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Hepatic UDP-glucuronyltransferase in Wistar and Gunn rats - in vitro activation by diethylnitrosamine
Vol. 32, No. 5, 1968
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REPATICGDP-GLUCURONYLTRANSFERASEINWISTARAND GTJNNRATS IN VITRO ACTIVATION BY DIETRYU?ITRGSAMINE I.
Stevenson, D. Greenwood* and J. I%%wen
Department of Pharmacology end Therapeutics, Dundee, Scotland.
University
of Dundee,
Received August6, 1968 Dutton (1966)
has previously
pointed out that it is difficult
the absolute UDP-glucuronyltransferase being little enzyme.
known of the in vitro Investigations
solubilised
trsnsferase
in different
regulating
The stimulatory
The results
there
of this
of DDPGA-
in this paper demonstrate, from Wistar and Gunn
by the carcinogen diethylnitrosamine
appears to be confined to rat liver
marked with Gunn rat liver deficiency
the activity
and by the presence, in many tissues,
in vitro
effect
tissues,
complicated by lack of studies on
that hepatic DDP-glucuronyltransferase
rats may be activated
trsnsferase
factors
are further
destroying pyrophosphatases. nevertheless,
activity
to assess
preparations
of this tissue
(DE%).
and is particularly
- to the extent that the reported (Scbmid et al, 1958;
Lathe and Walker,
1958) is no longer apparent when DENis included in the ass&v system. DEN-activation
occurs with both microsomal and solubilised
and may be demonstrated using either Kinetic affinity Materials
analysis of the activation
o-aminophenol or paracetamol as substrate.
indicates
that DENdoes not alter
the
of the enzyme for substrate. end Methods
Male adult Wistar or Gunn rats were used throughout, for liver
transferase
enzyme studies by stunning and cervical
*Present address:
I.C.I.
Pharmaceutical Division,
866
animals being killed
dislocation.
Alderley
Following
Park, Cheshire.
BIOCHEMICAL
Vol. 32, No. 5, 1968
rapid excision of the liver, DDPGAsnd digitanin
KCl.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
homogenateswere prepared in alkaline
isotonic
were purchased from the Sigma Chemical Co. and
l-k-UDPGA from the Radiochemical Centre, Amersham. For preparation for 30 min.
of microsomes, whole hamogenateswere first
Microsomes, sedimented from the resultant
for 60 min at 100,000 g, were Solubilisation
volume.
of Pogell and Leloir
(1961),
equal volume of 1% digitonin centrif'ugation
supernatant by spinning
resuspended in KC1 in one-fifth
of glucuronyltransferase
spun at 4,000 g
of the original
was achieved by the method
the microsomal suspension being stirred for 30 min at O".
The supernatant after
at 100,000 g for 60 min was used as solubilised
DDP-glucuronyltrsnsferase
activity
with an
transferase.
using c+minophenol as substrate was
determined by the method of D&ton and Storey (1962) using 4.9 x 104M DDPGA and spectrophotometric incubation O.lml
estimation of the product at 565 w.
volume of 0.6 ml contained either
The total
0.1 ml of lO$whole homogenate,
of microsomal suspension or 0.2 ml of solubilised
enzyme.
Results Activation
of rat liver
around 1.6 x 10% DEN. this concentration trsnsferase
It
UDP-glucuronyltransferase
is apparent from the results in Table 1 that,
of DEN, approximately j-fold
occurred, the degree of activation
homogenate, microsomal or solubilised
activation
preparations
did not result
found to affect
activation
at
of Wistar rat liver
being the samewith either
preparations.
homogenatesmore than 20-fold activation observed with liver
was found to be msximal
With Qunn rat liver
was obtained.
No stimulation
from species other than the rat.
was
The
from reduced UDPGAbreakdown, for DENwas not
UDFGApyrophosphatase and the activation
of transferase
was demonstrable in DDFGA-saturated systems. DEN-stimulated synthesis of g-sminophenylglucuronide as estimated calorimetrically
in the Dutton and Storey procedure was confirmed by another
method utilising
%-DDPGA (Fig. 1).
As is evident from comparison of
ChromatogramsA and B, synthesis of
4 C-g-aminophenylglucuronide in the
867
Vol. 32, No. 5, 1968
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1 In vitro
effect
of DEN on rat liver
CDP-glucuronyltransferase
glucuronide
(A)
Enzyme preparation
No DEN
1.
Wistar
2.
formed?
(B)
B/A
DEN (1.62 x 10-2M)
rat liver
Homogenate
0.49
1.44
2.9
Microsomes
0.052
0.168
3.2
Digitonin-solubilised enzyme
0.053
0.165
3.1
0.060
1.33
22.2
Cunn rat liver Homogenate
%esults are expressed as poles o-eminophenyl glucuronide formed/preparatian from 1 g wet wt liver/30 min. E%h value quoted represents the mean value of separate determinations on preparations frcun at least five animals.
presence of DEN was more than twice that when the carcinogen The product
formed in the presence of DEN was almost ccmpletely
subsequent incubation
with P-glucuronidase
of UDP-glucuronyltrensferase phenolphthalein approximately
or menthol b-fold
Results
as substrates
activation
2.
from kinetic
It is evident
g-aminophenol
(Chromatogram C).
could be detected
using the above radioactive
Eg.
was excluded. hydrolysed
No DIQT-activation
using ~nitrophenol,
but, with 4 mM paracetemol
of Wistar
by
rat liver
transferase
as substrate,
was observed
procedure. studies
on the solubilised
that additi~
(4.4 x 10 -!M,.
transferase
are shown in
of DEN does not change the K, for
similar
868
experimentswithvarying
DDPGA
BIOCHEMICAL
Vol. 32, No. 5, 1968
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
,-----(
I..*
.
. . .
Chromatogram A
Chromatogram B
Chromatogram C
11.
1
t
1
2
3
Origin Figure 1. DEN-activation using labelled UDPGA.
4
5
s
Diitance
1
8
0
I
5
rl
10
11
12
.
13
14
8
15
15
from origin (cm)
of CDP-glucuronyltransferase
- confirmation
Incubations contained g-eminophenol end buffer as used by Dutton and Storey (1962) together with lO$ of 1O~Wista.r rat liver homo ate in a total volume of SO*. The donor nucleotide was 4.9 x 10~4M rtf?C-UDPGA The 30 min incubations at 37“ were terminated by immersion (2LSmc/mM). of the incubation tubes in boiling water for 1 min. Following addition of 0.2nil water and centrifugation, 104 of the incubate was spotted in a km-wide band on a thin layer (250~) silica gel plate. After development over l&m in a methanol: H20 (9:l) solvent system, lcm zones were scraped off into vials containing $I ml dioxsne-naphthalene scintillator and counted in a Nuclear Chicago Scintillation Counter using the Channels Ratio Method of Quench Correction. ChromatogremsA and B were from incubates containing No DENsnd 1.62 x 10m2MDENrespectively. ChromatogramC was from an incubate containing DENand which had been subsequently incubated with P-glucuronidase. The value for the two left-hand columns in each chromatogram was 2 x I&. Standard o-aminophenyl glucuronide (!.....I) and glucuronic acid (I----I) ran as inzcated.
869
BIOCHEMICAL
Vol. 32, No. 5, 1968
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60
.I
s= v=
o-aminophenol AOD
565mp
xl0 I
Figure 2. Kinetics of the DEN-activation UDP-glucuronyltransferase.
-4
M
10min.
of solubilised
Wistar rat liver
Incubations uere carried out for 10 min in the presence of o--------d 1.6 x 1O-2M DEN. Conditions No DEN, 8.0 x lo-3M DEZiand 0-6 of asssy are as described iu the text witi the exception that the o-aminophenol concentration was varied as indicated and that the final DDPGAconcentration used was 4.9 x lo-3M.
concentrations
suggest that the I$,, for the nucleotide
is also unaffected by
DEN. Discussion Several agents are known to stimulate glucuronide synthesis in vitro. The stimulation
by ATP and UDP-N-acetylglucosamiue described by Pogell and
870
BIOCHEMICAL
Vol. 32, No. 5, 1968
Leloir
results
from reduced CDPGAdestruction,
and Ffeff, 1967) may be related Cn the other hand, activation
to its
1961) seems to involve
transferase
protein.
It
mechanism also operates
there
is no measurable
A DEN effect
or from interference activity
of this,
from activation
effect
(Pogell
on the UDP-glucuronylthat
of transferase, of trsnsferase
this although
for substrate.
of otherwise non-functional
of DEN on rat liver
the very marked activation activities
that no transferase there are previous
in Dunn rats
(Arias,
Clarification in Gunn rat liver
1961;
enzyme
transferase
drug-metabolising
transferase
of Wistar deficiency
exists
of apparently
Javitt,
1966).
activity.
studies
enzymes in general.
the microsomal
In support
normal glucuronide
synthesis
and of the precise factors
does not activate
It has recently
enzyme involved
are c-arable,
in Gunn liver.
on the in vitro
DEN in vitro
in
In the presence
and Gunn rat livers
reports
must await further
is interesting,
of the Gunn enzyme.
of the mechanism of DEN-activation
UDP-glucuronyltransferase
to inhibit
structure.
by albumin
with an endogenous system controlling
of DEN the transferase suggesting
microscznal
from the above results,
in DEN-activation
of EDTA (Halac
is also possible.
The specific particular
effect
change in the affinity
resulting
to alter trsnsferase
a direct
is likely,
while the effect
ability
of solubilised
and Leloir,
latter
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lesion
regulating microsomal
been shown, for example,
in hexobarbitone
metabolism
(Stevenson
Research Council
and the technical
and Greenwood, 1968). Acbowledgments The financial assistance addition,
support of the Medical
of Mrs. A. Reekie are gratefully
acknowledged.
wish to thank Dr. G. J. Dutton for much helpful
The authors, discussion.
References Arias, I. M., Diochem. Biopws. Pes. Commun., 6, 81 (1961). Dutton, G. J. in ~~Glucuronic Acid" (G. J. Dutton, ed.), p. 218, Academic Press, NewYork, 1966.
871
in
Vol. 32, No. 5, 1968
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Dutton, G. J. and Storey, I. Il. E. in "Methods in &~ymology~~ (S. P. Colowick and N. 0. Kaplan, eds.), Vol. V, p. 159, Academic Press, New York, 1962. Halac, E and Reff, A., Biochim. Biophys. Acta, Qp, 328 (1967). Javitt, N. B., dm. J. Physiol., 2&, b24 (1966). Lathe, G. H. and Walker M., Eiochem. J., 70, 705 (19.58). Pogell, B. M. and Leloir, L. F., J. Biol. Chem., 2& 293 (1961). S&mid, R., Axelrcd, J., Hammaker,L. and Swarm, R. L., J. Clin. Invest., 37, 1123 (1958). Stevenson, I. H. and Greenwood, D. T., Biochem. Pharmac., I& 842 (1968).
872
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REPATICGDP-GLUCURONYLTRANSFERASEINWISTARAND GTJNNRATS IN VITRO ACTIVATION BY DIETRYU?ITRGSAMINE I.
Stevenson, D. Greenwood* and J. I%%wen
Department of Pharmacology end Therapeutics, Dundee, Scotland.
University
of Dundee,
Received August6, 1968 Dutton (1966)
has previously
pointed out that it is difficult
the absolute UDP-glucuronyltransferase being little enzyme.
known of the in vitro Investigations
solubilised
trsnsferase
in different
regulating
The stimulatory
The results
there
of this
of DDPGA-
in this paper demonstrate, from Wistar and Gunn
by the carcinogen diethylnitrosamine
appears to be confined to rat liver
marked with Gunn rat liver deficiency
the activity
and by the presence, in many tissues,
in vitro
effect
tissues,
complicated by lack of studies on
that hepatic DDP-glucuronyltransferase
rats may be activated
trsnsferase
factors
are further
destroying pyrophosphatases. nevertheless,
activity
to assess
preparations
of this tissue
(DE%).
and is particularly
- to the extent that the reported (Scbmid et al, 1958;
Lathe and Walker,
1958) is no longer apparent when DENis included in the ass&v system. DEN-activation
occurs with both microsomal and solubilised
and may be demonstrated using either Kinetic affinity Materials
analysis of the activation
o-aminophenol or paracetamol as substrate.
indicates
that DENdoes not alter
the
of the enzyme for substrate. end Methods
Male adult Wistar or Gunn rats were used throughout, for liver
transferase
enzyme studies by stunning and cervical
*Present address:
I.C.I.
Pharmaceutical Division,
866
animals being killed
dislocation.
Alderley
Following
Park, Cheshire.
BIOCHEMICAL
Vol. 32, No. 5, 1968
rapid excision of the liver, DDPGAsnd digitanin
KCl.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
homogenateswere prepared in alkaline
isotonic
were purchased from the Sigma Chemical Co. and
l-k-UDPGA from the Radiochemical Centre, Amersham. For preparation for 30 min.
of microsomes, whole hamogenateswere first
Microsomes, sedimented from the resultant
for 60 min at 100,000 g, were Solubilisation
volume.
of Pogell and Leloir
(1961),
equal volume of 1% digitonin centrif'ugation
supernatant by spinning
resuspended in KC1 in one-fifth
of glucuronyltransferase
spun at 4,000 g
of the original
was achieved by the method
the microsomal suspension being stirred for 30 min at O".
The supernatant after
at 100,000 g for 60 min was used as solubilised
DDP-glucuronyltrsnsferase
activity
with an
transferase.
using c+minophenol as substrate was
determined by the method of D&ton and Storey (1962) using 4.9 x 104M DDPGA and spectrophotometric incubation O.lml
estimation of the product at 565 w.
volume of 0.6 ml contained either
The total
0.1 ml of lO$whole homogenate,
of microsomal suspension or 0.2 ml of solubilised
enzyme.
Results Activation
of rat liver
around 1.6 x 10% DEN. this concentration trsnsferase
It
UDP-glucuronyltransferase
is apparent from the results in Table 1 that,
of DEN, approximately j-fold
occurred, the degree of activation
homogenate, microsomal or solubilised
activation
preparations
did not result
found to affect
activation
at
of Wistar rat liver
being the samewith either
preparations.
homogenatesmore than 20-fold activation observed with liver
was found to be msximal
With Qunn rat liver
was obtained.
No stimulation
from species other than the rat.
was
The
from reduced UDPGAbreakdown, for DENwas not
UDFGApyrophosphatase and the activation
of transferase
was demonstrable in DDFGA-saturated systems. DEN-stimulated synthesis of g-sminophenylglucuronide as estimated calorimetrically
in the Dutton and Storey procedure was confirmed by another
method utilising
%-DDPGA (Fig. 1).
As is evident from comparison of
ChromatogramsA and B, synthesis of
4 C-g-aminophenylglucuronide in the
867
Vol. 32, No. 5, 1968
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1 In vitro
effect
of DEN on rat liver
CDP-glucuronyltransferase
glucuronide
(A)
Enzyme preparation
No DEN
1.
Wistar
2.
formed?
(B)
B/A
DEN (1.62 x 10-2M)
rat liver
Homogenate
0.49
1.44
2.9
Microsomes
0.052
0.168
3.2
Digitonin-solubilised enzyme
0.053
0.165
3.1
0.060
1.33
22.2
Cunn rat liver Homogenate
%esults are expressed as poles o-eminophenyl glucuronide formed/preparatian from 1 g wet wt liver/30 min. E%h value quoted represents the mean value of separate determinations on preparations frcun at least five animals.
presence of DEN was more than twice that when the carcinogen The product
formed in the presence of DEN was almost ccmpletely
subsequent incubation
with P-glucuronidase
of UDP-glucuronyltrensferase phenolphthalein approximately
or menthol b-fold
Results
as substrates
activation
2.
from kinetic
It is evident
g-aminophenol
(Chromatogram C).
could be detected
using the above radioactive
Eg.
was excluded. hydrolysed
No DIQT-activation
using ~nitrophenol,
but, with 4 mM paracetemol
of Wistar
by
rat liver
transferase
as substrate,
was observed
procedure. studies
on the solubilised
that additi~
(4.4 x 10 -!M,.
transferase
are shown in
of DEN does not change the K, for
similar
868
experimentswithvarying
DDPGA
BIOCHEMICAL
Vol. 32, No. 5, 1968
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
,-----(
I..*
.
. . .
Chromatogram A
Chromatogram B
Chromatogram C
11.
1
t
1
2
3
Origin Figure 1. DEN-activation using labelled UDPGA.
4
5
s
Diitance
1
8
0
I
5
rl
10
11
12
.
13
14
8
15
15
from origin (cm)
of CDP-glucuronyltransferase
- confirmation
Incubations contained g-eminophenol end buffer as used by Dutton and Storey (1962) together with lO$ of 1O~Wista.r rat liver homo ate in a total volume of SO*. The donor nucleotide was 4.9 x 10~4M rtf?C-UDPGA The 30 min incubations at 37“ were terminated by immersion (2LSmc/mM). of the incubation tubes in boiling water for 1 min. Following addition of 0.2nil water and centrifugation, 104 of the incubate was spotted in a km-wide band on a thin layer (250~) silica gel plate. After development over l&m in a methanol: H20 (9:l) solvent system, lcm zones were scraped off into vials containing $I ml dioxsne-naphthalene scintillator and counted in a Nuclear Chicago Scintillation Counter using the Channels Ratio Method of Quench Correction. ChromatogremsA and B were from incubates containing No DENsnd 1.62 x 10m2MDENrespectively. ChromatogramC was from an incubate containing DENand which had been subsequently incubated with P-glucuronidase. The value for the two left-hand columns in each chromatogram was 2 x I&. Standard o-aminophenyl glucuronide (!.....I) and glucuronic acid (I----I) ran as inzcated.
869
BIOCHEMICAL
Vol. 32, No. 5, 1968
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60
.I
s= v=
o-aminophenol AOD
565mp
xl0 I
Figure 2. Kinetics of the DEN-activation UDP-glucuronyltransferase.
-4
M
10min.
of solubilised
Wistar rat liver
Incubations uere carried out for 10 min in the presence of o--------d 1.6 x 1O-2M DEN. Conditions No DEN, 8.0 x lo-3M DEZiand 0-6 of asssy are as described iu the text witi the exception that the o-aminophenol concentration was varied as indicated and that the final DDPGAconcentration used was 4.9 x lo-3M.
concentrations
suggest that the I$,, for the nucleotide
is also unaffected by
DEN. Discussion Several agents are known to stimulate glucuronide synthesis in vitro. The stimulation
by ATP and UDP-N-acetylglucosamiue described by Pogell and
870
BIOCHEMICAL
Vol. 32, No. 5, 1968
Leloir
results
from reduced CDPGAdestruction,
and Ffeff, 1967) may be related Cn the other hand, activation
to its
1961) seems to involve
transferase
protein.
It
mechanism also operates
there
is no measurable
A DEN effect
or from interference activity
of this,
from activation
effect
(Pogell
on the UDP-glucuronylthat
of transferase, of trsnsferase
this although
for substrate.
of otherwise non-functional
of DEN on rat liver
the very marked activation activities
that no transferase there are previous
in Dunn rats
(Arias,
Clarification in Gunn rat liver
1961;
enzyme
transferase
drug-metabolising
transferase
of Wistar deficiency
exists
of apparently
Javitt,
1966).
activity.
studies
enzymes in general.
the microsomal
In support
normal glucuronide
synthesis
and of the precise factors
does not activate
It has recently
enzyme involved
are c-arable,
in Gunn liver.
on the in vitro
DEN in vitro
in
In the presence
and Gunn rat livers
reports
must await further
is interesting,
of the Gunn enzyme.
of the mechanism of DEN-activation
UDP-glucuronyltransferase
to inhibit
structure.
by albumin
with an endogenous system controlling
of DEN the transferase suggesting
microscznal
from the above results,
in DEN-activation
of EDTA (Halac
is also possible.
The specific particular
effect
change in the affinity
resulting
to alter trsnsferase
a direct
is likely,
while the effect
ability
of solubilised
and Leloir,
latter
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lesion
regulating microsomal
been shown, for example,
in hexobarbitone
metabolism
(Stevenson
Research Council
and the technical
and Greenwood, 1968). Acbowledgments The financial assistance addition,
support of the Medical
of Mrs. A. Reekie are gratefully
acknowledged.
wish to thank Dr. G. J. Dutton for much helpful
The authors, discussion.
References Arias, I. M., Diochem. Biopws. Pes. Commun., 6, 81 (1961). Dutton, G. J. in ~~Glucuronic Acid" (G. J. Dutton, ed.), p. 218, Academic Press, NewYork, 1966.
871
in
Vol. 32, No. 5, 1968
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Dutton, G. J. and Storey, I. Il. E. in "Methods in &~ymology~~ (S. P. Colowick and N. 0. Kaplan, eds.), Vol. V, p. 159, Academic Press, New York, 1962. Halac, E and Reff, A., Biochim. Biophys. Acta, Qp, 328 (1967). Javitt, N. B., dm. J. Physiol., 2&, b24 (1966). Lathe, G. H. and Walker M., Eiochem. J., 70, 705 (19.58). Pogell, B. M. and Leloir, L. F., J. Biol. Chem., 2& 293 (1961). S&mid, R., Axelrcd, J., Hammaker,L. and Swarm, R. L., J. Clin. Invest., 37, 1123 (1958). Stevenson, I. H. and Greenwood, D. T., Biochem. Pharmac., I& 842 (1968).
872