(CANCER RESEARCH 47, 5948-5953, November 15, 1987)
Regulation of A^-Nitrosodimethylamine Demethylase in Rat Liver and Kidney1
Jun-yan Hong, Jinmei Pan, Zigang Dong, Shu M. Ning, and Chung S. Yang2
Department of Biochemistry, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103-2757
this enzyme because any factors affecting P-450ac activity may
influence the carcinogenic effect of NDMA. For example, the
higher activity of hamster liver microsomes (compared to adult
rat liver microsomes) in activating NDMA to a mutagen (18,
19) is correlated to a higher concentration of P-450ac in the
former microsomes (18). In the present paper, we investigated
the regulation of P-450ac during development in neonatal rats
and during the induction of NDMA demethylase by acetone
and isopropyl alcohol. We also demonstrated that P-450ac is
present in rat kidney and regulated similarly to that in liver.
ABSTRACT
In previous work, the low k„,form of /V-nitrosodimethylamine
(NDMA) demethylase has been demonstrated to be due to a specific
form of cytochrome P-450 (designated as P-450ac) and to be the enzyme
required for the metabolic activation of NDMA. The present work deals
with the regulation of P-450ac in rat liver during development as well as
the mechanism of induction of P-450ac in rat liver and kidney by inducers.
NDMA demethylase activity was almost undetectable in the liver of
newborn rats, increased after day 4, and remained elevated throughout
the first 17 days of the neonatal period. The enhancement of NDMA
demethylase activity during development was accompanied by corre
sponding increases of P-450ac content and P-450ac mRNA levels as
determined by Western and slot blot analyses, respectively. No sex
differences with respect to this enzyme were observed in the developing
rats. Acetone treatment on late-term pregnant rats for 2 days resulted in
transplacental inductions of P-450ac and P-450ac mRNA in the newborn
rats. Pretreatment of young male rats and adult female rats with acetone
or isopropyl alcohol caused increases of NDMA demethylase activity
and P-450ac content in the liver but no significant change in the P-450ac
mRNA level. These facts suggest the possible existence of a posttran
scription regulatory mechanism under these induction conditions. The
presence of P-450ac in rat kidney was demonstrated by Western and
Northern blot analyses. The renal form of I'-451lac seemed to be regulated
in a fashion similar to the hepatic P-450ac regarding its response to
inducing factors such as fasting and acetone treatment.
INTRODUCTION
NDMA3 is a potent carcinogen in many animal species and
a suspected carcinogen in human beings. Metabolic activation
is required for NDMA to exert its carcinogenic effect (1-3).
The enzyme system responsible for the metabolic activation of
NDMA is commonly known as NDMA demethylase, an activ
ity displayed by a P-450-dependent monooxygenase system.
Previous work from this laboratory has demonstrated that the
low Km form of this enzyme activity is due to a specific form of
P-450, whereas the high Km value may be due to other P-450
isozymes (4, 5). This specific form of P-450 is inducible by
various factors such as fasting, diabetes, and pretreatment of
animals with acetone, isopropyl alcohol, ethanol, or pyrazole
(6-10). Recently, this protein was purified from acetone-in
duced rat liver microsomes and designated as P-450ac in this
laboratory (11). The cDNAs of both rat and human P-450ac
were isolated and sequenced (12). Judging from its known
properties, P-450ac is probably identical to the P-450J that was
purified from isoniazid-induced rat liver microsomes (13) and
is an orthologue of the ethanol-inducible I' 45flr M<„
from rabbit
liver (14).
In view of the important role that P-450ac plays in the
metabolic activation of NDMA and other carcinogens (15-17),
there is a need for a clear understanding of the regulation of
Received 4/29/87; revised 8/20/87; accepted 8/24/87.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked aiirertiwmrm in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Grants ES-03938 and CA-37037 from the N1H.
1To whom requests for reprints should be addressed.
3The abbreviations used are: NDMA, A'-nitrosodimethylamine; SDS, sodium
dodecyl sulfate; P-450, cylochrome P-450; P-450ac, a rat microsomal cytochrome
P-450 isozyme inducible by acetone and fasting; cDNA, complementary DNA;
poly(A)* RNA, polyadenylated RNA; SSC, 0.15 M NaCI-0.015 M sodium citrate,
pH7.0
MATERIALS
AND METHODS
Chemicals. Guanidine thiocyanate, NADP, glucose 6-phosphate and
glucose-6-phosphate dehydrogenase were obtained from Sigma Chem
ical Co. (St. Louis, MO); cesium chloride was from Pharmacia Fine
Chemicals (Piscataway, NJ); nitrocellulose paper was from Schleicher
and Scimeli (Keene, NH); [32P]dCTP (3000 Ci/mmol) was from New
England Nuclear (Boston, MA); and NDMA was purchased from
Aldrich Company (Milwaukee, WI). Reagents for electrophoresis and
immunoblot were obtained from sources described previously (18).
Animals. Sprague-Dawley rats were obtained from Taconic Farms
(Germantown, NY) and maintained in air-conditioned quarters with
12-h light-dark cycles. The average body weights of the male and female
rats were 100 and 260 g, respectively. The female rats were received
pregnant and their pups were used for the developmental studies.
Almost 1 month after giving birth, those females were weighed and
used for the induction studies. Generally, acetone or isopropyl alcohol
(25% solution in water) was administered by one intragastric intubation
at a dosage of 4 ml acetone or isopropyl alcohol/kg body weight. The
control animals received equal volumes of water only. The animals
received the treatment at different time points prior to sacrifice. The
rats were sacrificed within 1 h. For the study of developmental changes,
late-term pregnant rats were obtained and the neonatal rats were
sacrificed at different ages after birth. The pregnant rats used for the
transplacental experiment were given 50% acetone solution intragastrically on the 19th and 20th days of pregnancy at a dosage of 3.5 ml
acetone/kg body weight/day. During the experiment, the animals were
given a commercial laboratory chow (Ralston Purina Co., St. Louis,
MO) and water ad libitum. The fasting rats received no food but only
water. After sacrifice, the livers and kidneys were removed immediately,
frozen on dry ice, and stored at —80°C
until the preparation of RNA
and microsomes.
Microsome Preparation and Enzyme Assay. Rat liver and kidney
microsomes were prepared by differential centrifugation as described
previously (15). The protein concentration was determined by the
method of Lowry et al. (20). The microsomal NDMA demethylase
activity was determined by measuring the production of formaldehyde
as described previously (15).
RNA Preparation and Poly(A)* RNA Isolation. Total hepatic and
renal RNA was prepared from frozen tissues by the guanidinium/
cesium chloride method (21) and quantified by measuring the absorbance at 260 nm (one /426ounit = 40 MgRNA/ml). Poly(A)+ RNA was
isolated by oligodeoxythymidylate chromatography (21). Total RNA
and poly(A)* RNA were stored in 70% ethanol at -80'C until use.
cDNA Probe. pUC 18 plasm id containing P-450ac cDNA insert was
constructed as described (12) and was kindly provided by Dr. Frank
Gonzalez (Laboratory of Molecular Carcinogenesis, National Cancer
Institute, NIH, Bethesda, MD). The 1.6-kilobase cDNA insert was
excised from the vector by EcoRI digestion, separated by 1% agarose
gel electrophoresis, and recovered (22). 32P-labeled probe was prepared
by nick-translation using [32P]dCTP (21). Consistent with the previous
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REGULATION
OF NOMA DEMETHYLASE
report using the same cDNA probe (12, 23), Northern blot analysis in
the present study revealed that only a single 1.8-kiIobase mKN'Aspecies
was detected in both control and treated rats and in both male and
female rats at different ages.
Northern Blot and Slot Blot Analyses. RNA was either electrophoretically separated in 1% agarose gel containing 2.2 M formaldehyde
and blotted with nitrocellulose filter (21) or directly applied to the
nitrocellulose paper using a slot blot apparatus (Minifold II; Schleicher
and Schnell). The nitrocellulose Tillers were baked at 80'C for 2 h in a
vacuum. Prehybridization was carried out overnight at 42°Cin 5 x
Denhardt's solution (bovine serum albumin, 0.1%; Ficoll, 0.1% and
Table 1 Hepatic NDMA demethylase activity in developing rats
Liver microsomes were prepared from rats of different ages and NDMA
demethylase activity was measured. In the acetone-treated group, the pregnant
rats were treated with acetone in late pregnancy. Values are means ±SD. The
number of samples, n. is shown in parentheses. For the I-day-old groups, n
represents the number of samples analyzed. Each sample was pooled from 6 to 7
male rats or 2 to 3 female rats from the same litter. For other experiments, the
liver for each rat was analyzed as one sample.
demethylase activity
protein)SexF (nmol HCHO formed/min/mg
Age
(days)14817NDMA
polyvinylpyrrolidone, 0.1%), 5 x SSC, 0.1% SDS, 10 Mg/ml of dena
tured and sheared salmon sperm DNA, and 50% formamide. The filters
were hybridized in the same solution with the 32P-labeled probe. Fol
lowing overnight hybridization at 42"( ', the filters were washed twice
with 2 x SSC-0.1% SDS for 15 min each at room temperature and
then twice with 0.1 x SSC-0.1% SDS for 15 min each. The filters were
then exposed for varying lengths of time at -80°C to Kodak XAR-5
film with a Lightning Plus intensifying screen. The bands or slots on
the autoradiographs were quantified by a densitometer (Shimadzu
Dual-wavelength TLC Scanner, Model CS-930).
Anti-P-450ac Preparation and Western Blot Analysis. P~450ac was
purified from acetone-induced rat liver microsomes (II) and used for
the preparation of anti-P-450ac IgG from rabbit (18). For Western blot
analysis (18), microsomal proteins were separated by SDS-polyacrylamide gel electrophoresis and then transferred electrophoretically to a
nitrocellulose sheet using Hoefer's TF-52 Transphor (Hoefer Scientific
Instruments). The nitrocellulose sheet was then treated with anti-P450ac IgG followed by phosphatase-labeled goat anti-rabbit IgG. For
immunostaining, the sheet was developed with a mixture of 5 bromo
4-chloro-3-indolyIphosphate and nitroblue tetrazolium in Tris buffer
solution. A known amount of purified P-450ac was always carried
through the electrophoresis and staining steps to serve as the standard.
In the present study, since the amount of P-450ac was very low in liver
microsomes from newborn rats and in kidney microsomes, excess
microsomal proteins were used for the gel electrophoresis for those
samples and the immunostaining process was usually prolonged. Under
these conditions, several polypeptides smaller than P-450ac were visu
alized in the Western blot. They were most probably caused by nonspe
cific adsorptions. The possibility that some of these polypeptides were
produced via P-450ac degradation could not be fully excluded. Quantitation of the Western blot was carried out by the aforementioned
densitometer.
RESULTS
Developmental Changes in Hepatic P-450ac in Neonatal Rats.
Liver microsomes were prepared from neonatal male and fe
male rats of various ages, and NDMA demethylase activity was
determined. NDMA demethylase activity was almost undetectable in newborn rat livers but increased dramatically after day
4 and remained elevated at day 17 in both male and female rats
(Table 1). There were no differences in liver microsomal
NDMA demethylase activity between male and female neonatal
rats. Western blot analysis showed that the increase of NDMA
demethylase activity in developing rats was due to a correspond
ing increase in the P-450ac protein level in the liver microsomes
(Fig. 1). P-450ac protein was almost undetectable in female and
male day 1 newborns (Fig. 1, Lanes 2 and .V),even though more
microsomal proteins were used in the electrophoresis (65 ¿ig/
well for samples from day 1 rats versus 49 /¿g/wellfor samples
from older rats). However, P-450ac content in liver microsomes
increased appreciably at day 3 (Fig. 1, Lane 4 for females; Lane
10 for males) and increased further at day 8 (Fig. 1, Lanes 5
and 11) and day 17 (Fig. 1, Lanes /and 13) in both female and
male neonatals. Densitometry of the immunoblots indicates
that, using the samples from day 17 neonates as 100% (the
males and females differed by 15%), the average P-450ac con-
(n)0.01
(n)0.06
MF
±0.03 (S)
(1)0.27
ND°
±0.10(5)
(I)0.30
0.04
MF
±0.10(2)
(3)0.57
0.34
±0.16
0.29 ±0.40
(2)0.65
±0.02 (3)
0.63
(3)1.80
±0.14
MFMControl
2
3
4
(2)1.20
±0.23
±0.25 (2)
±0.51 (5)
1.69 ±0.35
1.83 ±0.38 (5)Acetone-treated
(2)°
ND, not detected.
1
(1)
5
6
7
8
9 10 11 12 13
14
Fig. l. Western blot analyses of liver microsomal proteins from developing
rats and transplacentally acetone-treated rats. Equal amounts of microsomal
protein from individual rats (see Table 1 for the number of rats) in each group
were pooled and subjected to electrophoresis. The amount of protein applied per
well is shown in parentheses. Lane 1, P-450ac standard; Lanes 2 to 7, samples
from female neonatal rats. Lane 2, control, day 1 (65 pg); Lane 3, treated
(transplacentally with acetone), day 1 (65 >ig); Lane 4, control, day 4 (49 jig);
Lane 5, control, day 8 (49 /ig); Lane 6, treated, day 8 (49 MÕ:K
and Lane 7, control,
day 17 (49 jig). Lanes 8 to 13, samples from male neonatal rats. Lane 8. control,
day I (65 pg); Lane 9, treated, day 1 (28 /ig); Lane 10, control, day 4 (49 fig);
Lane II, control, day 8 (49 /jg); /<""' 12, treated, day 8 (49 fig); and Lane 13,
control, day 17 (49 fig). Renal microsomal protein (244 pg) from young male rats
was run in Lane 14 for comparison.
tent in male and female liver microsomes was <5% in day 1
newborns and increased to 36 and 58% in day 3 and day 8
neonates, respectively. The identities of the lower molecular
weight polypeptide bands were not known, but they probably
were caused by nonspecific staining. In order to investigate the
mechanism for this developmental change in P-450ac, P-450ac
m KNA levels were examined by slot blot analysis. As shown in
Table 2, hepatic P-450ac mRNA increased about 5-fold at day
4 and this was followed by a further gradual increase during the
remaining period studied. There were no sex differences in P450ac mRNA levels in the animals studied herein.
Transplacental Induction of Hepatic P-450ac by Acetone. The
late-term pregnant rats were treated with acetone on the 19th
and 20th days of pregnancy to study the transplacental effect
of acetone on P-450ac. An increase of liver NDMA demethylase
activity was found in both male and female newborn rats, but
the induction no longer existed in the later neonatal period
(Table 1). The same types of changes were observed ¡nthe
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REGULATION
OF NOMA DEMETHYLASE
amount of P-450ac in liver microsomes as demonstrated by the
Western blot analysis (Fig. 1). The immunodetectable P-450ac
in liver microsomes from day 1 rats increased about 3-fold in
newborns transplacentally treated with acetone (Fig. 1, compare
Lane 3 to Lane 2 for female rats and Lane 9 to Lane 8 for male
rats). The effect of transplacental induction on P-450ac seemed
to disappear at day 8, at which time no significant difference
between the control and treated rats was observed. Similar
results were obtained in another set of experiments (data not
shown). The transplacental induction of P-450ac by acetone
was accompanied by an increase in the P-450ac mRNA level
(Table 2). Transplacental treatment with acetone caused a 2fold increase of P-450ac mRNA in newborn rats (statistically
significant, P < 0.01, when the male and female groups were
combined).
Effect of Acetone and Isopropyl Alcohol on Hepatic P-450ac
in Adult Rats. Previously, we demonstrated that liver NDMA
demethylase activity was induced in young male rats by pre
treatment with acetone and isopropyl alcohol (8). This was
confirmed in the present study. The induction of NDMA de
methylase activity was detectable 3 h after acetone or isopropyl
alcohol pretreatment in young male rats (Table 3), and the
enhancement of NDMA demethylase activity was accompanied
by an increase of P-450ac content in liver microsomes as
demonstrated by Western blot analysis (Fig. 2). Compared with
the untreated rats, the P-450ac content as determined by densitometry increased to 150% 3 h after treatment with acetone
or isopropyl alcohol; it increased to 200 and 300% after 6 and
12 h, respectively, and did not increase further at 24 h after the
treatment. In adult female rats, acetone pretreatment also
caused increases both in NDMA demethylase activity and in P450ac content (Table 3; Fig. 2). However, corresponding in
creases in P-450ac mRNA levels were not observed in slot blot
analysis. In Table 3, Experiment 1, P-450ac mRNA seemed to
be increased at 6 or 12 h after pretreatment with acetone or
isopropyl alcohol, respectively. Nevertheless, statistical analysis
showed that the differences were not significant (P > 0.05). The
repeated experiment (Experiment 2) with more animals re
vealed no differences in hepatic P-450ac mRNA levels between
control animals and acetone- or isopropyl alcohol-treated ani
mals at these two time points. Significant changes in P-450ac
mRNA levels were also not observed in adult female rats after
acetone treatment.
P-450ac in Rat Renal Tissue and Its Induction by Fasting and
Acetone Treatment. The existence of P-450ac in rat kidney was
demonstrated by Western blot analysis (Fig. 1, Lane 14), in
Table 2 Hepatic P-450ac mRNA level in developing rats
P-450ac mRNA level is shown from the scanning densitometry of slot blot
analysis. Total RNA used in slot blot was S ¿ig/slotfor rats at ages 1 and 4 days
and 2 pg/slot for rats at ages 8, 17, and 25 days. The values have been normalized.
Animal treatment and data presentation were the same as in Table 1. For the 1day-old groups, n represents the number of samples analyzed. Each sample was
pooled from 2 to 3 rats from the same litter.
level
area)SexFMFMFMF(arbitrary units, peak
Age
(days)14817mRNA
(n)1.9
(n)4.1
±1.1 (5)
1.0(4)10.6
1.7 ±
±1.8(6)
1.9(5)10.0(1)
3.8 ±
±2.7 (3)
(3)39.3
10.1 ±2.1
(2)32.9
8.2 ±2.8
±4.4 (3)
12.2(3)66.6
32.4 ±
±4.7 (3)
(1)46.4
47.6
±30.5 (5)
±35.1 (2)
54.6 ±37.3 (2)
MControl 50.3 ±16.4 (3)Acétone
Table 3 Effect of acetone and isopropyl alcohol treatment on P-450ac mRNA
level and NDMA demethylase activity in rat liver
Rats were treated with one dose of a 25% acetone (or isopropyl alcohol)
solution at a dosage of 4 ml of acetone/kg body weight. The P-450ac mRNA
content (in an arbitrary unit) is shown as mean ±SD of each group; n = number
of animals used in each group. NDMA demethylase activity is the mean of the
duplicate assays of each pooled sample.
P-450ac
mRNA
(arbitrary
protein)4.6
units)
demethylase
(nmol HCHO
formed/min/mg
microsomal
Experiment
1MaleControlAcetone3h6
1.03.9
±
1.46.7
±
3.03.9
±
2.34.9
±
1.73.8
±
h12
h24hIsopropyl
alcohol3h6h12
1.93.9
±
1.16.8
±
2.47.4
±
2.44.6
±
h24
hFemaleControlAcetone
2.46.6
±
2.04.7
±
h)Experiment (27
maleControlAcetone
2:
2.55.8
±
h)Isopropyl
(6
3.04.4
±
alcohol (12 h)n44444444443g88NDMA
±2.71.01.81.93.63.61.52.22.84.61.04.21.63.22.5
1
23
4
567
8 9 10 11 12
Fig. 2. Western blot analysis of liver microsomal proteins from rats pretreated
with acetone or isopropyl alcohol. Equal amounts of microsomal protein from 4
rats in each group were pooled and subjected to electrophoresis (6.5 pg/well).
Lane I, untreated male rats; Lanes 2 to 5, male rats sacrificed 3, 6, 12, and 24 h
after acetone treatment, respectively; Lanes 6 to 9, male rats sacrificed 3, 6, 12,
and 24 h after isopropyl alcohol treatment, respectively; Lane 10, female untreated
rats; Lane ¡I,female rats 27 h after acetone treatment; Lane 12, P-450ac standard.
which a specific polyclonal antibody against purified rat hepatic
P-450ac was used. The detection of this M, 52,000 protein
band by this anti-P-450ac IgG in kidney microsomes suggests
that renal P-450ac is the same as, or very similar to, hepatic P450ac. This was further supported by Northern blot analysis
which detected the same 1.8-kilobase mRNA species in both
liver (Fig. 3, Lanes 6 to 8) and kidney (Fig. 3, Lanes 1 to 5)
samples by using a P-450ac cDNA probe. Compared with
poly(A)+ RNA, total hepatic RNA seemed to have more RNA
degradation which was manifested by the "tailing" effect on the
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REGULATION OF NOMA DEMETHYLASE
12
3
4
56
78
9 10 11 12 13 14 15
Fig. 5. Western blot analysis of kidney microsomal proteins from adult female
rats after fasting or treatment with acetone. The amount of microsomal protein
used was 333 fig/well. Lane 1, P-450ac standard; Lanes 2 to 6, untreated rats;
Lanes 7 to III. rats 27 h after acetone treatment; Lanes 11 to IS, rats fasted for
48 h.
1
234
5678
Fig. 3. Northern blot analysis on P-450ac mRNA. Equal amounts of RNA
from individual rats in each group were pooled and subjected to electrophoresis
either directly or after oligodeoxythymidylate cellulose chromatography. The
amounts of RNA applied per well are shown in parentheses. Lanes 1 to 3, renal
poly(A)* RNA from female rats. Lane I, untreated (4 jm): Lane 2, 27 h after
acetone treatment (4 *ig); Lane 3, fasted for 48 h (4 fig). Lanes 4 and 5, renal
poly(A)* RNA from g-h-fasted (2.5 ng) and 16-h-fasted (5 Mg)male rats, respec
tively. Lane 6, total hepatic RNA from 16-day-old untreated female rats (20 ng).
Lanes 7 and 8, hepatic poly(A)* RNA (4 /ig each) from untreated 3-day-old female
and male rats, respectively.
Table 4 Effect of fasting on renai P-4SOac mRNA in male rats
Fasted rats were sacrificed at different time points and the total renal RNA
was prepared for slot blot analysis (5 jig/slot). Values are mean •SD from
determinations on individual samples, n = numbers of animals used in each
group.
mRNA content
Fasting
(arbitrary
units)2.3
(h)0
816
333P-450ac
±0.2
3.0 ±1.1
10.9 ±3.0"
14.0 ±4.1°
24n3
* Values significantly different (P < 0.05) from those for fasting 0 h.
Table 5 Effect of acetone or fasting on renal P-450ac mRNA infernale rats
Adult female rats were fasted for 48 h or treated with acetone (intragastrically;
10 ml of 25% solution/kg body weight) 27 h prior to sacrifice. The data presen
tation was the same as in Table 4.
P-450ac mRNA
n
(arbitrary units)
5.5 ±2.0
Control
7.0 ±I.I
Acetone
10.9
±4.1"
Fasting
* Values significantly different (P < 0.01) from the control.
1234
5
6 7
8
9 10 11 12 13 14 15 16
Fig. 4. Western blot analysis of kidney microsomal proteins from young male
rats fasted for different lengths of time. Kidney microsomes were prepared from
individual rats and were subjected to electrophoresis (333 *<gprotein/well). Lane
I, P-450ac standard; Lanes 2 to 4, rats not fasted; Lanes 5 to 7, rats fasted for 8
h; Lanes 8 to III. rats fasted for 16 h; Lanes //to 13, rats fasted for 24 h; Lanes
14 to 16, rats fasled for 48 h.
Northern blot analysis. The regulation of the renal P-450ac
gene by fasting and acetone pretreatment was also studied.
Renal P-450ac content was determined by Western blot analy
sis. In Fig. 4 (and Fig. 5), because of the overloading of kidney
microsomal proteins, the edges of the protein bands overlapped
with those from neighboring lanes. Nevertheless, the increases
of renal P-450ac content in rats treated with acetone or during
fasting were clearly demonstrated. P-450ac content in renal
microsomes increased 3-fold after a 16-h fasting in young male
rats (Fig. 4, Lanes 8 to 10) and remained at the elevated level
during a 48-h fasting (Fig. 4, Lanes 11 to 13 for 24-h fasting;
Lanes 14 to 16 for 48-h fasting). This was accompanied by 5to 6-fold increases in P-450ac mRNA in male rats after they
were fasted for 16 and 24 h (Table 4). Fasting for 48 h also
caused increases in both P-450ac contents (Fig. 5, Lanes 11 to
15) and P-450ac mRNA levels in the kidneys of adult female
rats (Table 5). Fasting-induced P-450ac mRNA was also con
firmed in the Northern blot analysis by using renal poly(A)'f
RNA (Fig. 3). The increases of P-450ac mRNA were observed
in female rats fasted for 48 h (Fig. 3, Lane 3) and in male rats
fasted for 16 h (Fig. 3, Lane 5). Pretreatment of female adult
rats with acetone caused a 2.5-fold increase in renal P-450ac
content (Fig. 5, Lanes 7 to 10) but did not produce a significant
change in the P-450ac mRNA level in the kidney (Table 5).
DISCUSSION
Previously, we demonstrated
that a P-450ac-mediated
NDMA demethylase is important in the metabolic activation
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REGULATION
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of NDMA to a reactive species, presumably a methylating
agent, that causes DNA methylation (IS), hepatotoxicity (24),
and V79 cell mutagenesis (25). In the present work, we observed
that this carcinogen-metabolizing enzyme undergoes changes
during development. With newborn rats, the liver microsomes
appeared to have no or very low activity for metabolizing
NDMA, but such activity was detectable after day 4 and was
much higher at days 8 and 17. The increase of NDMA demethylase activity was accompanied by corresponding increases in
P-450ac contents and P-450ac mRNA levels. This result is
consistent with our previous work showing the neonatal devel
opment of aniline hydroxylase and P-450ac (12). The increase
in P-450ac mRNA accumulation during development has been
attributed to the transcriptional activation of the P-4SOac gene
as determined by nuclear run-on analysis (12). Several other
forms of cytochrome P-450 also become elevated during devel
opment (26-29). In most of the cases, the developmental
changes are believed to be regulated by hormones. In the case
of P-450ac, however, metabolic changes such as the production
of ketone bodies may be involved. From the present and pre
vious studies, we may summarize that, in rats, NDMA demethylase, i.e., P-450ac, begins to develop after birth, reaches a
plateau at about day 30, and subsequently decreases with age
(12, 18, 30). Rat liver microsomes from 90-day-old male rats
had only about one-third as much NDMA demethylase activity
as those from 25-day-old rats (18). Such developmental and
age-dependent alteration of this enzyme activity is expected to
be reflected in the metabolic activation of NDMA in animals
of different age groups. The relationship between such changes
in NDMA demethylase activity and NDMA carcinogenesis
remains to be further elucidated.
Although acetone and isopropyl alcohol are known inducers
of NDMA demethylase activity and P-450ac, the present study
demonstrates that the increase in P-450ac in young and adult
rats cannot be accounted for by the accumulation of P-450ac
mRNA. This observation is consistent with the previous result
showing that P-450ac was induced without a significant eleva
tion of its mRNA level when acetone was administered in
drinking water (5%, v/v) to young rats for 10 days prior to
sacrifice (12). Hence, the mechanism of P-450ac induction by
acetone and isopropyl alcohol in young and adult rats appears
to be different from that by fasting and diabetes; both of the
latter conditions produced significant P-450ac mRNA accu
mulations (23, 31). Acetone appears to induce the synthesis of
P-4SOac by enhancing a posttranscriptional event, which could
be due to an increase in the stabilization of P~450ac protein or
an increase in the translational efficiency of P-450ac mRNA,
or both.
Acetone pretreatment of late-term pregnant rats leads to an
increase of P-450ac in their newborns. The mechanism of this
transplacental induction by acetone is unknown. Since P-450ac
mRNA was also induced in the newborn rats, the mechanism
for the transplacental induction appears to differ from the
induction of P-450ac by acetone in adult rats. Although it has
been reported that phénobarbital and 3-methylcholanthreneinducible P-450 isozymes can be induced by transplacental
treatment (29, 32-34), our report is the first to identify the
transplacental induction of P-450ac, a form which represents a
different P-450 gene family (12).
The present study clearly demonstrates that P-450ac exists
in the rat kidney. This renal P-450ac is very similar to, if not
identical to, the hepatic form, as judged by Northern hybridi
zation and Western blot analysis. This is further supported by
the fact that the regulation of renal P-450ac by fasting and
acetone was similar to that in the liver. The roles of renal and
other extrahepatic P-450ac in the metabolic activation of
NDMA remain to be further investigated.
ACKNOWLEDGMENTS
The authors thank Yu C. Huang for help with the preparation of
poly(A)+ RNA; and M. Lee and H. Lu for their assistance in part of
the study. We are indebted to Drs. F. J. Gonzalez and H. V. Gelboin
(Laboratory of Molecular Carcinogenesis, National Cancer Institute,
NIH) for the P-450ac cDNA probe and helpful discussions.
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Regulation of N-Nitrosodimethylamine Demethylase in Rat Liver
and Kidney
Jun-yan Hong, Jinmei Pan, Zigang Dong, et al.
Cancer Res 1987;47:5948-5953.
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