Altered proline uptake by mouse liver cells after chronic exposure to ethanol and its metabolites - Gut
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Gut: first published as 10.1136/gut.25.2.138 on 1 February 1984. Downloaded from http://gut.bmj.com/ on February 15, 2021 by guest. Protected by copyright. Gut, 1984, 25, 138-144 Altered proline uptake by mouse liver cells after chronic exposure to ethanol and its metabolites C L MENDENHALL, A CHEDID, AND CAROL KROMME From the Department of Medicine, University of Cincinnati Medical Center, and the VA Medical Center, Cincinnati, Ohio; and Department of Pathology, University ofHealth Sciences, Chicago Medical School, North Chicago, Illinois, USA SUMMARY A cloned mouse liver cell model (NCTC 1469) treated with either ethanol or one of its metabolites, acetaldehyde or acetate, was used to study proline uptake. Beginning with the stationary phase of cell growth, 14C-proline uptake was markedly accelerated by both ethanol and acetaldehyde (p
Gut: first published as 10.1136/gut.25.2.138 on 1 February 1984. Downloaded from http://gut.bmj.com/ on February 15, 2021 by guest. Protected by copyright.
Altered proline uptake after chronic exposure to ethanol and its metabolites 139
tion was used. the cells were predominantly epitheloid in type but
A Multiple Automated Sampler Harvester II some macrophages and fibroblastic cell types were
(MASH II, Microbiological Associates) was used to present (Fig. 1). Biochemical characteristics of
harvest the culture plates, transferring the contents hepatocytes were also maintained in that significant
of each well onto filter paper discs and washing the amounts of a-fetoprotein, 25 ngm/ml, were released
cells free of unincorporated isotope. The use of the into the medium and alcohol dehydrogenase activity
microtitre plates and MASH II as opposed to tissue was present sufficient to generate 1 21 nmol of
culture flasks allowed a large number of cultures to NADH/min/107 cells. Albumin synthesis, however
be evaluated concurrently for each experiment. could not be detected.
Viability was checked for each treatment by trypan Cell growth was not significantly altered by
blue exclusion as well as by DNA synthesis which ethanol or its metabolites as measured by cell
was monitored by pulsing with 1 uCi 3H-thymidine number/well, and 3H-thymidine uptake/104 cells
for one hour before harvesting. Proline uptake was (Fi. 2 and 3).
determined by the addition of 7 x 102 ,Ci 14C- 1 C-proline uptake was evaluated in both controls
proline to each culture well one hour before and treated cells. Uptake represented intracellular
harvesting. Filter discs from the MASH containing labelling in both the free amino acid pools as well as
washed cells were counted in 10 ml of toluene a small amount incorporated into protein. One hour
containing 2,5-diphenyloxazole (PPO), 1,4-bis-(2- cell exposutre with r4C-proline before harvesting,
phenyloxazole)-benzene (POPOP) and Triton X resulted in less than 10% incorporation in acid
using a Beckman LS-230 Liquid Scintillation precipitable protein. Hence, most of the observed
System. Quench calibration was accomplished by changes are occurring within the intracellular free
the external standard-channels ratio method. proline pool. As shown in Figures 4 and 5, 14C-
Proline pool sizes were measured in the following proline uptake was significantly increased by both
manner on both control and ethanol-treated ethanol and acetaldehyde treatments during the
cultures. At the end of eight days, four untreated stationary phase of cell growth. The addition of
flasks or five ethanol treated flasks were trypsinised, acetate to the medium in physiological concentra-
combined, washed and counted yielding 55-3x 106 tions did not alter proline uptake. The greatest
control cells and 75.2 x 106 ethanol-treated cells. The increase was observed with acetaldehyde on day
cells were homogenised in sulphosalicyclic acid for eight, 306-1% of control; while ethanol produced a
deproteinisation, and thienylalonine for an internal 181-2% increase and acetate was essentially
standard. The homogenate was centrifuged and identical to controls.
50 ,ul of the supernatant was analysed on a Beckman At this time, the intracellular proline pool sizes
121 MB Amino Acid Analyser. were not significantly different, 78-2 nmoles/107 cells
A second set of experiments was also performed and 54 5 nmoles/107 cells, controls vs ethanol-
to evaluate ethanol's effects independent of its treated. In a second experiment in which 4-methyl
oxidation product, acetaldehyde. In these studies, pyrazole was added to the medium concomitantly
either 50 mM ethanol or 10 ,um acetaldehyde in with ethanol, the ethanol effect was markedly
combination with 0 24 mM 4-methyl pyrazole was diminished from 187.2% of control to 112.4% of
added to the cultures in order to significantly inhibit control (Fig. 6). In absolute numbers, this was a
alcohol dehydrogenase. Cells were harvested on the reduction from 95 1 DPM/cellxlO-s with ethanol to
seventh day at a time when differences between 57X1 DPM/cell x 105 with ethanol plus 4-methyl
controls and treated were maximal. Viability, 3H- pyrazole (p=0-0008) as compared to 50 8 DPM/
thymidine and 14C-proline incorporation were cellxlO-5 in untreated controls.
measured as described below. Data analyses Changes in DNA synthesis (3H-thymidine
between controls and treatment groups were accom- uptake) as well as viability were not significantly
plished by use of the Student's t test. altered by this dose of 4-methyl pyrazole. Treatment
with combined acetaldehyde and 4-methyl pyrazole
Results when compared with acetaldehyde alone resulted in
a slight increase in proline uptake which was not
The chronic addition of ethanol so as to maintain a significantly different. Both treatments significantly
culture concentration comparable with that seen in stimulated uptake when compared with controls.
human serum after mild and heavy alcohol
consumption, 10 mM and 50 mM (46 mg% and 230 Discussion
mg%) did not alter cell viability, 94 to 96% by
trypan blue exclusion. This is in agreement with Using '4C-proline, the effects of chronic ethanol
Shields et al'6 and Walker et al. 17 Morphologically ingestion have been reported to produce increasedGut: first published as 10.1136/gut.25.2.138 on 1 February 1984. Downloaded from http://gut.bmj.com/ on February 15, 2021 by guest. Protected by copyright.
140 Mendenhall, Chedid, and Kromme
Fig. 1 Phase contrast micrograph viewed through an inverted microscope showing cloned cells from mouse liver, NCTC
1469 (American Type Culture Collection). Cell morphology is predominately epitheloid but somefibroblastic cell types are
present. 1 cm represents 50 microns.
35-
30-
< 25-
x
a= 20-
Fig. 2 Mouse liver cell ")
replication monitored by @ 15-
directly counting cell number.
Although control cultures 0
*-a were typically lower 10-
than ethanol 0-0 or
acetaldehyde 0-0,
differences were significant only 5.
on day 9. * indicates
significance, pGut: first published as 10.1136/gut.25.2.138 on 1 February 1984. Downloaded from http://gut.bmj.com/ on February 15, 2021 by guest. Protected by copyright.
Altered proline uptake after chronic exposure to ethanol and its metabolites 141
900- incorporation of the isotope into liver collagen and
an increase in the activity of collagen proline
800
hydroxylase activity in both rats and baboons.4 5
Similar studies in mice have also shown an increase
in collagen and proline hydroxylase activity.6 In
700 e addition, an increase was observed in the incorpora-
I tion of radiotagged sulphur into glycosaminoglycans
600 of collagen. Both the increase in enzyme activity and
glycosaminoglycan synthesis were observed acutely
I"500
18 hours after a single dose of ethanol (6 gm-kg), but
i hepatic collagen was not detectably increased until
the third month of treatment.6
0
2:400- In human studies, Leevy and Chen7 using auto-
radiographs prepared from the liver of alcoholic
patients with cirrhosis, also observed increased
300-
100 f ." 4C-proline uptake which was interpreted as
200-
increased synthesis. Unfortunately, the ethanol-
induced increase in collagen synthesis has not been
universally observed. Mezey and associates8 found
100- increased deposition of chemically detectable
collagen without significant changes in parameters
I I I I I
associated with hepatic collagen synthesis. Evidence
0 2 4 6 8 10 12 of decreased collagen degradation, however, was
Time (days) observed. Similarly, Henley and associates9 using
combined alcohol in drinking water plus a choline
Fig. 3 Mouse liver cell replication monitored by deficient diet to potentiate collagen formation,
3H-thymidine uptake in controls *-@, ethanol treated showed increased collagen deposition in the ethanol
0-0, and acetaldehyde O-O cultures. 3H-thymidine animals but could not show an increase in synthesis.
uptake was consistently higher in control cultures during the From this, they concluded that an inhibition of
growth phase. * and * * indicate significance, pGut: first published as 10.1136/gut.25.2.138 on 1 February 1984. Downloaded from http://gut.bmj.com/ on February 15, 2021 by guest. Protected by copyright.
142 Mendenhall, Chedid, and Kromme
300' i for reasons other than an ethanol effect alone.
Our studies indicate that '4C-proline uptake is
significantly accelerated by treatment with either
ethanol or acetaldehyde, while acetate did not
produce a significant effect. The peak production
occurred during the stationary phase of cell growth,
days six to 12, and did not appear to be dose related.
200- Maximal increases during this period were 306*1%
of controls after acetaldehyde and 181.2% after
ethanol (pGut: first published as 10.1136/gut.25.2.138 on 1 February 1984. Downloaded from http://gut.bmj.com/ on February 15, 2021 by guest. Protected by copyright.
Altered proline uptake after chronic exposure to ethanol and its metabolites 143
hydroxyproline via proline hydroxylase. 8 Mezey E, Potter JJ, Slusser RJ, Abdi W. Changes in
As ethanol is oxidised reversibly to acetaldehyde hepatic collagen metabolism in rats produced by
principally by alcohol dehydrogenase, the inhibition chronic ethanol feeding. Lab Invest 1977; 36: 206-14.
of this enzyme by 4-methyl pyrazole in combination 9 Henley KS, Laughrey EG, Appelman HD, Flecker K.
with ethanol treatment should give primarily an Effect of ethanol on collagen formation in dietary
cirrhosis in the rat. Gastroenterology 1977; 72: 502-6.
ethanol effect independent of its metabolites. 10 Kershenobich D, Fierro FJ, Rojkind M. The relation-
Therefore to evaluate ethanol and acetaldehyde ship between the free pool of proline and collagen
effects independently, ethanol treatment was content in human liver cirrhosis. J Clin Invest 1970; 49:
combined with 4-methyl pyrazole. With this 2246-9.
combination the increase in 1 C-proline uptake seen 11 Phang J, Finerman GAM, Singh B, Rosenberg LE,
with ethanol alone was not significantly reduced. Berman M. Compartmental analysis of collagen
These findings suggest that acetaldehyde and not synthesis in fetal rat calvaria. I. Pertubations of proline
ethanol represents the principal stimulator of transport. Biochim Biophys Acta 1971; 230: 146-59.
proline uptake. They do not exclude by direct 12 Dunn MA, Rojkind M, Warren KS, Hait PK, Rifas L,
measurement the possibility of changes in redox Seifter S. Liver collagen synthesis in murine schisto-
somiasis. J Clin Invest 1977; 59: 666-74.
state contributing to this effect. As each mole of 13 Rojkind M., Diaz de Leon L. Collagen biosynthesis in
ethanol undergoing oxidation by alcohol dehydro- cirrhotic rat liver slices: a regulatory mechanism.
genase ultimately generates 2 moles of NADH, Biochim Biophys Acta 1970; 217: 512-22.
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change than acetaldehyde. Rather than paralleling and sensitiive method for the simultaneous quantitation
redox changes, however the experimentally of ethanol and acetaldehyde in biological materials
observed ethanol-effect on proline uptake was using head-space gas chromatography. J. Chromatogr
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15 Richards RG, Mendenhall CL, MacGee J. A simple
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fibroblasts in vitro. Scott Med J 1974; 10: 125-7.
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