R 39
Lack
of
in
Viva
Binding
to
DNA of
Piroctone-olamine
P.
Sagelsdorff,
W.K.
Lutz
and Ch.
Schlatter
Institute Swiss F ederal Institute CH-8603 cf
of
Toxicology, and University Switzerland of Zzrich,
Technology
Schwerzenbach,
Report
prepared
for
HOiCHST
PHARMA FORSCHUNG TOXIKOLOGIE Fostfach 80 03 2'0 am Maln 80
D-6320
Frankfurt
Date Sept.
of 16,
isslre: 1983
DNA Binding
Assay
with
Piroctone-olamine
we
4
Materials
and Methods
Test
compound.
[6-14C)Piroctone-olamine
monoethanolamine
salt
(I)
(I-hydroxy-4-mlethylmonoethanolamine specific pyridone layer radioactivity ring. salt)
6-(2',4',4'-trimethylpentyl)-?(lH)-pyridone was of prepared 33.4 by the Hoechst radiolabel shown to (Merck) company with a the by thin
mCi/mmol. purity
The was
was in be 98.5% with
The radiochemical on polyamid vol.)
chromatography
11 F,54
rats were between
plates
methanol/ethanolamine Animals. Kissleg, .held (No. libitum 4 per Young male
(99+1 SPF Uistar weights cage
obtained 150 were and
from 170
Ivanovas, g. They were chow ad for
FRG, ,with macrolone
ranging
on saw dust. Mijhle access
They
fed
laboratory
71-343-7, and they
Klingenthal had free
AG, Kaiseraugst, to tap water for
Switzerland) one week
acclimatisation. Treatment. 50 % aqueous by The subcutaneous animais of 0.4 were 35 mg/kg b.w, (3.5 mCi/kg) (12 in single order in the vol.) hours to after under a teflon was mg/ml), to glass Piroctone-olamine, was simulate metabolism air the to administered 100% dermal cages a trap dissolved to three in rats
l,?-propanediol injection placed
absorption. where with an air
stream
l/min
transported (I+4 48
exhaled collect the ether
ethanolamine/methanol Isolation were were -. bled excised of chromatin. heart
expired
['4C]O?. the The animals livers
administration, anaesthesia.
by open
puncture in
and homogenized at 4'C. Crude detergent [SageTsdorff ?O-30 ~'$000
Potter-Elvehjem-type prepared (BDH by precipitation Ltd., Poole
homogenizer with the
chromatin Nonidet et mg protein dpm/ml.
non-ionic England)
P 40 al., per
Chemicals This pellet, was
BH12 4NN, about . 3-3
1983];
containing
the
mg DNA and contained
g liver,
washed until
Suspension
DNA Binding
Assay
with
Piroctone-olamine
we
5
Isolation homogenized Urea in
of
DNA and further
chromatin in a Waring pH 6.8.
protein. blender
The chromatin in 1 % (w/v) was
pellet SDS,
was 10 m M EDTA, with by 8 M
0.24
M Nazp04,
The homogenate (CIP) column,
deproteinized
chloroform/isoamyl adsorption precipitation [Sagelsdorff protein) The amount was of on
alcohol/phenol a hydroxylapatite with et a?., ethanol to
and the
dialysis, specific purified
DNA was purified and repetitive radioactivity DNA (less succinate, of was than
constant
I983 2. The highly in 8 m M CaCl?,
O.?
"6:
dissolved
?D m M sodium
pH 6.0. 30 at performed counter analysis 2'60
DNA was determined of I mg DNA/ml.
by assuming Scintillatjon in a Packard
an absorbance counting scintillation
nm for after Tricarb v' of
a solution
addition
460 CD, labelled Chromatin
of 10 m l Insta-Gel
equipped with
and calibrated
for the
acetone The
automatic
samples. protein in was 1X (w/v) precipitated SDS (five with times). for the the Folin from from the CIP extract was The
and
redissolved to of
last
solution
diluted amount Control together ccxnoarison of the
0.1 5 SDS and protein
1 ml was used with
scintillation reagent. an untreated total to count show that
counting.
was determined (1) treated historical
experiments. with with the
DNA was isolated ones.
animal - upon the
held
The respective - was used without external
controls
work-up with
DNA samples
was performed
contamination
radiolabels.
(?) The chromatin
incubated the first for chromatin animal. was
pellet
isolated at
from the
the
liver
of
an untreated suoernatant from this the main
rat of a in
was
15 minutes
4OC with step of the net
radiolabelled of the
precipitation radioactivity from
DNA preparation after in DNA only
treated incubation exDeriment isolation
The
the
DNA isolated
vitro
deducted all was
DNA radioactivity into
so that procedure
radiolabel accounted
introduced for.
during
the
LAM
;ino:ng
Assay
Wlin
Plroctone-o;mine
page
6
Results
and
Discussion
The injection respectively. 4 percent At and
excretion amounted
of to
radiolabel 6+? percent
within and
24 hours ?9_tlO arL bier in
after in
subcutaneous urine and faeces, additional respectively. the liver DNA
percent, the
Between and 24 percent
?4 and 48 hours were excreted
administration and isolated the faeces, from results. Under the was activity converted in were to
urine was
this
time for
DNA and chromatin [14C]-labelling. at that the
protein Table limit minute
counted were
1 compiles of detection.
The
samples preliminary DNA-bound samples units Table for the of
radiolabelled assumption
this
DNA radioactivity the specific and
due of the
to the DNA
Piroctone-olamine were the divided 'Covalent by the Binding
metabolites, dote
administered Index, CBI', O.O??
molar to .
as defined and 0.013
a footnote calculated
1 [Lutz, three
1979 J. Values treated rats.
of
0.016,
Radioactivity treated interactions the test with
on the a radiolabelled of the test with
DNA isolated substance compound DNA is in with
from is
an animal not necessarily Non-covalent the of
that
has due
been to covalent of
DNA.
interaction of the
compound experiments was
unlikely th e third A more
because column likely incorporation molecules the for is ['4CJ03 taken
data Table
respective that this
control contribution
given not is
1 show for the
measurable. the biosynthetic to Such j14C;D,,
reason of to
observed if pool of that 6 of
DNA radioactjvity the compound acid is
radioactivity enter the
degradable
small is
able case
nucleic are the Table
precursors. -lo yield
obviously because,
with the
compounds caroon
cegraoed purine.bases
instance, from
adenine the
and guanine data on the
a CO, molecule. after as exhaled amount not broken little within may as 0.01 48
2 surrunarizes of the
exhalation On average,
administration oercent . hours to inat in of the
[14CJpiroctone-olamine. dose This
radioactivity form of [14C JO?,
administered extremely and
was small does js
be due mean during
radiochemical the pyridone
impurities moiety of
administered ?iroctone-oiamine
necessarily down
metabolism. Due to the the fincing :hac the ;iLC,)Z, is formed in after the administration DNA must Control mignt in part of be with
radiolabe?:ed
samole,
radioactivity
due to biosynthetic incorporation 07 raaioiacel. Li4-? i,meIn2no; show :hzI this source of racioac:ivi:;/
experiments fui?y
DNA Binding
Asgay
with
Piroctone-olamine
we
7
account biosynthetic
for
the
DNA radioactivity: into liver
Exhalation DNA were
of
['4CJ0,
and after oral
incorporation iof 45 hIours ac'tivity 15 mg/kg after
determined rats.
administration from the liver specific
b.w.['4Cjmethanol administration, to
to male was clearly
DNA isolated and
radiolabelled of 4?B. The
corresponded
an apparent to 85% of
CBI the
corresponding radioactivity For and
[:14CJexpiration dose, exhaied of the as
accounted ['4C
administered
JO? within
radioactivity
48 hours. [14C into expired
an estimation
relationship
between
JO, exhalation
DNA, the ratio of was calculated liver DNA.
biosynthetic DNA activity basis of of ratio range
incorporation-of (CBI methanol the [14C]0, in units) data data
specific on the ,
per to
('4CJ0, be 428:85%
the
= 503 for
Multiplication with in the this
obtained
with
Piroctone-olamine Index of about 0.05, in i.e. CBI
resulted as the
an apparent
Binding
same
observed
DNA radioactivities
expressed
units. Contamination observed the.protein that of DNA with but given protein in in protein could also contribute on the to the basis fact ci small of
DNA radioactivity radioactivity of result the above
a quantitative in the Table 1 is
analysis conplicated of high
by the activities
precipitation must on
presence
molecules Based radioactivity is not due
overestimations. it is highly probable of that the DNA
results after but of
detected to DNA binding ?rxursors protein.
administration to biosynthetic
[14CJPiroctone-olamine incorporation of ccntmination of
radiclabelled the DNA with
DNA synthesis
and pernaps
to
If one disregards
radioactivity as due . to on covalent low
the
above
explanation hypothetically Piroctone-olamine with known
for
the the to
origin values DNA,
of given the The
the in Table wculd 1
DNA and takes binding upon of
figures strongest
be extremely hepatocarcinogenic like :s >/inyl aflatoxjn
ii0
comparison with
carcinogens. initiating a CBI
compounds 5:' or
a genotoxi.c, exhibit
mode of in the
action, of
dimethylnitrosamine, heparocarcinogens a CBI of a few
order and
i0'. chioride
.!+? .fSerate s;‘ ~w such :-'I?. as The
li ke l-acetyiaminofluorene nundrea, ana weak genotoxic to
of
carcinogens ieve? cf
N,N-dimethyl-4-aminoazobenzene maximum possible is :oo DNA-binding low action. to consider
bind zb'lity
DNA on CgI
?irXzone-oizzl activity S.D.a
Net activityS.D.b [dpmpl
Specific
[dpm/mg$! [CBI units? tl S.D. Control Cruae protein
activity
S.D.
0.4
kO.1 0 .C!S +0.005
Data chromatin [dpm/mgj
157
133
113
a
The total variability (statistical counting error and fluctuations due to vial, scintillation cocktail, counter, external radiation) for a DNA sample containing little radioactivity was assumed tc be equal to the variability of DNA samples isolated from untreated animals held together with the treated ones. On the basis of 36 background a respective standard deviarion of 3.73 s:alues compiled for 12 years, cpm was calculated. The standard deviation for a net radioactivity in a vial therefore was taken as: 7 . : ~0.;3T+(().73,‘ \,,~ )? = 0.76 cpm - r.cl.n =‘
5
The 'imit
2 ;evei The
,-?T
zf
of de:ect'cn 7 s-2 . The kindin
ior radioactivity counting efficienfy Index Sauna
appliE0
in a vial was 75% as:
was calculate:!
on
c
Ccvaient
:
$3:)
is
defined
VI.
piy] cnemicz iii;l? c I cnernlct; ?i$
/ ma1 D!lA nuc?ecr:de ; Kg soay wslgnr 3.2-108
l
CBI
=
dpm / mg DNA / kg booy neight
DNA Binding
A&say
with
Piroctone-olamine
page
10
Table
7.
Exhalation
administration
of
['4CJlabelled of
CO;, after
subcutaneous to 3 male rats
[14CJPiroctone-olamine
Period [h after
of
collection application]
Cumulative Fraction Rat 1 of
exhalation dose ? [10e5_! Rat 3 .o 4.6 a.4 11.0 13.0 3
Rat 2.8 3.6 6.1 8.0 9.7
o-
4
2.4 3.0 4.6 5.6 6.5
4 - 10 10 - 20 30 - 30 30 - 48