and between acetone and hepatic N-nitrosodimethylamine demethylase activity in male S-D rats. Charbonneau et al. (1986)
correlated acetone dose/blood level and CCl4 hepatotoxicity
in male S-D rats but did not examine relationships among
acetone, CYP2E1, and CCl4 toxicity. Our findings of substantially higher blood acetone, hepatic CYP2E1, 14CCl4 covalent
binding, and serum enzymes in fasted rats rather than in
nonfasted rats were anticipated. It was surprising, however,
that circadian rhythms in the two sets of animals generally
paralleled one another. Although we did not monitor beyond
one 24-h fasting period, other researchers have found diurnal
rhythms in lipid metabolites to persist during an additional
24 to 48 h of fasting (Ahlersova et al., 1985; Escobar et al.,
1998).
Acetone may not be formed in sufficient amounts during
the rat’s 12-h inactive period to account solely for diurnal
CYP2E1 induction. Concentrations of AA and ␤HB are typically higher than acetone levels, so these two ketone bodies
could augment CYP2E1 induction by acetone. Results of limited in vivo (Barnett et al., 1992) and in vitro (Zangar and
Novak, 1998) studies indicate that neither ␤HB nor AA induces CYP2E1. Johansson et al. (1988) demonstrated that
starvation for 3 days exerted a synergistic effect with acetone
on induction of CYP2E1 and CCl4 metabolism. These researchers believed that this synergistic effect of fasting could
be the result of an increased rate of CYP2E1 gene transcription or mRNA stabilization, possibly due to action of stress
hormones. Plasma levels of a number of hormones, including
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 16, 2017
and 6:00 AM in rats fed ad libitum. Fasting, stress, and
exercise each stimulate lipolysis to satisfy the body’s need for
energy. Excess fatty acids can be converted to ␤-hydroxybutyrate (␤HB) and acetoacetate (AA), the latter undergoing
decarboxylation to acetone. Acetone is a potent inducer of
CYP2E1 and CCl4 metabolism (Johansson et al., 1988). Acetone, a substrate for CYP2E1, is believed to induce CYP2E1
primarily by binding to it and thereby protecting it from
cAMP-dependent phosphorylation (Ronis et al., 1991). Acetone formation during maximal lipolysis each day likely contributes to diurnal induction of CYP2E1. Mlekusch (1982)
and Ahlersova et al. (1985) observed sharp peaks in plasma
AA and ␤HB like that we saw for acetone at the onset of the
active cycle of rats. We observed that CYP2E1 activity and
covalent binding of 14CCl4 were also maximal or near-maximal during this time. Clark and Powis (1974) reported a peak
in aniline hydroxylase activity within 30 min of i.p. injection
of female rats with acetone. Such a rapid increase is compatible with the mechanism of ligand stabilization of CYP2E1.
It was of interest in the current study that blood acetone
levels diminished so rapidly after reaching their zenith, but
CYP2E1 activity remained high. The acetone bound to
CYP2E1, however, is not measured by our analytical GC
headspace technique. As noted previously, the binding prolongs the residence time of the CYP2E1. After metabolism of
its substrate (i.e., acetone), CYP2E1 is degraded quickly.
CYP2E1 has a half-life of 4 to 6 h or less in rat liver (Roberts
et al., 1994). Ingested CCl4 reaches the liver, undergoes metabolic activation, and damages the liver in a matter of minutes (Rao and Recknagel, 1968; Sanzgiri et al., 1997). Thus,
there appears to be time during a 24-h period for the necessary sequence of events to occur and to be reversed for subsequent diurnal cycles.
Acetone plays an important role in potentiation of CCl4
hepatotoxicity in fasted subjects. Sato and Nakajima (1985)
280
Bruckner et al.
urnal variations and exhalation of acetone peaks before
breakfast (Wildenhoff, 1972). Thus, people may be more susceptible to CCl4 and other chemicals that undergo CYP2E1mediated activation when exposed to the chemicals during
their initial waking hours.
Information gained from the current study of the circadian
rhythmicity of CCl4 hepatotoxicity may have a number of
applications. The phenomenon may well apply to a variety of
VOCs and other lipophilic, low molecular weight compounds
that are metabolically activated by CYP2E1 to cytotoxic or
mutagenic metabolites. The toxic and carcinogenic potency of
such chemicals could vary with the (clock) time of exposure.
Thus, such chemicals should be evaluated at the time(s) of
maximum sensitivity of the test animal. Light/dark cycles
and treatment times should be considered in designing experimental protocols. P450 isozymes other than CYP2E1
may also be found to exhibit diurnal rhythms.
Acknowledgments
We appreciate the efforts of Libby Moss and Mary Eubanks in
preparation of the manuscript.
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