The Effect of Cysteine on the Metabolic Changes Produced by
Two Carcinogenic N-Nitrosodialkylamines
in Rat Liver
I. J. MIZRAHIANDP. EMMELOT
(Department
of Hiochemistry,
Antoni tränLeeun-enhaek-Huis:
Amsterdam,
The Netherlands
Cancer Institute,
The \etherlanrls)
SUMMARY
Of the two N-nitrosodialkylamines studied, intravenous injection of diethylnitrosamine (DENA) produced a smaller inhibition of the in vitro incorporation of amino acids
into the proteins of the microsomal-soluble fraction of rat liver than did administra
tion of dimethylnitrosamine (DMNA). The liver glycogen was reduced in amount to a
greater extent by DMNA than by DENA.
Pretreatment of the rats with excess cysteine for 2 days markedly reduced both the
percentage inhibition of amino acid incorporation and the glycogenolysis produced by
DMNA. No such effects of cysteine were observed in the case of DENA. The microsomal N-demethylase of the cysteine-livers, which probably initiates the conversion
of DMNA to its actual toxic derivative, diazomethane, was markedly reduced in ac
tivity as compared with the enzyme of normal liver. Such was, however, not the case
with the microsomal N-deethylase, which initiates the conversion of DENA to diazoethane.
The later findings may explain the differential effect of cysteine on the metabolic
changes produced by the two N-nitrosodialkylamines in rat liver and provide strong
evidence for the mechanism by which the latter compounds are converted to their toxic
and carcinogenic derivatives.
Cysteine pretreatment also secured the survival of more than 50 per cent of the rats
which received a lethal dose of DMNA.
The results are discussed, and it is concluded that both the toxic and the carcino
genic effects of the N-nitrosodialkylamines are due to the in situ formation of dia/oalkanes, which are alkylating agents and appear to interact primarily with the endoplasmic reticulum of the liver cells.
The hepatic carcinogen DMNA1 produces toxic
changes in the liver of rats within a few hours after
its administration. Inhibition of amino acid incorporation (12, 36), loss of glycogen, accumulation
of lipide, and changes in the fine structure of the
ER have been observed (19, 20-22). Since the
ethyl homolog, DENA, has recently been reported
(3, 53) to be a stronger hepatocarcinogen but to be
less toxic than DMNA, it became of interest also
to study the effect of DENA on rat liver.
Previous work had led to the conclusion (12)
•
Abbreviations
used: DMNA
= dimethylnitrosamine
nitrosodimethylamine);
DENA = diethylnitrosamine
trosodiethylamine);
ER = endoplasmic
reticulum;
ribonucleicacid; s-RNA = soluble RNA.
Received for publication October 4, 1961.
(N(N-niRNA =
that DMNA per se was not toxic but that it acted
only after being converted to a toxic derivative,
Taking into account that N-demethylation was
probably involved (12, 36), it was considered
likely to trap the intermediate (N-hydroxymethyl-) or final reaction product (diazomethane)
of the metabolic conversion of DMNA by pretreating the animals with excess cysteine. If suecessful, the toxic effects of DMNA should be absent from the livers of cysteine-treated rats.
Cysteine was indeed found to counteract the toxic
manifestations
of DMNA,
«Ct. However,
case of DENA,
HO such effect was obtained
ill the
which
in itself appeared
to be less
including
its
lethal
ef-
tox¡c than DMNA. An explanation for the differential effect of cysteine in regard to the two N-
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research.
340
Cancer Research
Vol. 22, April 1962
and 7 the cysteine-treated rats were killed 15 hours
after receiving the last, i.e., fourth dose of cysteine.
For the amino acid incorporation experiments
(Tables 1, 3, and 5) the excised livers were homog
enized in the medium X of Zamecnik and Keller
MATERIALS AND METHODS
(18, 67), 2.5 ml. medium being used per 1 gm. of
Male albino rats of the inbred strain R-Amster
fresh weight of liver. In the experiments of Table 4
the bicarbonate of the latter medium was replaced
dam, weighing ca. 200 to 300 gm., were used un
by Tris buffer (0.035 M, pH 7.8). The postmitoless indicated otherwise. L-Cysteine, 150 mg. dis
solved in 1.5 ml. distilled water (pH 7.4), was in
chondrial fraction was prepared by centrifugation
jected subcutaneously twice daily, in the early for 15 minutes at 9000 X g. Then 0.7 ml. of the
morning and late afternoon, on 2 consecutive days. 9000 X g supernatant with additions as described
(18), including 58.5 mamólesDL-leucine-1-C14con
In all experiments food was present ad libitum.
taining
375,000 counts/min, to give a final %'olume
Care was taken that the rats used in an experi
ment were of the same age and approximately of of 1.0 ml., was incubated in duplo for 60 minutes
at 37°C. The radioactivity contained in 10 mg. of
the same body weight.
the isolated proteins was measured in a gas-flow
TABLE1
counter, correction being made for background
INHIBITION
OFTHEin VUnINCORPORATION
OFLEUCINE- and self-absorption. The results were expressed
1-C" INTOTHEPROTEINS
OFTHE MICROSOMAL-SOLUeither as percentage inhibition of amino acid incor
BLEFRACTION
FROM
LIVEROFRATSGIVENINJECTIONS poration produced by the N-nitrosodialkylamines
(Tables 1 and 5) or as the m/imoles leucine-C14
OFDMNAANDDENA
Results are expressed as per cent inhibition in relation
(Table 4) incorporated into the proteins per flask
to the amino acid incorporation of liver preparations from rats
(or per mg. microsomal RNA). Microsomes were
receiving saline which were run simultaneously in each ex
spun down for 60 minutes at 105,000 X g, and the
periment. These preparations contained from 179 to 191counts/
min/mg protein. Each value listed was calculated from the
protein and RNA of the pellet and the protein of
data obtained with liver preparations from one rat, treated
the supernatant were determined according to
as indicated, and incubated in duplo.
Lowry et al. (42) and Schneider, as modified by
Scott et al. (55). The soluble fraction used in the
INHIBITIONExp.
li CENT
experiments of Table 3 consisted of the 105,000 X
DOSE(MO/KGBODY
g supernatant. Liver glycogen was measured by
WT.)DMNA,
1<<?)762046154867Exp.
2(9)758391254668Exp.
3(9)618286101729Exp.
4(c?1)532532
the anthrone method. The production of formalde
hyde and acetaldehyde from DMNA, DENA, and
100DMNA,
4-monomethyIaminoazobenzene by the postmito50DMXA,
chondrial liver fractions (15 min. at 12,000 X firin
100DENA,
140DENA,
phosphate buffer) was measured according to
200DENA,
LaDu et al. (39). Total volume was 5 ml., contain
50DENA,
ing an amount of fraction derived from 250-750
100DENA,
¿00HOURSBETWEENINJECTIONANDSACRIFICE5202055202020l'i
mg. of wet weight of liver in the various experi
ments and 25 Amólesof the nitrosodialkylamines
or 10 /¿moles
of the azo dye. The latter was added
DMNA and DENA, dissolved in 0.1 ml. saline, in 0.05 ml. ethyl alcohol. Blanks were subtracted.
in the quantities mentioned under "Results,"
Incubation was carried out for 60 minutes at
were injected into the tail vein. Controls received 37°C. All experiments were carried out in duplo,
saline only. Both untreated rats and rats treated
the results obtained being in close agreement.
with cysteine received these injections. In the ex
RESULTS
periments listed in Tables 3, 4 and 6, and Chart 2,
the N-nitrosodialkylamines were administered to
A COMPARISON
OF THE INHIBITIONSOF AMINO
the cysteine-treated rats on the early morning fol
ACIDINCORPORATION
PRODUCEDBY DENA
lowing the 2d day of cysteine treatment, together
ANDDMNA
with another—i.e., the fifth, dose of cysteine. The
Table 1 lists a number of our experiments in
animals were killed 5 hours later (Tables 3, 4, and which the in vitro incorporation of leucine-1-C14
6) or 3-9 hours later (Chart 2). The fifth dose of into the proteins of the postmitochondrial frac
cysteine was omitted in most of the experiments of tions (microsomes plus soluble fraction), obtained
Table 5, the rats being sacrificed 3-20 hours after from rats treated with DENA and DMNA, were
receiving DMNA. In the experiments of Tables 2 compared (for further data cf. also Table 4). The
nitrosodialkylamines came from experiments in
which the N-dealkylation of these compounds by
liver microsomes was studied. A preliminary ac
count of part of this work has been published (21).
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research.
MiZRAHi AND EMMELOT—Cysteine and Hepatic Injury
inhibition of amino acid incorporation
produced
by DENA was much smaller than that produced
by DMNA. In the many experiments carried out
with DMNA no difference in response between
males and females has been observed. In the pres
ent experiments,
however, the impression was
gained that the amino acid incorporation system
of the males was somewhat more susceptible to
DENA than that of the females. In a separate se
ries of five experiments with male and female rats
of about the same age and body weight per experi
ment, the average inhibition of amino acid incor
poration
in the liver preparations
of males
amounted to 35 per cent (range: 24-50 per cent)
and that of females to 20 per cent (range: 15-25
per cent) 5 hours after receiving 200 mg DEN A/kg
body weight. In three additional experiments with
male rats of the inbred strain U, 100 mg DMNA/
kg produced an inhibition of amino acid incorpora
tion of 66, 61, and 59 per cent, whereas 200 mg
DENA/kg
caused an inhibition of 30, 35, and 29
per cent, respectively, 4 hours after injection.
ever, be excluded that the acetaldehyde produced
from DENA in the experiments of Table 2 was
partly due to the enzymic conversion of the second
ethyl group, thus resulting in less diazoethane than
was actually accounted for by the acetaldehyde
measured. Since this argument may, however, also
be applied to the case of DMNA, the tentative
conclusion might be drawn that the quantitative
difference in effects of the two N-nitrosodialkyl
amines is due to the difference in toxicity of the
two reaction products, diazomethane
and diazo
ethane (compare below).
TABLE 2
N-DEALKYLATION OF DMNA AND DENA BY THE MlCROSOMAL-SOLUBLEFRACTION FROM LIVERS OF NOR
MAL AND CYSTEINE-TREATED RATS
In each experiment, carried out in duplo, two normal and
two cysteine-treated
rats were used. Results are expressed
per amount of fraction derived from 1 gm. of fresh weight of
liver. The last entry (marked *) represents the results of an
experiment in which whole homogenate
was used (expressed
per 1 gm. of fresh weight of liver).
N-DEALKYLATIONOF DENA ANDDMNA
BY LIVER MICKOSOMES
The LD50 of DENA (m.w. = 102) has been re
ported (53) to be 210 mg/kg, death following 3-14
days after injection. By contrast, injection of 50
mg DMNA/kg
(m.w. = 74) led to the death of all
our rats within 3 days.
It is of interest that this difference in toxicity
between the two N-nitrosodialkylamines
is also
reflected in the degree of inhibition of amino acid
incorporation
and in the liver glycogen content
(see below). The quantitative
difference between
the effects of DMNA and DENA may be due to
one of the following two reasons: If, as appears
probable from the present investigations, DMNA
and DENA are converted to their actual toxic
derivatives by two separate enzymes of the "drugmetabolizing" type (9), then either less of the toxic
derivative might be produced from DENA than
from DMNA or the toxicity of the two derivatives
might differ. Liver microsomes are known to be
capable of dealkylating
both N-methyl and Nethyl groups, and N-demethylation
to formalde
hyde may, but need not, occur about twice as fast
as N-deethylation
to acetaldehyde (26). However,
the results showed that not less but rather more
acetaldehyde
was produced from DENA than
formaldehyde
from DMNA by the liver microsomes (Table 2). N-Dealkylation
of one of the
alkyl groups of the N-nitrosodialkylamines
gives
rise to N-nitrosomonoalkylamines
which readily
yield (28) the corresponding
diazoalkanes
(com
pare reactions 1 and 2 in Chart 1). It cannot, how
341
PRODUCED
PRODUCED
BYNormal
FROMDENA
BYNormal
FROMDMXA
RATS0"
o"
0"cf9
liverprepn.Cysteineliverprepn.(/¿moles)0.36
liverprepn.ales)0.39
liverprepn.(M>D0.550.39
0.37
0.39
0.300.30
0.440.26
0.18
0110.09
0.430.27
9
0.37
0.04
99FORMALDEHYDE
0 «*0.15
0.37
0.36
0.31
0.30
0.08*ACETALDEHYDE
0 37*Cysteine0.39*
DIFFERENTIAL EFFECT OF CYSTEINE AD
MINISTRATIONON THE Toxic EFFECTS
PRODUCED BY DMNA AND DENA
The effect of cysteine on rat liver.—Administra
tion of 150 mg. cysteine twice daily for 2 days to
rats had a marked effect on the composition and
metabolism of the liver. The amount of microsomal material obtained from the cysteine-livers
was consistently found to be increased, as com
pared with that from normal liver. Soluble protein
also increased, but soluble UNA did not. Glycogen
decreased by 10-30 per cent. The water content of
the cysteine-livers was similar to that of normal
liver, but the liver weight (per 100 gm. body
weight) was frequently higher in the animals re
ceiving cysteine. The microsomal-soluble
fraction
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research.
Cancer Research
342
Vol. 22, April 1962
obtained from the cysteine-livers incorporated
from 1.4 to 2.2 as much leucine-C14 into its pro
teins per mg. of microsomal UNA as did the cor
responding fraction of normal livers. Changes in
the activities of a number of microsomal mem
brane-bound enzymes were also apparent after the
cysteine treatment. The fine structure of the ER of
the cysteine-liver cell was markedly changed. A
preliminary account of these observations has been
presented.2
know whether the pool of endogenous leucine has
changed or not. Otherwise, a change in the incor
poration of C14might simply be due to a difference
in dilution of the added labeled compound. In the
present investigation this problem was approached
by measuring the labeling of the s-RNA by amino
acid. The soluble fractions (105,000 X g supernatants) were prepared from 10 gm. of normal and
cysteine-liver obtained from controls and rats
treated with DMNA (100 mg/kg, 5 hours). In
cubation was carried out with 140 m jumólesDLFORMATIONOF C' ^LABELEDLEUCYL-S-RNA
leucine-1-C14 and 250 jumólesadenosine triphosFor the correct interpretation of amino acid-C14 phate for 15 minutes at 37°C. Leucyl-C14-s-RNA
was isolated by the phenol method and purified by
incorporation into proteins under different condi
tions of the biological material, it is imperative to gel filtration on Sephadex (6, 7). Table 3 illustrates
DMNA
DIAZOMETHANE
.N-N
xC2H4OH
/H
-»CH3CHO+O.N-N-C2H5-
-C2H4N2
(2)
DIAZOETHANE
DENA
CHART 1.—N-dealkylation of N-nitrosodialkylamines by
liver microsomes. The enzymes of the "drug-metabolizing"
type (9), dependent on reduced triphosphopyridine nucleotide
(TPNH2) and oxygen, catalyze the reaction. The monoalkylnitrosamines are unstable and rearrange to diazoalkanes. The
formation of diazoalkanes was first suggested by Rose (cf. 36).
The toxieity (and probably also the carcinogenicity) of the
X-nitrosodialkylamines is governed by the following param
eters: (a) The rate of formation of the diazoalkanes.—No diazo
compound is formed when the hydrogens at the o-C atom of
one or both of the alkyl chains are missing (tert. C atom). In
the aryl series, the lack of carcinogenicity of N-nitrosodiphenylamine (3) is a case in point. The rate of enzymic Ndealkylation may decrease with increasing chain length
OC2H6! [26]). When, however, both alkyl groups are elimi
nated no diazoalkane can be formed. Since the N-dealkylating
enzymes are located in the lipoprotein membranes of the ER,
a good lipide solubility of a compound will favor its conversion.
The finding that of the water-soluble N-dialkylnitrosamines
DENA is de-ethylated at least as rapidly as DMNA is de-
methylated may be connected with the better lipide solubility
of the former compound. (6) The rate of reaction of the diazo
alkanes with tissue constituents and water.—Increasing the
number of C atoms in the alkyl chain leads to a greater reactiv
ity of the diazoalkanes as a result of an electron-repelling
effect. In aqueous medium diazoalkanes are converted to
alkyldiazonium ions. Diazoethane will thus form diazonium
ions at a faster rate than does diazomethane. However, of the
carbonium ions CHt and CzIIt subsequently arising from the
alkyldiazonium ions, the former are more reactive than the
latter. As recently pointed out by us (reference in footnote 4)
the intermediary monoalkylnitrosamine (alkyldiazohydroxide)
may also form alkyldiazonium ions directly (through addition
of H+ and splitting off of HSO)instead of yielding a diazoalkane
first. Compounds containing one tert. C-atoni linked to N
(compare D. F. Heath, Nature, 192:170, 1961) may be con
verted in this manner; the tert. carbonium ions thus arising are
more stable than n-carbonium ions, (o) The qualitative changes
produced in tissue constituents (compare "Discussion").
TABLE 3
one out of four experiments which yielded identical
LACKOFEFFECTOFCYSTEINE
AND/ORDMNAADMINIS results. Both the amounts of s-RNA recovered and
TRATION
ONTHEFORMATION
OFLEUCYL-CIJ-S-RNA their specific activities were the same regardless of
BYTHESOLUBLE
FRACTIONS
OFRATLIVER
the previous treatment applied to the animals.
Methods mentioned in the text. DMNA: 100 mg/kg, 5
hours.
ratsNone
Treatment
offreshs-RNA/gm
of
RNA)537
weight
liver)0of
61
(saline)
543
0 59
Cysteine (saline)
518511(nig
DMNACysteine,
0.600.59
DMNALeucyl-C"-a-RNA(counts/mm/mg
EFFECT OF CYSTEINEADMINISTRATION
ON THE
INHIBITIONOF AMINOACIDINCORPORATION
PRODUCEDBY DMNA ANDDENA
a) Normal and cysteine-treated rats were in
jected intravenously with saline, DMNA (100
2 Emmelot, P.; Mizrahi, I. J.; Naccarato, R.; and Benedetti,
E. L. Changes in Function and Structure of the Endoplasmic
Reticulum of Rat Liver Cells after Administration of Cysteine.
J. Cell Biol., 12-.177-H1, 1963.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research.
MiZRAHi AXD EMMELOT—Cysteine and Hepafic Injury
mg/kg) or DEN A (200 ing/kg). As in the former
experiments, the cysteine-treated rats received an
other dose of 150 mg. cysteine subcutaneously, to
gether with the N-nitrosodialkylamine or the sa
line. The animals were killed 5 hours later. The
postmitochondrial
fractions, containing microsomes and cell sap, were obtained by centrifugation of the liver homogenates and incubated with
leucine-CH. The counts/min/mg of the isolated
proteins were multiplied by the amount of protein
(in mg.) found by chemical means, to give the
total C14activity present in the proteins. Because
of these values the m/anoles leucine-C14 incorpo
rated into the proteins of each flask were calcu
lated. Table 4 lists the results of five such experi
ments. It is shown that, whereas the absolute in
hibition of amino acid incorporation produced by
343
DMNA (mamóles leucine-C14 less incorporated)
was but little less in the cysteine- than in control
liver preparations, the percentage inhibition was
much less in the former than in the latter cases.
By contrast, both the absolute and percentage in
hibition produced by DENA were more pro
nounced in the cysteine- than in the control liver
preparations. Although DMNA was more active
than DENA in the control livers, the reverse ap
peared to be true in the cysteine-livers.
6) Table 5 illustrates the percentage inhibition
of amino acid incorporation into the proteins of the
microsomal-soluble fraction produced by DMNA
under different experimental conditions. In these
experiments the rats were treated with cysteine
for 2 days, as usual. DMNA (100 mg/kg) was ad
ministered on the morning of the 3d day, but no
TABLE4
EFFECTOFCYSTEIXEADMINISTRATION
ONTHEINHIBITIONOFAMINOACIDINCORPORATION
IN THE
POSTMITOCHONDRIAL
FRACTIONS
OFLIVERSFROMRATSRECEIVINGDMNA ANDDENA
Conditions and calculation of data as described in the text. The mamólesleucine-C14incorporated into protein/mgof microsomal RNA in the first experiment amounted to 0.75, 0.35, 0.56, 1.21, 0.90, and 0.77, respectively. Comparable results were obtained
in the other experiments of the present series.
PROTEINExp.10.920.440.701.961.491.34Exp.«0.960.410.691.891.461.22Exp.S1.050.500.711.941.270.95Exp.40.880.440.591.851.480.90Exp.50.810.400.621.891.581.01Av.
LECCDiE-C" INCORPORATED INTO
INHIBITIONAbso
X- \ITHOSODIALKYLAMIXEDMXADENADMNADENACYSTEINE—-+++MjlMOLES
exp.,1-50.920.440.661.901.441.06Av.
lute*0.480.260.460.84Percent53882444
* Mamólesleucine-C14less incorporated.
TABLE 5
INHIBITIONOFAMINOACIDINCORPORATION
INTOTHEPOSTMITOCHONDRIAL
FRACTIONS
OFLIVERS
OFNORMALANDCYSTEINE-TREATED
RATSAFTERADMINISTRATION
OFDMNA
Cysteine treatment mentioned in the text. In experiments d and e an additional 150 mg. cysteine was given
at the time of injection of DMNA (100 mg/kg) and 5 hours afterwards. In each of the experiments normal and
cysteine-liver preparations of rats given injections of saline were included (compare Table 1); the counts/min/mg
protein of these preparations are listed in parentheses. Experiment e was carried out with LE-hybrid rats ((?)',
the liver preparations of these rats consistently showed a lower protein radioactivity than those of R-rats. Other
experiments of the type listed under d and e gave values intermediate between those listed in this table.
INHIBITIONExp.a57
HOURS
BETWEENINJECTION
ANDSACRIFICE3310102020DMNA++++++CYSTEDÕE—+—+—+P£B
CENT
(183)25
(173)12
(181)25
(447)66
(269)66
(392)74
(185)66
(184)66
(180)16*(323)Exp.e80
(125)40*(25S)
(420)76
(272)Exp.C52
(183)90
(173)89
(391)Exp.b48
(37l)Exp.d51
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research.
344
Cancer Research
extra cysteine was given. The animals were killed
after 3, 10, and 20 hours. Experiments a-c (Table
5) show that the protection afforded by cysteine
against the DMNA inhibition gradually disap
peared. When, however, extra cysteine (150 mg.)
was administered at the time of injection of
DMNA and after another 5 hours, the protective
effect of cysteine remained apparent 10 hours after
injection of DMNA (Exps. d and e, Table 5).
Longer periods were not studied. These results in-
Vol. 22, April 1962
stimulatory effect on the amino acid incorporation
system of the normal liver preparations (increase
in protein labeling from 4 to 14 per cent). A similar
result was obtained in five of the experiments with
the liver preparations of the DMNA-treated rats.
In three experiments a slightly higher stimulation
of incorporation of amino acid into the proteins of
the DMNA- than into those of the normal liver
preparations was produced by the sulfhydryl com
pounds, resulting in a decrease of about 10 per cent
in the DMNA inhibition. A significant counterac
tion of the DMNA inhibition by added cysteine
and glutathione was encountered in only one ex
periment, the DMNA-inhibition being reduced
from 65 to 35 and 40 per cent by addition of
cysteine and glutathione, respectively.
EFFECT OF CYSTEINEADMINISTRATION
ON THE
Loss OF LIVER GLYCOGENPRODUCED
BY DMNA ANDDENA
Administration of DMNA (100 mg/kg) leads to
a rapid loss of glycogen from rat liver. Chart 2
illustrates the time course of this phenomenon.
Although the initial rate of glycogen disappear
ance differed in individual experiments, the bulk
of the glycogen was always lost in the period from
3 to 10 hours after injection of DMNA. Individual
or different batches of rats appeared to vary to
some extent in their susceptibility toward DMNA,
as shown by the initial rate of glycogen disappear
ance, inhibition of microsomal protein synthesis,
and the macro- and microscopic damage to the
liver. Moreover, the normal liver-glycogen content
appeared to vary among the rats; the values cen
tered around approximately 6 and 8 per cent of
wet weight of liver. Since the loss of glycogen from
the liver cells resulting from the administration of
DMNA occurred in a nonuniform manner,3 certain
cells being more affected than others, the glycogen
O
3579
determinations were always carried out with two
HOURS AFTER DMNA
samples of 200 mg. of wet weight of minced liver
CHAHT2.—Time course of glycogen disappearance from
each.
normal and cysteine-livers after administration of DMNA.
Cysteine administration in itself also resulted in
DMNA: 100 mg/kg. Conditions as in Table 6. • •
some loss of glycogen from the liver (10-30 per
normal rats; O
O, cysteine-treated rats.
cent decrease). However, as shown in Chart 2 and
Table 6, no or little glycogenolysis was produced
dicated that cysteine, or a cysteine metabolite,
by DMNA in the livers of rats pretreated with
had to be present in the liver in order to counteract
cysteine. The loss of liver glycogen following injec
the toxic effect of DMNA.
c) Nine experiments were carried out in which tion of DENA was smaller than that following
DMNA. However, whereas cysteine treatment
the effect of the in vitro addition of cysteine or re
duced glutathione (20 Amóles/1ml final medium) protected against the glycogenolysis produced by
on the incorporation of leucine-1-C14 into the pro
DMNA, it had no such effect in the case of DENA.
teins of the microsomal-soluble fraction from livers Thus, an exact parallel existed between the results
of normal and DMNA-treated (3-5 hours) rats obtained on liver glycogen and on amino acid in
corporation.
was studied. In all experiments the sulfhydryl
3 Histochemical observations by Dr. E. L. Benedetti.
compounds were found to lack any significant
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research.
MiZRAHi AND EMMELOT—Cysteine and Hepatic Injury
EFFECT OF CYSTEINE ADMINISTRATIONON THE
N-ÃœEALKYLATIONOF DMNA AND DENA
BY LlVER
MlCROSOMES
a) The above findings suggested that cysteine
was not active in protecting4 against the interac
tion of the DENA derivative with the processes of
amino acid incorporation
and glycogen metabo
lism—i.e., at 61 and 62 of the following scheme:
345
ment may explain the reduced toxicity of DMXA
under the latter condition. The finding that the
absolute inhibition of amino acid incorporation
produced by DMNA (Table 4) was only slightly
less in the cysteine- than in the normal liver prepa
rations may now be accounted for. Not only were
these microsomes increased in amount per unit
weight of liver but calculation of the m/anoles
amino acid incorporation
N-nitrosodialkylamines
—
* toxicderivatives
62 glycogen metabolism
For this reason the possibility was considered that
cysteine administration
led to an inhibition of the
conversion of DMNA, but not of DENA, to its
toxic derivative (at a of the above scheme). As
shown in Table Z the experimental evidence was
fully in favor of this interpretation.
The livermicrosomal N-demethylase
converting DMNA to
formaldehyde
was markedly reduced in activity
after cysteine administration.
At least 50 per cent
less formaldehyde was produced by the cysteineliver microsomes than by the normal liver microsomes derived from 1 gm. of fresh liver. In view of
the increase in microsomal protein (40 per cent and
higher), the specific activity of the N-demethylase
was reduced by far more than 50 per cent after
cysteine treatment. However, the N-deethylase of
the cysteine-liver microsomes, converting DENA
to acetaldehyde, showed the same activity as that
of the normal liver microsomes derived from 1 gm.
of fresh liver.
Table 7 shows that the N-demethylase
convert
ing the methyl group of 4-monomethylaminoazobenzene to formaldehyde was also inhibited after
cysteine administration.
The nature of the inhibi
tion of the microsomal N-deme thy lase (s) by cys
teine is under further investigation.
So far, the
addition of cysteine to normal liver preparations
incubated with either DMNA or 4-monomethylaminoazobenzene
has also been found to result in
a decreased recovery of formaldehyde.
The pre
liminary finding that reduced glutathioiie was not
active in the latter respect tends to eliminate the
possibility that sulfhydryl groups reacted with the
N-methylol derivative or competed for formalde
hyde with the formaldehyde trapping agent semicarbazide used in the assay.
6) The decreased activity of the N-demethylase
of the liver microsomes following cysteine treat4Protection by cysteine might be due to shielding tissue
SH-groups or trapping the alkylating agent.
leucine-C14 incorporated into the proteins per mg.
microsomal RNA also showed that the cysteineliver preparations were from 1.4 to 2.2 as active as
the normal liver preparations.
Thus, in the cys
teine liver a smaller amount of toxic DMNA de
rivative (diazomethane)
had interacted
with a
more abundantly
present system that per unit
TABLE 6
EFFECTOFCYSTEINEADMINISTRATION
ONTHELIVER
GLYCOGEN
OFRATS5 HOURSAFTERINJEC
TIONOFDMNA ANDDENA
Rats were treated with cysteine on 2 consecutive days.
The next morning DMNA (100 mg/kg) or DENA (200 mg/kg)
was injected intravenously and another dose of cysteine subcutaneously. Controls received saline. Each determination
was carried out in duplo with 200 mg. of fresh weight of liver
mince. Listed are the average values and, in parentheses,
the range and the number of rats used.
GLYCOGENCONTENT(PER CENTop FRESHWEIGHTOF LIVER)
CYS
TEINE—+Saline7.2
(5. 6-8.
(8)6.2
5)
(4.2-5.5)(4)3.8
(4. 6-8.0) (8)DMNA3.6(2.7-5.5X8)6.0(4.2-7.8)(8)DENA4.8
(2.7-4.6)(4)
amount of synthetic sites showed an increased ca
pacity to incorporate amino acids in vitro as com
pared with the case of normal liver. This situation
may be envisaged to result in a reduction of the
percentage inhibition but not necessarily in a re
duction of the absolute inhibition produced by
DMNA.
In the case of DENA the same amount of
diazoethane
was formed in situ irrespective of
cysteine treatment,
and its interaction with the
cysteine-activated
microsomal amino acid incor
poration system may account for the increase in
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Cancer Research
346
the inhibition of the latter
with that of normal liver.
system
as compared
COUNTERACTIONOF THE LETHAL EFFECT
OF DMNA BY THE PRIOR ADMINIS
TRATION OF CYSTEINE
Rats (cf ) receiving a dose of 75 mg DMNA/kg
body weight succumbed within 40 hours. With 50
mg DMNA/kg, death followed from 30 to 60 hours
after the injection. As shown in Table 8 pretreat
ment of the rats with cysteine either markedly
prolonged (four of thirteen rats) or secured the
survival (seven of thirteen rats) of rats treated
with a lethal dose of DMNA. In these experiments
DMNA was injected intravenously
at the same
time as the last dose of cysteine was administered,
i.e., in the late afternoon of the 2d of 2 days of
cysteine treatment.
Thus, cysteine appears to protect also against
the over-all toxicity of DMNA. In this connection
it is of interest to note that, although the absolute
inhibition of amino acid incorporation
produced
by DMNA was the same in the control- and
cysteine-liver preparations,
more amino acid was
incorporated
in the preparations
from cysteine-
Vol. 22, April 1962
livers of rats receiving DMNA than in those from
normal liver (Table 4).
Preliminary observations with the electron mi
croscope carried out by Dr. E. L. Benedetti of this
Institute have shown that the cysteine regimen,
though in itself causing definite alterations in the
fine structure of rat liver cells, markedly counter
acted the manifestation of the changes in liver fine
structure (swelling of ER and lipide accumulation)
resulting from DMNA.
INHIBITIONOF AMINOACIDINCORPORATION
INTOTHE PROTEINSOF LIVER SLICES
PRODUCEDBY DMNA in Vitro
Next to the inhibitory effect of injected DMNA
on the in vitro incorporation of amino acids into
the proteins of the isolated liver microsomal-soluble fraction, addition of DMNA to normal liver
slices also leads to an inhibition of amino acid in
corporation into the slice proteins (12, 36). In the
present experiments it was investigated
whether
(a) DMNA could interfere with amino acid incor
poration into liver slices other than those from
intact adult rats and (6) a differential inhibition by
DMNA of amino acid incorporation into the vari-
TABLE 7
EFFECTOFCYSTEINE
ADMINISTRATION
ANDADDITION
ONTHEN-DEMETHYLATION
OF4MONOMETHYLAMJNOAZOBENZE.NE
BYTHEPOSTMITOCHONDRIAL
FRACTION
FROMLIVER
Each flask contained an amount of fraction derived from 400 mg of fresh weight of liver. Listed
in parentheses is the formaldehyde release per 10 mg. of microsomal protein; ¿0/itnoles cysteine added
in vitro per 5 ml. medium as indicated.
flG.
FORMALDEHYDE
LCTKBNormal
Normal, cysteine in vitro
Cysteine, in viroEip.
117.
ï17.7+.2
323.5+
3+. 2 (25. 2)
7.4+. 2 (10.8)
11. 7+ .3 (12.0)Bip.
(25.3)
9.7+. 3 (14.2)
11. 7±.5 (11 3)Eip.
11.0+.6
15.S+.3
.8 (36.7)
(17.2)
(14.1)
TABLE 8
ous cellular organdÃ-es of rat liver slices could be
observed.
a) Hypophysectomized rats and newborn mice.—
Leucine-1-C14 incorporation
into the proteins of
liver slices from adult hypophysectomized
rats (10
THECYSTEINE-TREATED
SURVIVORS IN"
AFTER40hr.44570hr.43590hr.434140hr.324ÃŽ5months322
GROUP
days after operation) was inhibited after preincuBODYWEIGHTRATS(APPROX.
bation of the slices with DMNA (12.5 pinoles, 400
mg. of wet weight of liver slices, 2.5 ml. KrebsGM.)290250200DMNA(MC/KO)757550No.RATS4*4*5tNo.
Ringer phosphate buffer, 100 per cent oxygen).
This inhibition (25-37 per cent) was somewhat less
than that observed with liver of pair-fed controls
(38-50 per cent). In similar experiments with liver
slices
from newborn mice (1-24 hours after deliv
* None of the four untreated rats survived 40 hours.
ery) DMNA was found to inhibit the amino acid
f None of the five untreated rats survived 60 hours.
incorporation
for 40-60 per cent. The postmito\ Two rats died 10 days after injection of DMNA.
COUNTERACTION
OFTHELETHAL
EFFECTOFDMNA
BYADMINISTRATION
OFCYSTEINE
TORATS
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research.
MizRAHi AXD EMMELOT—Cysteine and Hepatic Injury
chondrial fraction obtained from the livers of these
newborn mice (in all, 241 animals were used) pro
duced 0.36 /¿molesformaldehyde/postmitochondrial fraction derived from 1 gm fresh weight of
liver/hour.
The corresponding
fraction derived
from the livers of young rats (2 weeks old) was at
least as active. The results obtained with the new
born animals were somewhat unexpected, since it
has been shown (37) that the corresponding en
zyme which X-demethylates
monomethyl-4-aminoantipyrine is not active in newborn mouse liver.
However, experiments carried out by Dr. J. A. J.
Brouwers some years ago in this laboratory had
failed to show an enhancement of the N-demethylation of DMNA after the injection of methylcholanthrene and benzpyrene (cf. 16). The micro-
347
DISCUSSION
Metabolic conversion of y-nitrosodialkylamines
to diazoalkanes.—Arguments
have been presented
previously (12) for the view that DMNA in itself
is not toxic but that it acts as such only after being
converted to a reactive derivative by the enzymes
of the "drug-metabolizing"
type (9) present in the
microsomal membranes of liver (reaction 1, Chart
1). Support for this view can also be found in the
work of Magee and co-workers (36, 46). Additional
strong support is provided by the present results.
Although we previously considered that the Nhydroxymethyl
intermediate and/or formaldehyde
derived from DMNA by the N-demethylase
were
responsible for the biological effects of DMNA, the
view (36) that the alkylating agent diazomethane
TABLE 9
In Vitro INHIBITIONPRODUCEDBY DMNA OFAMINOACID INCORPORATION
INTOTHEPROTEINSOFLIVERSLICEFRACTIONS
300 Mg. of wet weight of rat-liver slices, suspended in 2.5 ml. Krebs-Ringer
phosphate buffer, were gently shaken in the
presence of DMNA (7.5 /imoles) at 87°C. for 60 minutes in Exps. 1 and 3 and for 40 minutes in Exp. 2. After this period of
preincubation
DL-leucine-1-C14 (58.5 m/imoles) was added from the side-arm of the flasks, and incubation was allowed for another
60 minutes. Atmosphere 100 per cent O?. Controls (no DMNA added) were treated similarly. The slices were spun down and sus
pended in sucrose solution (Exps. 1 and 3:0.25 M sucrose containing 10~3 M ethylenediaminetetraacetate;
Exp. 2: 0.88 M sucrose).
After homogenization
the nuclear (¿V),mitochondria! (J/t) and microsomal (Mie) fractions were isolated as follows. Exp. 1 and
3: 10 min. at 800 X g (yielding: Ar), 15 min. at 7,000 X g (->Mt) and 60 min. at 105,000 X g (-*Mic). Exp. 2: 15 min. at 800 X g
(—».V),
15 min. at 10,000 X g (—»3/0,15min. at 20,000 X g (yielding a mixture of small Mt and large Mie, not included in table,
labeling was intermediate between that of Mt and Mie, per cent inhibition by DMNA was 33) and 90 min. at 105,000 X g (—»3/Ã-c).
FRACTIONNMtMieCONTROLCounts/minprotein5005471280DMNACounts/minprotein346404370Per
rentintiibition2!)Õ672ControlCounts/minprotein346244650•DMNACounts/minprotein259184380Per
centinhibition232443CONTROLCounts/minprotein5235971120DMNACount!/
centinhibition353637
minprotein341384708Per
somal enzyme which metabolizes DMNA thus ap
pears to differ from related ones, such as those
metabolizing monomethyl-4-aminoantipyrine
and
various methylated amino azo dyes, by being not
inducible, at least in our R-strain rats. This result
is in accordance with the findings of Conney et al.
(16), who observed a selective action of benzpyrene
on the microsomal N-dealkylation
of various com
pounds.
6) Differential inhibition of amino acid incor
poration into nuclei, mitochondria, and microsomes
of liver slices.—In the experiments shown in Table
9 the nuclear, mitochondrial, and microsomal frac
tions were isolated from liver slices which had been
preincubated with DMNA for 1 hour, followed by
addition of leucine-1-C14 and incubation during
another hour. In two of four experiments the in
corporation
of leucine into the proteins of the
microsomal fraction was found to be inhibited to a
greater extent than that of the other fractions.
is the actual toxic and carcinogenic derivative is
much more impressive. A similar role may be at
tributed to the diazoethane derived from DENA
(reaction 2, Chart 1).
The biological properties of DMNA are closely
similar to those of the alkylating agents. Both
types of compounds are highly toxic, mutagenic,
carcinogenic, and inhibitory to amino acid incor
poration (15, 17). The mutagenic effect of DMNA
on Drosophila, 10-12 per cent sex-linked recessive
lethal mutations being obtained after injection of
8 fig. DMNA into adult males, was kindly brought
to our attention by Professor A. M. Clark of the
University of Tasmania. Diazomethane
is also a
strong poison showing mutagenic (8) and carcino
genic (54) effects. Some carcinostatic
alkylating
agents have been shown (38, 56) to produce a de
crease of the diphosphopyridine
nucleotide con
tent of susceptible tumors, and it is of interest that
DMXA (100 mg/kg) has been found (22) to have a
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research.
348
Cancer Reitearch
similar effect in rat liver 20 hours after injection.
Administration
of cysteine is known to protect
against a lethal dose of nitrogen mustard (52, 58,
59). The protection may be due to any one or both
of the two effects mentioned in footnote 3. The
present experiments
do, however, suggest that
cysteine had no such effect in the case of the diazoethane formed in sifu.5
The primary site at which the N-nitrosodialky!amine derivatives interact.—The inhibition of amino acid incorporation and the glycogenolysis pro
duced by DMNA in liver are accompanied
by
changes in the fine structure of the rough (granu
lar) and smooth (agranular) ER of the liver cells
(19, 20). The particles of the rough ER harbor the
protein synthetic sites, and the smooth ER is prob
ably involved in the deposition and mobilization
of glycogen (13, 20, 48). The ER seems to be the
primary target of DMNA. Since diazomethane is
formed from DMXA at or close to the ER mem
branes, which contain the enzymes initiating the
latter conversion, it is probable that susceptible
groups of the phospholipides
and protein of the
membranes are methylated.
An interaction with
acid groups of the phospholi pides might be respon
sible for the observed vacuolization of the rough
ER in the liver cells of rats receiving DMNA (19,
20) ; Alexander and Lett (1) have previously pro
posed that certain alkylating agents interact in the
above manner. Moreover, if there exists a func
tional relationship between membranes and associ
ated ribonucleoprotein
particles as regards protein
formation (14), a change in the structure of the
membranes
might affect the latter process ad
versely (62). Next to this, a direct interaction be
tween diazomethane and sulfhydryl, phosphate, or
other susceptible groups of co-factors, enzymes,
and RNA templates of the particles may be en
visaged to lead to an inhibition of amino acid in
corporation. The experimental evidence is strongly
in favor of the conclusion that the reactive
DMNA derivative inhibits amino acid incorpora' However, the possibility may exist that diazoethane did
react with cysteine to form S-ethylcysteine which, like Sethylhomocysteine (ethionine), might have a similar toxic ef
fect as diazoethane. Recent experiments have shown that intraperitoneal administration of H.5 mg. cysteamine to male rats,
followed 1 hour later by 10 mg. cysteamine intravenously and
100 mg DMNA/kg or 200 mg DENA/kg, completely abol
ished the inhibitory effects 4 hours after injection of the latter
compounds on the liver microsomal amino acid incorporation.
When the intraperitoneal injection of cysteamine was omitted,
the inhibitory effect produced by DENA was still completely
abolished, whereas that produced by DMNA was only par
tially prevented. Since cysteamine administration did not
affect the liver microsomal N-dcalkylating enzymes, the
present results are suggestive of a general protective effect of
SH-groups against diazoalkanes (P. Emmelot; I. J. Mizrahi;
and K. Kriek. Nature, in press).
Vol. 22, April 1962
tion i»the postmitochondrial
fraction following its
in vivo interaction with the microsomes. The in
hibition of amino acid incorporation
remains ap
parent when normal cell sap is incubated with the
microsomes from DMNA-treated
livers. The for
mation of amino acyl-s-RNA, which occurs in the
cell sap as the introductory step in protein synthe
sis, is not inhibited by DMNA (Table 2 and [12]).
Glutathione-sulfhydryl
groups have recently been
found to play an important role in the incorpora
tion of the amino acyl moiety of the carrier RNA
into microsomal protein (30). However, in view of
the absence of a general effect of added glutathione
on the depressed amino acid incorporation system
of the livers of rats treated with DMNA, the con
clusion is warranted that in case diazomethane
does interact with these sulfhydryl groups their
blockage is not the rate-limiting factor in amino
acid incorporation. This leads to the further con
clusion that the methylation by diazomethane
of
other molecular groups, very probably of the mi
crosomes, is responsible for the inactivation of the
amino acid incorporation system. In this connec
tion it is of interest that Farber and Magee (23)
have recently reported in abstract form that the
guanine moiety of the liver RNA was methylated
after administration
of DMNA; alkylating agents
are known to alkylate the base moiety of nucleic
acids (10, 11, 40).
Carcinogenic effects of the N-nitrosodialkylamines.—DENA is less toxic than DMNA but
produces a higher incidence of liver tumors at an
earlier time (3, 53). This result is not necessarily in
disaccord with the view that both the toxic and
carcinogenic effects of the N-nitrosodialkylamines
are due to the formation of diazoalkanes. The ex
perimental evidence suggests (Table 2) that at
least as much diazoethane is formed from DENA
as is diazomethane from DMNA. This leads to the
conclusion that diazoethane is less toxic but more
carcinogenic than diazomethane.
Various ethylating agents are known to exert a greater mutagenic
effect but a smaller toxic (or nucleic acid-inactivat
ing) effect than methylating agents (2, 11, 25, 41,
50, 51). This is apparently because of the type or
the extent of change produced by these agents in
nucleic acids6 and possibly also in other biological
compounds
as far as toxicity
alone is concerned.
6Differences may exist between the rate of alkylation of
phosphate groups vs. that of the bases (50), the rate of transalky lation from alkylated phosphate groups to bases (£),the
base-pairing capability of nucleic acids containing alkylated
purines, and the stability of nucleic acids carrying alkylated
phosphate groups or alkylated bases (chain fission). Experi
ments are presently being carried out in this laboratory by
Drs. E. Kriek and W. Bont on the effect of diazoalkanes on sRNA and highly polymerized RNA from liver microsomes.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research.
ANDËMMELOT—Cysteiiie
and Hepatic Injury
It is suggested that such differences may also be of
importance in carcinogenesis.
DMNA and DENA give rise not only to liver
tumors but also to kidney and lung tumors (45,
66). This result seems to be in disaccord with the
diazoalkane theory, since the enzymes of the
"drug-metabolizing"
type, which dealkylate
DMNA and DENA are, as far as is known, specific
for liver. However, lung and kidney might take up
an as yet unknown carcinogenic product (such as
an alkylated nucleic acid fragment or S-alkyl
cysteine) released into the blood stream by the
liver. Moreover, enzymes are known which oxidatively remove N-methyl groups (of sarcosine and
dimethylglycine) according to a mechanism differ
ent from that of the liver-microsomal N-dealkylases
—e.g.,by dehydrogenation instead of "hydroxylation," but leading to the same qualitative change
in the molecule (26, 43). In the latter connection it
is of interest that preliminary experiments have
shown that homogenates of lung and kidney and
kidney mitochondria produced minute amounts of
formaldehyde and acetaldehyde from DMNA and
DENA.7
Argus has recently reported (3) that various
water-soluble hepatocarcinogens cause the denaturation of protein, DENA being more active in
this respect than DMNA. The author considered
carcinogenesis and protein denaturation to be re
lated processes. The observations on the toxicity
of the two N-nitrosodialkylamines and their spec
ificity of action are, however, hard to reconcile
with this view. Moreover, the present paper af
fords another explanation for the quantitative dif
ference in the carcinogenic effects of these two
compounds.
The results obtained so far indicate that the
liver ER is the primary site affected by the Nnitrosodialkylamines. Since the possibility may be
considered that interaction of the diazoalkanes
with the ER initiates the carcinogenic process, it is
of much interest that many results (4, 20, 24, 27,
31-36, 48, 49, 57, 64, 65) of recent years suggest
that interaction of such diverse hepatocarcinogens
as CC14,thioacetamide, aminoazo dyes, and amines
with the ER (membranes) is a general phenome
non. The ER membranes, which are topographi
cally related to both the nuclear outer membrane
and the cell membrane, contain tissue-specific fac
tors (60, 61) which are lost during carcinogenesis
7Lung and kidney homogenates yielded 1.5-2.5 jig formal
dehyde and acetaldehyde/gm fresh tissue/hour. Mitochondria
prepared from 1 gm. of fresh weight of decapsulated kidney and
thoroughly washed in 0.95 Msucrose produced 1.8-2.3 (and in
onecaseevenll ¿ig.)
and 5.7-7.8 ng. formaldehyde from DMNA
and henadryl (diphenhydramine), respectively, during 45 min.
349
(63). In addition, protein synthetic functions,
which are normally securing the differentiated
function of the cell, are lost to a smaller or larger
extent in the tumor cell. Moreover, the ER is less
developed in hepatomas (5, 29) than in liver, and
even in the Morris 5123 rat hepatoma, which is
functionally highly differentiated, a decreased
quantity of Ell has been noted (47). It remains,
however, to be decided whether these changes are
the cause or the effect of the neoplastic character
of the cells.
Although the present study has been concen
trated on the liver cytoplasm, the possibility
should not be disregarded that the nucleus also is
affected by DMNA. Nuclear damage may be
brought about either by an alkylated metabolite
which is formed in the cytoplasm but reaches the
nucleus in the course of time—a view which may
be of interest in regard to carcinogenesis—or by
diazoalkane formed at the nuclear outer mem
brane. The latter is considered to constitute part
of the ER-membrane network which contains the
N-dealkylating enzymes. In vivo formation of nu
clear proteins has been found (44) to be inhibited
by DMNA to about the same extent as that of the
microsomal ones. However, our in vitro experi
ments (Table 9) have shown that a differential
effect may be obtained.
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The Effect of Cysteine on the Metabolic Changes Produced by
Two Carcinogenic N-Nitrosodialkylamines in Rat Liver
I. J. Mizrahi and P. Emmelot
Cancer Res 1962;22:339-351.
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