•
Alcohol & Alcoholism, Vol. 28, No. SIB, pp. 105-108, 1993
Elsevier Science Ltd
Medical Council on Alcoholism
Printed in Great Britain. All rights reserved
0735-0414/93 $6.00 + 0.00
Pergamon
0735·0414(93)E0014·3
ABNORMAL ETHANOL METABOLISM IN LONG-EVANS CINNAMON
RATS, A MUTANT STRAIN DEVELOPING SPONTANEOUS HEPATOMA
MASAHIRO NAKAJIMA,* JUNJI KATO,* YUTAKA KOHGO,t SHINICHI KATSUKI,*
NORIAKI INUI,* MAS AMI OHYA,* NORITOSHI TAKEICHI:j: and YOSHIRO NIITU*
*Department ofInternal Medicine (Section 1), Sapporo Medical University School of Medicine, West-16, South-I, Chuo-Ku,
Sapporo 060, Japan; tLaboratory of Cell Biology, Cancer Institute, Hokkaido University School of Medicine, West-7, North-IS,
Kita-ku, Sapporo 060, Japan
Abstract - The Long-Evans Cinnamon (LEC) rat is a mutant strain established from Long-Evans rats.
LEC rats display hereditary hepatitis and spontaneous hepatocellular carcinoma (HCC), We first tried to
examine effects of ethanol consumption on the development of HCC, and fed a Lieber's liquid diet
containing 5% ethanol to LEC rats. However the rats died within 2 weeks because of acute alcohol
intoxication. In LEC rats, the concentration of ethanol and acetaldehyde in blood was significantly
higher, and liver alcohol dehydrogenase activity was slightly lower and acetaldehyde dehydrogenase
activities were remarkably suppressed compared to those of Wistar rats, These results suggest that LEC
rats have hereditary deficiencies of ethanol and acetaldehyde metabolizing enzymes.
Long-Evans Cinnamon (LEC) rats have been
established from a closed colony of Long-Evans
rats. LEC rats suffer from spontaneous hepatitis
with jaundice developing around 4 months after
birth, followed by death in 40% of rats due to
fluminant hepatitis. The remaining rats recover,
but exhibit chronic hepatitis and develop
cholangiofibrosis or hepatocellular carcinoma
(HCC) (Sasaki et aI., 1985; Yoshida et al., 1987;
Takeichi et al., 1988).
Patients with alcoholic liver cirrhosis rarely
develop HCC while they are drinking heavily, but
HCC may emerge sometimes after complete
abstinence from ethanol (Lee, 1966). It is
suggested that ethanol drinking may contribute to
the development of HCC; however, the detailed
mechanism remains obscure.
We first tried to elucidate the effects of ethanol
on the development of HCC. Unexpectedly, all
LEC rats died within 2 weeks after feeding on the
ethanol-containing liquid diet. This suggests that
abnormal metabolism of ethanol may exist in LEC
rats. In the present paper, we therefore measured
the blood concentration of ethanol and
t Author to whom correspondence should be addressed.
105
acetaldehyde after intraperitoneal administration
of ethanol, and examined enzymes related to
ethanol metabolism, such as alcohol dehydrogenase ((ADH) and acetaldehyde dehydrogenase
(ALDH) in the liver of LEC and Wistar rats.
MATERIALS AND METHODS
Animals'
Male LEC rats were maintained under
conventional conditions at the Center for
Experimental Plants and Animals of Hokkaido
University (Sapporo, Japan). Wistar rats were
obtained from Charles River Japan, Inc. (Autsugi,
Japan). We used 6-week-old LEC rats, and 6week-old Wi star rats as controls. The alcoholdosed group was fed a diet containing 5% ethanol
(Lieber et aI., 1963) and the control group was fed
the same diet except containing sucrose with the
equivalent amount of calories as ethanol (Oriental
Yeast Co., Tokyo, Japan). Both groups were fed
50 rn1 per day.
Histological examination
The liver tissue was fixed with 10% formalin in
phosphate-buffered saline and processed routinely
for light microscopy. Paraffin-embedded sections
were stained with hematoxylineosin.
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INTRODUCTION
106
M. NAKAJIMA et al.
Measurement of ethanol and acetaldehyde
concentration in blood
Alcohol dehydrogenase activity
The liver was homogenized with a Dounce
homogenizer in 0.25 M sucrose containing 5 roM
Tris-HC1, pH 7.2, and 0.5 mM ethylenediaminetetraacetic acid (EDTA). The homogenate
was centrifuged at 480 g for 10 min, and the
resulting post-nuclear supernatant fraction was
centrifuged at 10,000 g for 10 min. T4e resulting
cytosolic fraction was subjected to determine
ADH activity.
The assay mixture contained 70 mM
NaOH-glycine buffer, pH 9.6, 0.67 mM NAD+
(Boehringer Mannheim, Germany), and 10 mM
ethanol. The reaction was started at room
temperature by addition of the coenzyme and the
initial rate of reduction to NADH was measured
spectrophotometrically at 340 nm (Koivula et at
1975).
Acetaldehyde dehydrogenase activity
The liver was homogenized in 0.25 M sucrose
containing 5 roM Tris-HCI, pH 7.2, and 0.5 mM
EDTA. The homogenate was then centrifuged at
480 g for 10 min, and the resulting post-nuclear
supernatant fraction was subjected to determine
the total and low Km AKDH activities. The assay
mixture contained 50 mM sodium pyrophosphate,
pH 8.8, OSmM NAD+, 0.1 roM pyrazol (Sigma
Chemical Co., S1. Louis, U.S.A.), 5 mM
RESULTS
The survival of LEC and Wi star rats after
ethanol intake was analyzed by the Kaplan-Meier
method (Kaplan and Meier, 1958). After ethanol
intake, LEC rats began to appear intoxicated as the
movements became slow and unsteady. As shown
in Fig. 1, all the alcohol-dosed LEC rats died
within 2 weeks. The average length of survival of
LEC rats was 7.0 days. All the Wistar and LEC
rats fed with control diets and all the Wistar rats
fed with an ethanol diet survived without any
intoxication.
Table 1 demonstrates the concentration of
ethanol and acetaldehyde in blood after
intraperitoneal administration of ethanol. Both
ethanol and acetaldehyde concentration were
--
(...
__
'.. _ _.rci.'.r........."".............-""--. ..-""'_.....--...- ...-.......- .............-..,-...
-- ....
--. ..
- ...
--..,,-....
--. ..
-. ..
--.tR.C(_1nII
..
I
..
.~~~~~--~~~~----~~------~
IS
•
"
Fig. 1. Survival curves of LEe and Wistar rats.
The rats (n = 15) were fed with or without ethanol-containing
Lieber's liquid diets_
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Three LEC and three Wi star rats were
intraperitoneally administered with a dose of 2 g
ethanol/kg body weight. At 0.5, 1, 2, and 3 hr
following administration of ethanol, 0.2 ml of
each rat's blood was drawn from the jugular vein.
Each sample was analyzed quantitatively for
ethanol and acetaldehyde by gas chromatography
with use of head space analysis (Peter et al.,
1982). The samples were treated with 0.6 N
perchloric acid (Wako Pure Chemical Industries,
Ltd, Osaka, Japan) to minimize artefactual
formation of acetaldehyde during the preparation.
In brief, each blood sample was added to 1 rnl of
0.6 N perchloric acid made in ice-cold saline and
was centrifuged at 4000 g for 10 min at 4 ·C, 0.5
ml of the supernatant was heated at 65°C for 30
min, and a 1.0 m1 volume of head space gas was
analyzed by gas chromatography (Hitachi G-3000,
Tokyo, Japan).
acetaldehyde (Nacalai Tesque, Inc., Osaka, Japan)
and 2 )lM rotenone (Sigma) for the total ALDH
activity. To determine low Km ALDH, 50 ~M
acetaldehyde was used as substrate in the assay
(Tottmar et al.• 1973). The reaction was started by
addition of the substrate. Pyrazol was added to
inhibit ADH and rotenone to inhibit mitochondrial
NADH oxidase. This was assayed spectrophotometrically with acetaldehyde as substrate by
measuring the reduction of NAD+ at 340 urn
(Tottmar et al., 1973). One unit of activity is
defined as the amount of enzyme catalyzing the
formation of 1 ~mole of NADH per min under the
above conditions.
LONG-EVANS CINNAMON RATS
Table I. Blood ethanol and acetaldehyde concentration after
ethanol administration in LEC and Wistar rats
Ethanol concentration
(mgldl)
Wistar
0.5 hr
Ihr
2hr
3hr
174.0± 6.0
148.3 ± 1.5
114.6±9.0
102.3 ± 10.0
LEC
Table 2. ADH, total ALDH, and low Km ALDH activity in the
liver of LEC and Wistar rats
Acetaldehyde concentration
(11M)
Wistar
LEC
203.6 ± 10.2* 9.1 ± 1.5 13.8 ±2.0
200.0±5.0* 20.1 ± 5.2 26.4 ± 1.4
195.3 ± 26.0* 30.1 ± 5.1 38.0 ± 2.1 *
160.6 ± 9.5* 32.6 ± 4.4 43.2 ± 0.3*
*p < 0.05, compared to Wistar rats (Student's t-test).
Wistar
LEC
ADH
(units!g liver)
TotaiALDH
(units!g liver)
Low Km ALDH
(units!g liver)
12.58 ± 0.18
6.58 ± 0.15*
9.17 ± 0.14
2.13 ± 0.35**
3.91 ± 0.35
0.80 ±0.1**
*p < 0.001, compared to Wistar rats; **p < 0.0001, compared
to Wistar rats (Student's t-test).
was mainly responsible for the impainnent of the
total ALDH activity.
DISCUSSION
The LEC rats display hepatitis and HCC
spontaneously. The production of radicals induced
by copper and iron, which accumulate abnonnally
in liver tissue around 12 to 13 weeks after birth, is
regarded as an important cause (Ono et aI., 1991;
Kato et ai., 1993). We administered ethanol to
LEC rats and tried to examine the influence of
ethaIlol on the development of HCC using Wistar
rats as control animals. However, all the LEC rats
died within 2 weeks after feeding with the ethanolcontaining diet, suggesting that the LEC rats have
some potential for abnormal ethanol metabolism
(Fig. 1).
One possibility was that the LEC rats became
malnourished. However, an insufficient intake was
excluded because no significant differences in
Fig. 2. Light microscopic findings of the hemotoxylin-eosin stained liver of a LEC rat at 7 days after ethanol
intake (400x).
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significantly higher in LEC rats than in Wistar
rats.
Histological observations on the liver of LEC
rats which died on the 7th day after ethanol intake
are shown in Fig. 2. The liver tissue showed a
slight swelling of hepatocytes but did not show
any other abnonnalities, including inflammation,
fatty change, and necrosis.
Table 2 demonstrated the ADH activity and the
total and low Km ALDH activities. The ADH
activity in the Wistar and LEC rats were 12.53 ±
0.18 units/g liver and 6.58 ± 0.15 units/g liver,
respectively. The value in the LEC rats was as low
as 50% of that in the Wistar rats. The total ALDH
activity in the Wi star and LEC rats was 9.17 ±
0.14 units/g liver and 2.13 ± 0.35 units/g liver,
respectively. The value in LEC rats was as low as
23% of the activity in the Wi star rats. The
decrease of low Km ALDH activity in LEC rats
107
108
M. NAKAJIMA et al.
Acknowledgements - We thank Mr Masashi Ichinoseki of
Tomakomai Clinical Chemistry Laboratories for generous help
with gas chromatography analysis, and Mrs Kanako Kaga for
preparation of the manuscript. This work was supported in part
by Grants-in-aid from the Ministry of Education, Japan.
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body weights or dietary intake were noted in any
groups (data not shown). The second possibility
was the hepatic failure due to ethanol ingestion.
However, this assumption was denied because the
tissue specimens of liver of LEC rats were almost
normal and serum levels of aspartate
aminotransferase (AST) and alanine aminotransferase (ALT) were only slightly increased
(data not shown). Thus, we consider that neither
malnutrition nor hepatic failure are causes of
death.
We then examined the concentration of ethanol
and acetaldehyde and whether the hepatic
clearance was impaired. It was noteworthy that
significantly high concentrations of ethanol and
acetaldehyde were observed in LEC rats compared
with Wistar rats, suggesting that the activity of
ethanol metabolism-related enzymes in the livers
is decreased. While ADH activities in the LEC
rats were slightly impaired, the total ALDH
activities were decreased remarkably in LEC rats,
to about a quarter of the value in the Wistar rats.
By using a low concentration of substrate, the
decrease of total ALDH activity was mainly due to
the lack of low Km ALDH. Therefore, it is
suggested that the LEC rats were unable to
metabolize acetaldehyde because of partial ALDH
deficiency.
Within a few days after ethanol intake, LEC
rats showed apparently intoxicated states. This
phenomenon could be explained by the fact that
the concentration of acetaldehyde in the blood
became too high. Presumably, death may be
caused by a suppression of the central nervous
system by the intoxication. These rats may be
regarded as a good model for studying acute
alcoholic intoxication or genetic racial differences
of acetaldehyde elimination in humans (Goedde et
ai., 1979). However, further studies are needed to
know what kind of molecular abnormalities are
present in LEC rats.