EXPERIMENTAL AND MOLECULAR PATHOLOGY 43, 364-374 (1985)

Protective Effect of Tryptophan and Cysteine against Carbon

Tetrachloride-Induced Liver Injury’
DING WANG,* ETHEL VERNEY, AND HERSCHEL SIDRANSKY
Department of Pathology, The George Washington University Medical Center, Washington, D.C.
20037 and ‘Department of Pathology, Institute of Traditional Chinese Medicine,
Inner Mongolia, China

Received May 20, 1985, and in revised form August 20, 1985

The effects of the administration of tryptophan and/or cysteine on carbon tetrachloride

(CC&)-induced hepatic injury were investigated. Rats received Ccl, (1 ml/kg ip) followed 6
hr later by tryptophan (300 mg/kg) and/or cysteine (950 mg/kg) via stomach tube and rats
were killed after 24 hr. Treatment with tryptophan, cysteine, or both reduced the degree of
hepatic necrosis observed histologically. While Ccl, caused polyribosomal disaggregation
and decreased [r4C]leucine incorporation into liver proteins in vitro and in t,ivo, treatment
with tryptophan, cysteine, or both caused a shift in polyribosomes toward heavier aggre-
gation and protein synthesis was increased. Serum activities of lactic dehydrogenase (LDH),
glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and y-glutamyl-
transpeptidase were markedly increased after CCI, alone but after subsequent treatment
with cysteine or with tryptophan and cysteine appreciable decreases occurred. Glutathione
concentration decreased but total amount remained constant in the livers of CC&-treated
rats while subsequent treatment with cysteine alone or together with tryptophan elevated
both levels of glutathione. Using isolated hepatocytes, CCI, caused decreases in cell via-
bility, in release of LDH, and in [r4C]leucine incorporation into protein. Treatment with
CCI, and tryptophan and/or cysteine revealed that cysteine alone or with tryptophan im-
proved cell viability and decreased LDH release of the cells. while tryptophan alone or
with cysteine improved protein synthesis. Upon cytologic evaluation, the isolated hepato-
cytes revealed membrane distortions after Ccl, alone but these were less marked after Ccl,
plus tryptophan, cysteine, or both (most improvement). Thus, tryptophan and cysteine act
in a beneficial manner against Ccl,-induced hepatic injury in the rat. o 1985 Academic PWSS. IK.

INTRODUCTION
It has been demonstrated in an earlier study from our laboratory (Sidransky et
al., 1977) that the administration of L-tryptophan to rats treated with Ccl, im-
proved hepatic polyribosomes and protein synthesis. This suggested that tryp-
tophan has a beneficial effect on the liver in regard to Ccl,-induced hepatic injury.
De Ferreyra et al. (1983a), have reported, using a different model concerned
with CC&-induced liver necrosis in the rat, that cysteine but not tryptophan ex-
erted a weak preventive effect while both together exerted a marked protective
effect against CC&-induced liver necrosis. As a follow-up to our own previous
investigations into how tryptophan affects the liver in normal animals (Sidransky,
1976, 1985) as well as in animals treated with hepatotoxins (Sidransky, 1985;
Sidransky et al., 1982), we have now studied how tryptophan and cysteine would
act in relation to CC&-induced liver injury in the rat. The effects of tryptophan,
cysteine, or both on CC&-induced injury was assessed in the livers of rats in vivo
and also in isolated rat hepatocytes in vitro.

1 This work was supported by United States Public Health Service Research Grant AM-27339 from
the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases.

364
0014-4800/85 $3.00
Copyright 0 1985 by Academic Press, Inc.
All rights of reproduction in any form reserved.
 TRYPTOPHAN AND/OR CYSTEINE AND Ccl4 LIVER INJURY 365

MATERIALS AND METHODS

Male and female rats of the Sprague-Dawley strain (Microbiological Associ-

ates, Bethesda, Md.), weighing 216 to 231 g, were used. The animals, maintained
in an air-conditioned room, were fed a commercial diet (Wayne Lab-Blox, Allied
Mills, Inc., Chicago, Ill.) before the experiments were begun. All animals were
fasted overnight (from 5:00 PM to 8:00 AM) before the experiments. The following
morning the rats were divided into groups, each of which contained two to five
rats. Control rats were injected intraperitoneally with olive oil (0.5 ml/l00 g body
wt) while the experimental rats were injected intraperitoneally with a 20% (v/v)
solution of Ccl, in olive oil at a dose of 0.5 ml/100 g body wt. Six hours later
groups of rats were tube-fed L-tryptophan (30 mg in 3 ml water1100 g body wt),
L-cysteine (95 mg in 3 ml water/l00 g body wt), or a mixture of both containing
the same amounts as above but in 3 ml water/l00 g body wt. Rats had access to
water but no feed. All animals were killed by decapitation 24 hr after Ccl, ad-
ministration.
Rats were weighed at time of killing. In all experiments livers were removed
and weighed. Blood serum was collected for enzyme assays in some experiments.
Aliquots of liver were taken for specific analyses and for histological examination.
Postmitochondrial supernatants of livers were prepared by the method previ-
ously reported in earlier studies (Sidransky ef al., 1968). Size distribution of
deoxycholate-treated postmitochondrial supernatants was determined by layering
12-ml sucrose gradients [0.3 to 1.1 M sucrose containing TKM (0.05 M Tris (pH
7.4), 0.025 M KCl, and 5 mM MgCl,)] and by centrifuging in an ultracentrifuge
at 38,000 rpm for 90 min at 2°C and was analyzed as described earlier (Sidransky
et al., 1968). The degree of hepatic polyribosomal aggregation under the different
experimental conditions was evaluated from the patterns obtained with sucrose
density gradients by calculating the relative distribution of monomers-dimers to
total ribosomes. In in vitro incorporation experiments, incorporation of L-
[t4C]leucine, uniformly labeled, into proteins using postmitochondrial superna-
tants or microsomes (with a constant cell sap) was measured using methodology
described earlier (Sidransky et al., 1968). In in vivo experiments L-[‘4C]leucine
(290 mCi/mmole), 5 kCi/lOO g body wt, was injected intraperitoneally I hr before
killing and incorporation into liver and serum proteins was measured. In some
experiments, [t4C]orotic acid (61 mCi/mmole), 5 @i/100 g body wt, was injected
intraperitoneally 2 hr before killing and incorporation into hepatic RNA was mea-
sured (Sidransky et al., 1980). Hepatic total glutathione (GSH) levels were de-
termined using homogenates in 3 vol of 0.25 M sucrose in TKM. After depro-
teinization with 3% metaphosphoric acid, the supernatants were assayed by the
method of Tietze (1967), as modified by Griffith (1980).
Isolated hepatocytes were prepared from livers of fasted (24 hr) rats by the
two-step collagenase perfusion technique according to Seglen (1973, 1976a). Vi-
ability was determined using the trypan blue exclusion test. The isolated hepa-
tocytes were diluted to 1 x IO6 cells/ml in a suspension buffer (Seglen, 1973)
which contained a complete amino acid mixture (Seglen, 1976b). In addition, L-
cysteine, L-tryptophan, or both were added. Ccl,, 1 pi/ml of incubation mixture,
and L-[U-r4C]leucine (348 mCi/mmole) to give a final concentration of 0.0625 FCi/
ml, were added. Additions were made while flasks were on ice. Thereafter, the
cells were incubated at 37°C for 60 min in a gyratory shaker in an atmosphere of
366 WANG, VERNEY, AND SIDRANSKY

TABLE I
Effect of Tryptophan, Cysteine, or Both on Liver Weight and Necrosis Induced by Ccl,
Liver
Weight Degree of
Treatment” (g/100 g body wt) necrosis6
Control (18) 2.98 k 0.11’ (6)0
CCI, (18) 4.26 ? O.Ogd (6) 3.3 k 0.36
Ccl, + tryptophan (18)4.38 f 0.20” (6) 2.1 2 0.36’
CCI, + cysteine (18) 4.02 * 0.13d (6) 1.8 2 0.19f
CCI, + tryptophan + cysteine (16) 4.13 ? O.Ogd (6) 1.2 k 0.18/
y Rats were fasted overnight and then given Ccl, intraperitoneally as a 20% (v/v) solution in olive
oil at a dose of 5 ml/kg the following morning. Tryptophan (30 m&00 g body wt), cysteine (95 mgl
100 g body wt), or both were tube-fed 6 hr after Ccl,. Rats were killed 24 hr after Ccl,. Two or three
rats per group were used in each experiment.
b 1 = light (<25%) necrosis: 2 = moderate (25-50%) necrosis; 3 = marked (50-75%) necrosis;
and 4 = intense (>75%) necrosis.
c Number of rats in parenthesis. Means ? standard error.
d P < 0.01 (compared to control group).
e 0.05 > P > 0.01 (compared to CCI, group).
f P < 0.01 (compared to Ccl, group).

5% CO, and 95% 0, in stoppered 25ml Erlenmeyer flasks. For the evaluation of
protein synthesis, [i4C]leucine (0.18 p,mole/liter; 62.5 tKi/liter) was added to each
incubation flask and incorporation into protein was measured. Aliquots at begin-
ning of incubation and after 60 min were taken for measurement of radioactivity
in protein. Protein was assayed by the method of Lowry et al. (1951). For mea-
surement of lactic dehydrogenase (LDH) leakage, 100 ~1 samples were taken at
the beginning and after 60 min incubation of cells. The samples were centrifuged
at 600 t-pm for 5 min and LDH activity was measured using the supernatants
(Henry et al., 1960). This method was also used to obtain serum LDH activity.
Serum enzymes [glutamate oxalacetate transaminase (GOT), glutamate pyru-
vate transaminase (GPT), and y-glutamyltranspeptidase (GGT)] were assayed
using a SMA 12/60 (Technicon Instruments).
RESULTS
In three early experiments, L-tryptophan and L-cysteine were made up into
acidic solutions with HCl (pH =I) as described by De Ferreyra et al. (1983b),
and De Toranzo et al. (1983) and the solutions (3 ml/IO0 g body wt) were tube-
fed to the experimental groups of rats. Earlier we had observed that highly acidic
solutions of L-tryptophan were not as effective as L-tryptophan in water alone in
stimulating hepatic polyribosomal aggregation and protein synthesis. Therefore,
we determined the effects of tryptophan and cysteine under both conditions.
Using the highly acidic solutions of tryptophan and of cysteine, the degrees of
necrosis (histologically graded 0 to 4 according to severity) were as follows based
upon 7- 12 rats per group of three experiments: control, 0; Ccl,, 3.6 & 0.17; Ccl,
+ tryptophan, 2.5 + 0.39; Ccl, + cysteine, 3.0 ? 0.35; and Ccl, + tryptophan
+ cysteine, 2.2 + 0.28. The results of the subsequent experiments using unacid-
ified solutions of tryptophan and of cysteine are summarized in Table I. Such
solutions were also used in all subsequent experiments. The effects of the amino
acids singly or combined were greater when using unacidified than when using
 TRYPTOPHAN AND/OR CYSTEINE AND CCld LIVER INJURY 367
acidified solutions. The improvements based upon using histologic gradings, when
acidified solutions rather than unacidified solutions, respectively, compared with
Ccl, alone were as follows: Ccl, + tryptophan, 30 and 36%; Ccl, + cysteine,
16 and 45%; and Ccl, + tryptophan + cysteine, 39, 63%.
The liver weights of rats treated with Ccl, and followed by treatment with
tryptophan, cysteine, or both 6 hr later and 18 hr before killing are summarized
in Table 1. Rats that received Ccl, had significant increases in liver weights in
comparison to the control group. Similar increases in liver weights were observed
in rats treated with Ccl, in comparison to controls by Korsrud ef al. (1972),
where they observed significant increases in liver lipid in the CC&-treated rats.
In one experiment the percentage dry weights of livers of the control and exper-
imental groups were assayed and they revealed as follows: control, 29.2%; Ccl,,
30.1%; Ccl, + tryptophan, 32.5%; Ccl, + cysteine, 31.7%; and Ccl, + tryp-
tophan + cysteine, 31.6%. Histological evaluation of the livers of the control and
experimental groups are also summarized in Table I. The livers of the groups that
received Ccl, plus tryptophan, cysteine, or both appeared significantly better
(less hepatic necrosis) than did those of the Ccl, alone group. In one experiment,
a group of rats was killed at 6 hr after Ccl,, the time when tryptophan, cysteine,
or both were usually administered, and another group of rats was killed at 24 hr
after Ccl,. Livers of rats killed after 6 hr revealed histologically minimal (graded,
0.3) necrosis while livers of rats killed after 24 hr revealed marked (graded, 3.3)
necrosis of the liver. This indicates that the necrosis, visible histologically, de-
velops gradually and that the changes become most marked in the period between
6 and 24 hr after Ccl,, the time during which the tryptophan, cysteine, or both
have their protective effects (Table I). Rats of both sexes showed similar changes.
In another series of experiments hepatic polyribosomal aggregation and protein
synthesis (in vitro and in vivo) were evaluated in the control and experimental
groups of animals. Rats that received Ccl, alone revealed significantly greater
hepatic polyribosomal disaggregation than did control rats (Table II). Treatment
with tryptophan, cysteine, or both following Ccl, induced some improvement in
the hepatic polyribosomal profiles over the group that received Ccl, alone.
Table II summarizes the findings dealing with in vitro and in vivo hepatic protein
synthesis in the control and experimental groups. While treatment with Ccl, alone
caused a decrease in [14C]ieucine incorporation into hepatic proteins when mea-
sured in vitro or in vivo, treatment with Ccl, followed by tryptophan, cysteine,
or both caused some improvement (increases) in in vitro and in vivo hepatic
protein synthesis.
In consideration that alterations in pool sizes of the precursor (leucine) could
influence in vivo [‘4C]leucine incorporation into hepatic proteins, acid soluble
radioactivities were determined and considered as crude indicators of precursor
pool sizes. When acid precipitable counts per liver were corrected for acid soluble
counts per liver, the differences between groups were similar to those summarized
in Table II. Also, in three experiments, [14C]leucine incorporation into serum
proteins were measured. The specific activities (cpm/mg protein) of the serum
proteins, expressed in terms of percentage, were as follows: control, 100%; Ccl,,
51.7 2 24.2%; Ccl, plus tryptophan, 83.1 2 17.1%; Ccl, plus cysteine, 106.4 t
6.9%; and Ccl, plus tryptophan and cysteine, 71.8 ? 28.4%.
In two experiments [14C]orotic acid incorporation into hepatic RNA was as-
sayed in the control and experimental groups. The results, expressed as specific
368 WANG, VERNEY, AND SIDRANSKY
TABLE II
Effect of Tryptophan, Cysteine, or Both on Liver Polyribosomes and Protein Synthesis in
Rats Treated with Ccl,
[14C]Leucine incorporation into proteins
In vitro (cpm/mg RNA) using
Status of
polyribosomes Postmitochondrial In vivo
(monomer-dimerskotal supernatants Microsomes (cpm/mg protein)
Treatment ribosomes x 100) (%) (76)
Control (4) 47.5 2 2.13O (3) 100 (3) 100 (3) 100
CC], (3) 66.7 ” 5.78b (3) 74.3 zt 2.37’ (3) 72.8 ” 6.48 (3) 66.7 -t- 10.8
CCI, + tryptophan (4) 49.0 2 2.43d (3) 123.5 2 9.60’ (3) 109.8 e 9.83d (3) 107.7 I!Z Il.6
Ccl, + cysteine (4) 48.5 f 1.85” (3) 110.8 t 6.86p (3) 107.9 rf- 3.98p (3) 113.3 -t 2.1b.”
Ccl, + tryptophan
+ cysteine (4) 52.4 k 2.24 (3) 108.4 + 8.%d (3) 102.5 + 9.42 (3) 110.0 ? l.9b.d
a Number of experiments in parenthesis. Means f standard errors. In each experiment. 2-3 rats
were used in each group.
b 0.05 > P > 0.01 (compared with control group).
c P < 0.01 (compared with control group).
d 0.05 > P > 0.01 (compared with Ccl, group).
e P < 0.01 (compared with Ccl, group).

activities (cpm/mg RNA), revealed an 8% decrease in the Ccl, compared with

the control group; treatment with Ccl, and tryptophan alone caused a 5% in-
crease; and treatment with Ccl, and cysteine alone or with cysteine plus tryp-
tophan caused a 33 and 40% increase, respectively, over that of the CC& group.
Table III summarizes the activities of four serum enzymes, LDH, GOT, GPT,
and GGT, in control and experimental groups. In all cases the serum enzyme
activities revealed significant increases in rats treated with Ccl, over that in the
control group. Ccl,-treated rats that subsequently received tryptophan revealed
minimal drops in the activities of serum GOT and GGT but a significant drop in
serum GPT in comparison with the levels of the Ccl, group. Addition of cysteine
caused significant decreases in the activities of serum LDH, GOT, and GPT but

TABLE III
Effect of Tryptophan, Cysteine, or Both on Serum Enzymes of Rats Treated with Ccl,
Serum activities0
Ikatment LDH GOT GPT GGT
Control (10) 3.98 f 0.31b (13) 376 2 17.3 (13) 53 + 1.6 (13) 1.9 k 0.60
CC], (11) 49.97 f 4.41’ (14) 17,504 k l613? (14) 8213 2 579” (14) 12.1 ? 2.47’
Ccl, + tryptophan(11) 47.37 k 5.04c (14) 15,136 k 2261’ (14) 5220 2 71Fd (13) 9.4 ? 2.73’
Ccl, + cysteine (11) 19.13 2 4.49C.d (14) 10,427 k 1069“,d (14) 4661 ? 368’,d (14) 7.7 2 1.73’
Ccl, + tryptophan
+ cysteine (11) 15.30 k 2.73r,d (16) 9,262 2 l178C,d (16) 3946 2 420c,d (16) 4.6 ? 1.55f
u LDH = lactic dehydrogenase; GOT = glutamate oxaloacetate transaminase; GPT = glutamate
pyruvate transaminase; and GGT = y-glutamyltranspeptidase.
b Number of rats in parenthesis. Means f standard errors.
c P < 0.01 (compared to control group).
d P < 0.01 (compared to Ccl, group).
e 0.05 > P > 0.01 (compared to control group).
f0.05 > P > 0.01 (compared to Ccl, group).
 TRYPTOPHAN AND/OR CYSTEINE AND CC14 LIVER INJURY 369
TABLE IV
Effect of Tryptophan, Cysteine, or Both on Hepatic Glutathione Levels of Rats Treated with Ccl,
Liver glutathione

Number of umoleig ~mole/liver/100 g

Treatment experiments liver body wt
Control 5 9.8 2 1.05” 29.9 ‘- 3.93
ccl, 5 6.7 + 0.316 29.2 2 0.61
Ccl, + tryptophan 5 8.0 L 0.60 34.8 2 2.56
CCI, + cysteine 5 11.3 ?z 1.25’ 46.8 t 5.36b.d
CCI, + tryptophan
+ cysteine 5 10.0 2 0.59’ 40.4 t 4.04d

(1Means k standard errors.

b 0.05 > P > 0.01 (compared to control group).
c P < 0.01 (compared to Ccl, group).
d 0.05 > P > 0.01 (compared to CCI, group).

not in GGT in comparison with the Ccl, group. Addition of both tryptophan and
cysteine caused significant decreases in the activities in all four serum enzymes
in comparison to the Ccl, group. In one experiment, one group of rats was killed
at 6 hr after Ccl, while another group of rats was killed at 24 hr after Ccl, and
serum enzymes were assayed. The results for the 6- and 24-hr groups, respec-
tively, were as follows: GOT, 3538, 16,625; GPT, 1913, 6175; and GGT, 3, 8.
Thus, even at 6 hr after Ccl,, appreciable increases in enzyme activities of GOT
and GPT in comparison to control levels were present (Table III).
The liver glutathione levels in the control and experimental animals are sum-
marized in Table IV. Treatment with Ccl, caused a significant decrease in the
concentration but not in total levels of glutathione in the liver when compared to
the control group. Treatment with cysteine alone or with tryptophan following
Ccl, treatment induced an increase (concentration and total levels) of glutathione
over that in the Ccl, group.
Table V summarizes the findings dealing with isolated rat hepatocytes which
were treated in vitro with Ccl, alone or with Ccl, and tryptophan, cysteine, or
both. In a few preliminary studies, we investigated the amounts of L-cysteine or
L-tryptophan that needed to be added to obtain high levels of [i4C]leucine incor-
poration into proteins. Cysteine (0.005-1.0 mM) and tryptophan (0.1 mM) gave
maximal incorporation. Therefore, levels of 0.1 mM for L-cysteine and for L-
tryptophan were used in the subsequent in vitro experiments. Viability of the
hepatocytes after incubations were evaluated by trypan blue exclusion exami-
nation. Decreased viability of hepatocytes after Ccl, treatment was apparent. No
improvement occurred after the addition of tryptophan but significant improve-
ment occurred after the addition of cysteine alone or after tryptophan and cysteine
(Table V). Consistent with the viability data were the results obtained in studies
wherein LDH activity released into the medium was assayed (Table V). Hepa-
tocytes treated with Ccl, and cysteine or with Ccl, and cysteine plus tryptophan
when compared with those treated with Ccl, alone revealed a drop in enzyme
release.
In vitro [*4C]leucine incorporation into proteins was assayed using hepatocytes
of the control and experimental groups. Ccl, treatment caused a significant drop
in protein synthesis, while marked improvements were observed in the groups
370 WANG, VERNEY, AND SIDRANSKY
TABLE V
Effect of Tryptophan, Cysteine, or Both on Isolated Hepatocytes Treated in Vitro with Ccl,
[‘4C]Leucine [‘4C]Orotate
LDH incorporation incorporation
release into protein into RNA
Treatment Viability % % %

Control (4) 74.5 k 1.38“ (4) 100 (4) 100 (3) 100
ccl, 55.0 k 1.83’ 247.1 2 32.3b 51.0 k 8.4b 78.3 k 13.7
Ccl, + tryptophan 55.3 2 7.04’ 276.0 2 26.6b 122.8 i 20.1’ 88.5 r 14.3
CCI, + cysteine 66.0 t 1.63c,d 150.5 k 20.9b,’ 70.1 2 8.1 87.1 2 8.4
CCI, + tryptophan
+ cysteine 68.5 k 2.24’ 127.2 2 9.6b,’ 122.8 t 21.1’ 102.6 2 7.9

a Number of experiments in parenthesis. Means 2 standard errors.

b P < 0.01 (compared to control group).
’ 0.05 > P > 0.01 (compared to control group).
d P < 0.01 (compared to Ccl, group).
’ 0.05 > P > 0.01 (compared to CCI, group).

that also received tryptophan alone or together with cysteine (Table V).
[14C]Orotic acid incorporation into RNA of hepatocytes revealed no significant
changes among the control and experimental groups (Table V).
A cytologic study on isolated liver cells after incubation was undertaken in an
attempt to detect early morphologic alterations that were induced in vitro by Ccl,
alone or together with tryptophan, cysteine, or both. In three experiments, 100
viable hepatocytes from each group after incubation for 60 min were examined
and they were graded according to alterations in the cell surfaces. Each viable
cell was graded as follows: 0, normal with no protrusions; 1, blebs involving one
quadrant of the cell surface were present; 2, blebs involving two quadrants; 3,
blebs involving three quadrants; and 4, blebs involving entire cell surface (all
quadrants). These results are summarized in Table VI. Differences in cell viability
(Table VI) similar to those indicated in Table V were observed. The isolated
hepatocytes exposed to Ccl, that remained viable at the end of incubation re-
vealed cell membrane protrusions in 40.4% of the cells. Fewer defects were noted
with Ccl, and cysteine treatment (26.3%) and with Ccl, and tryptophan and
cysteine treatment (12.8%). Also the degree of the defects according to the
grading (Table VI) were most severe after Ccl, alone and least severe after treat-
ment with Ccl, along with tryptophan and cysteine where the changes observed
were similar to those found in the control group of hepatocytes.
DISCUSSION
De Ferreyra ef al. (1977, 1983a), and De Toranzo et al. (1983), have for many
years been concerned with Ccl,-induced hepatic necrosis and its prevention by
selected amino acids. Although aspartic acid, cysteine, and tyrosine were effec-
tive when given as late as 6 hr after Ccl,, the protective effects were no longer
evident when observations of Ccl,-induced necrosis were made at 72 hr, except
for cysteine, which retained its protective potential (De Toranzo et al., 1983).
More recently they reported that cysteine given 6 hr after Ccl, exerted a weak
preventive effect on Ccl,-induced liver necrosis while tryptophan did not (De
Ferreyra et al., 1983a). However, when both amino acids were given together a
marked protective effect was observed (De Ferreyra et al., 1983a). Based upon
 TRYPTOPHAN AND/OR CYSTEINE AND Ccl, LIVER INJURY 371
TABLE VI
Effect of Tryptophan, Cysteine, or Both on Incubated Hepatocytes Treated in Vitro with Ccl,
Cytologic grading’

Viabilityh 0 I 2 3 4
Treatment” % (%‘c)

Control 14.3 87.8 10.8 1.8 0.3 0.8

ccl, 52.0 60.0 24.3 3.8 3.5 8.8
Ccl, + tryptophan 63.7 65.5 23.0 4.5 1.5 5.5
Ccl, + cysteine 65.0 73.8 18.5 2.3 0 5.5
CCI, + tryptophan
+ cysteine 69.0 87.3 10.0 1.0 1.0 0.8
n Isolated hepatocytes were incubated for 60 min with Ccl, alone or with the addition of tryptophan,
cysteine. or tryptophan and cysteine (Methods are described in text). Values are the means of four
experiments.
b Viability of isolated hepatocytes was evaluated after 60-min incubations. Viability at 0 time was
82.3%.
c Cytologic grading of isolated hepatocytes after 60 min incubation was conducted as follows: 0,
normal cells with no protrusions; I, blebs (involving 1 quadrant) per cell; 2, blebs (involving 2 quad-
rants) per cell: 3, blebs (involving 3 quadrants) per cell: and 4. extensive blebs (involving entire 4
quadrants of cell surface) per cell.

these findings they speculated that cysteine may influence glutathione synthesis
which in turn may lead to the protection of cell membranes against peroxides
and free radicals (Meister, 1981; Reed and Beatty, 1980). Also, they speculated
that since tryptophan stimulates hepatic protein synthesis cysteine may also be-
come involved in this process to the benefit of the liver. The current study was
directed toward obtaining more information in regard to how cysteine and tryp-
tophan act in preventing Ccl,-induced hepatic necrosis.
In the present study tryptophan itself has been observed to have a beneficial
effect against Ccl,-induced hepatic necrosis (Tables I and II). This finding disa-
grees with those of De Ferreyra et al. (1983a), who described no improvement
due to tryptophan alone. This discrepancy may be related in part to their use of
acidified solutions of amino acids in many of their experimental studies (De Fer-
reyra et al., 1983b; De Toranzo et al., 1983). They mention that in many of their
experimental studies the amino acids used were dissolved in water adjusted to
pH 21 with HCl (De Ferreyra et al., 1983b; De Toranzo et al., 1983). We found
that acidified solutions of tryptophan or cysteine were less effective than the
solutions of each amino acid in water alone.
In view of the histopathologic findings of appreciable Ccl,-induced necrosis in
liver within 24 hr (Table I), it is not surprising that a number of chemical changes
were observed in the liver and serum. However, whether to ascribe these chem-
ical changes predominantly to the necrosis per se, to the injury of surviving liver
cells, or to both is not possible. In an attempt to determine how Ccl, may affect
viable liver cells directly, isolated rat hepatocytes were exposed to Ccl, alone or
with tryptophan, cysteine, or both for 60 min. These in vitro studies revealed that
following exposure to Ccl, there was a decrease in hepatocyte viability, an in-
crease in LDH activity extracellulary, and a decrease in [‘4C]leucine incorporation
into protein (Table V). Addition of tryptophan improved (increased) hepatic pro-
tein synthesis but did not enhance hepatic viability or prevent the release of LDH.
On the other hand, addition of cysteine improved hepatocyte viability and de-
372 WANG, VERNEY, AND SIDRANSKY

creased LDH release without appreciably increasing protein synthesis. However,

the addition of both tryptophan and cysteine improved all three parameters. In
comparing these results with those of in v&o studies (Table II), the overall effects
were similar but hepatic protein synthesis was less significantly increased due to
tryptophan in the in vivo studies (Table II) than in the in vitro studies (Table V).
This may be attributed in part or totally to the hepatic necrosis that had already
been induced in vivo by the Ccl,.
Other investigators (Perrissoud et al., 1981; Stacey and Fanning, 1981; Tyson
et al., 1983) have described the morphologic alterations in isolated rat hepatocytes
when they were incubated with Ccl,. Perrissoud et al. (1981), reported that the
addition of Ccl, to isolated rat hepatocytes rapidly affected the plasma mem-
brane, inducing blebs on the cell surface. Our current findings revealed that in
vitro treatment with tryptophan and cysteine diminished the appearance of sur-
face blebs due to Ccl, (Table VI) as well as the extracellular release of LDH
(Table V). Thus, under our in vitro experimental conditions, the addition of tryp-
tophan and cysteine was capable of reducing the degree of injury in isolated rat
hepatocytes due to CCL,, effects similar to those reported in the in vivo studies
(Tables I-III).
The sensitivity of alterations in the levels of several serum enzymes in detecting
CC&-induced liver damage in rats has previously been evaluated (Balazs et al.,
1961; Korsrud et al., 1972; Rees and Sinka, 1960; Zimmerman et al., 1965). In
our own study (Table III) it appeared that the following serum enzymes, according
to decreasing sensitivity, increased in activity in the CC&-treated rats: GPT, GOT,
LDH, and GGT. These findings with the first three enzymes are consistent with
the results reported by Korsrud et al. (1972). In regard to the degree of improve-
ment after treatment with tryptophan plus cysteine following Ccl,, the following
serum enzymes, according to decreasing degree of enzyme activity, were ob-
served: LDH, GGT, GPT, and GOT (Table III). Thus, the evaluation of serum
enzyme activities in relation to degree of improvement revealed some differences
in sensitivity compared to that observed with the hepatotoxin itself. Yet, in gen-
eral, all four enzymes revealed parallel responses.
The ability of tryptophan, cysteine, or both in particular to reduce the extent
of CC&-induced necrosis (present after 24 hr) when given 6 hr after Ccl, admin-
istration is of special interest. In the unprotected rats the intensity of the necrotic
process becomes marked at 24 hr while at 6 hr after Ccl, only a minimal degree
of necrosis is visible histologically. Thus, the improvement due to tryptophan and
cysteine suggests that they influence some process(es) which enable cells to sur-
vive rather than to die. Conceivably, tryptophan may act at the level of the nuclear
membrane, as suggested by other earlier studies (Sidransky, 1985), in influencing
protein synthesis and survival. Based upon an earlier study (Sidransky ef al.,
1977) and also on results from this study (Tables II and V), it appears that tryp-
tophan improves hepatic protein synthesis after Ccl, treatment. One aspect of
the mechanism which may be responsible for this improvement is that tryptophan
has been found to stimulate the availability of cytoplasmic poly(A)-mRNA after
Ccl, treatment (Sidransky et al., 1977). Likewise, tryptophan acts in this way in
rats after treatment with hypertonic NaCl which causes hepatic polyribosomal
disaggregation and decreased hepatic protein synthesis (Sidransky et al., 1976).
Indeed, tryptophan has been reported to improve hepatic protein synthesis after
treatment with a variety of hepatotoxic agents (Sidransky et al., 1982).
 TRYPTOPHAN AND/OR CYSTEINE AND Ccl4 LIVER INJURY 373

Cysteine is known to influence the level of glutathione in the liver. In our

present study the administration of cysteine alone or with tryptophan after Ccl,
treatment induced an increase in hepatic glutathione levels (Table IV). However,
the CC&-induced suppression of hepatic glutathione was minimal (Table IV). Al-
though it is therefore difficult to attach much significance to cysteine’s action in
increasing hepatic glutathione levels after Ccl,, it is possible that even small
changes (increases) may play a role in protecting cell membranes, as is suggested
by the results dealing with changes in serum enzyme levels (Table III) and in
LDH release (Table V). Indeed, cysteine appears to play an important role in the
maintenance of cell membrane permeability and in cell survival (Table V).
De Ferreyra rt ul. (1983a), has attributed the potentiating effect of tryptophan
given together with cysteine in diminishing the CC&-induced liver necrosis to the
stimulatory effect on hepatic protein synthesis. Earlier Tateishi et al. (1977). re-
ported that tryptophan administered to fasted rats suppressed the increase of
hepatic glutathione levels due to cysteine by possibly diverting the cysteine to
hepatic protein synthesis, De Ferreyra et ~1. (1983a), speculated that stimulation
of protein synthesis might be one mechanism by which cysteine might exert its
beneficial effects. However, in our present experimental study, tryptophan did
not suppress hepatic glutathione levels when given alone or together with cysteine
to Ccl,-treated rats (Table IV). Only tryptophan but not cysteine stimulated he-
patic protein synthesis in isolated rat hepatocytes treated with CCI, (Table V) yet
each of them did appear to stimulate hepatic protein synthesis when rats were
treated in GYO with CCI, (Table II). These findings suggest that, while tryptophan
has a stimulatory effect throughout, cysteine may be capable of stimulating he-
patic protein synthesis only following hepatic injury due to Ccl,.
At the present time it appears that tryptophan and cysteine each may have
specific actions which are protective against Ccl,-induced hepatic necrosis. While
the specifics of the actions of each of these amino acids need further elucidation,
it is clear that they are able to potentiate each other. Thus, while further explo-
ration is necessary as to mechanism(s), it is already evident based upon experi-
mental studies that the administration of tryptophan and cysteine might be con-
sidered as possible effective treatments for liver damage induced by acute ex-
posure to Ccl, in man.
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