(CANCER RESEARCH 52, 1510-1514, March 15, 1992]
Detection of Polycyclic Aromatic Hydrocarbon-DNA Adducts in White Blood Cells
from Coke Oven Workers: Correlation with Job Categories1
Steinar 0vreb0,2 Aage Haugen, David H. Phillips, and Alan Hewer
Department of Toxicology, National Institute of Occupational Health, P.O. Box 8149 Dep, 0033 Oslo 1, Norway [S. 0., A. H.], and The Haddow Laboratories, Institute
for Cancer Research, Cotswold Road, Belmont, Sutton, Surrey SM2 5NG, Great Britain [D. H. P., A. H.]
ABSTRACT
Polycyclic aromatic hydrocarbon (PAII)-DNA adducts were quanti
tatively determined by ultrasensitive radioimmunoassay (USERIA) and
"P postlabeling in 128 DNA samples from WBCs of 68 coke oven
workers and a local control group of 13 workers. Forty-four samples had
a detectable adduct level by USERIA, with a mean of 0390 fmol adducts/
jig DNA (12.9 adducts/108 nucleotides) in the exposed group compared
to a mean of 0.316 fmol adducts/jig DNA (10.4 adducts/108 nucleotides)
in the control group. The mean adduct level with 32P postlabeling was
0.05 fmol/MgDNA (1.67 adducts/108 nucleotides) for the exposed group
and 0.046 fmol/pg DNA (1.54 adducts/108) nucleotides for the control
group.
Based on job description the workers were divided in 4 groups: control,
low-, medium-, and high-exposure group. Both methods produced a
positive corrélationcoefficient between estimated exposure and PAHDNA adduct levels. The significance levels determined with Kendall rank
correlation were P = 0.0145 for USERIA and P = 0.0594 for 32P
postlabeling. Adduct levels determined by 32P postlabeling showed a
correlation with tobacco smoking in the control group. No significant
correlation between PAH-DNA adduct levels measured by USERIA and
l:l' postlabeling was found. These results show that these methods
recognize different parts of the complex exposures in a coke oven plant.
INTRODUCTION
Workers in coke oven plants are exposed to a variety of
chemicals, in particular, the PAHs.3 PAHs are implicated as
causative agents of cancer of the skin and lung and may play a
role in the epidemiology of occupationally induced cancers.
Elevated cancer risk has been found in coke oven plants (1-3).
Benzo(a)pyrene and other PAHs are metabolically activated
to electrophilic intermediates which bind to DNA and subse
quently exert their mutagenic and or carcinogenic effect (4).
Several assays for the determination of DNA adducts have been
developed, such as immunoassay (5, 6), SFS (7, 8), and 32P
postlabeling (9). These assays have been used to determine
increased levels of BPDE-DNA adducts in the WBCs of occu
pationally exposed workers in coke oven plants (7, 10-12) and
foundries (13-15) and of roofers (13). In an early study of 30
aluminum plant workers, only one WBC DNA sample showed
the presence of detectable PAH-DNA adducts by SFS (8), but
recently there have been 2 reports describing moderately in
creased adduct levels using more sensitive methods (16, 17).
Although the influence of smoking on adduct levels in WBCs
from healthy nonoccupationally exposed individuals has not
been evident in all studies (18-22), in DNA samples from other
Received 8/28/91; accepted 12/27/91.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by North Atlantic Treaty Organization Grant
880679 and Grant 42.70.08 from the Nordic Council of Ministers.
2To whom requests for reprints should be addressed.
3 The abbreviations used are: PAH, polycyclic aromatic hydrocarbon; BPDE,
(±)-r-7,f-8dihydroxy-r-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene;
USERIA,
ultrasensitive radioimmunoassay; PNPP, ^-nitrophenylphosphate;
SFS, syn
chronous fluorescence spectrophotometry;
PBS, phosphate buffered saline;
ELISA, enzyme-linked immunosorbent assay.
human tissues higher adduct levels have been found in smokers
compared to nonsmokers (23, 24).
The purpose of this work was to evaluate the relationship
between PAH exposure and adduct levels in WBCs from oc
cupationally exposed coke oven workers and to study the influ
ence of exogenous and host variables such as smoking, use of
protective mask, age, and number of years employed. This
investigation is a follow-up to the earlier study by Haugen et
«/.(IO).We have increased the number of subjects studied and
included the 32P-postlabeling assay in addition to USERIA. We
report here a
job categories,
from workers.
samples when
correlation between exposure levels, defined by
and PAH-DNA adduct levels in WBCs obtained
Smoking was a confounding factor in the control
measured with 32P postlabeling.
MATERIALS AND METHODS
Chemicals. Microtiter plates polyvinyl chloride fastbinder 6695 were
purchased from Costar, Europe, Ltd., Badhoevedorp, The Netherlands.
[3H]PNPP and Econofluor II were obtained from New England Nu
clear, Boston, MA. PNPP, RNase A, proteinase K, and calf thymus
DNA were obtained from Sigma Chemical Co., St. Louis, MO. Alkaline
phosphatase-conjugated goat anti-rabbit IgG F(ab)2 was obtained from
Jackson Immunoresearch Laboratories, Inc., Avondale, PA. High- and
low-modified BPDE calf thymus DNA was kindly provided by Dr.
Ainsley Weston. The modification level was 36 pmol/^g DNA (high
modified) and 3.35 fmoI/Mg DNA (low modified). Reagents and mate
rials for 32Ppostlabeling were from previously described sources (25).
Subjects. Blood samples were obtained from 68 healthy coke oven
workers and 13 office employees at the plant. Samples were collected
twice, in January and June of the same year. The workers were divided
in groups based on job description (Table 1). The topside coke oven
workers were assigned to the high-exposure group, the side-oven work
ers to the medium-exposure group, and the maintenance workers and
other workers to the low-exposure group.
All participants completed a self-administered questionnaire. The
workers were asked to indicate smoking habit and mask use. Age and
first employment date were obtained from the factory's files. Data from
the questionnaire are summarized in Table 1.
Samples. Samples of blood (30 ml) were collected in heparinized
Vacutainer tubes. The blood samples were kept on ice and transported
by air to our institute. Buffy coat cells were immediately isolated and
frozen. DNA was isolated essentially according to the procedure of
Gupta (26). DNA concentration was determined by absorbance meas
urements at 260 nm and fluorescence measurements with Hoechst
33258 (27).
USERIA. A polyclonal rabbit antibody (MP 33, kindly provided by
Dr. Miriam Poirier) raised against BPDE-modified calf thymus DNA
was used. This antibody cross-reacts with several other PAH-DNA
adducts (28). Therefore, all values are given as PAH-DNA adducts,
although we have used BPDE-modified DNA as a standard.
The USERIA method was done essentially as previously described
(13, 29) except we used double-stranded standard and sample DNA in
the assay.
The DNA samples and standards were sonicated for 3 s (sonicator
W-10; Ultrasonics, Plainview, NY). The microtiter plates were coated
with highly modified BPDE-DNA (36 pmol/jig) in lux PBS and dried
over night at 37°C.Plates were washed twice in 1x PBS and incubated
l h with Ix PBS containing 0.05% Tween-20 and 2% horse serum
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PAH-DNA ADDUCTS IN HUMANS
(heat inactivated). Samples and standards were preincubated with an
tibody against BPDE-modified DNA for 20 min at room temperature
before addition to the plates and incubation at 37°Cfor 1 h. The total
DNA concentration was 10 ^g/well, and the samples were analyzed in
triplicate with a coefficient of variation normally <10%. Finally, the
plates were incubated for l h at 37*C with goat anti-rabbit antibody
conjugated with alkaline phosphatase. After incubation for 4 h at 37'C
with [3H]PNPP, an aliquot was withdrawn and mixed with Ix PBS
and Econofluor II scintillation liquid and counted in a liquid scintilla
tion counter.
All samples were analyzed twice, and a sample was considered
positive for PAH-DNA adducts if one parallel exhibited >30% inhibi
tion and the second was >25%. High-modified BPDE-DNA was used
as the standard throughout the whole study, but one sample of lowmodified BPDE-DNA was included on each plate. Fig. 1 shows a
comparison of low- and high-modified BPDE-DNA. The slope of the
low-modified BPDE-DNA is lower than for high-modified BPDEDNA. All samples were therefore recalculated using the following
formula:
logic
^„„.formed
= —¿
(logio
•¿^'high-modified)
synthesized [^-"PJATP was determined by measuring the kinase-catalyzed incorporation of radioactivity into a known amount of ilAMI'
(31). Values fell within the range 492-1198 Ci/mmol for homemade
material and 2031-4069 Ci/mmol for commercial material. The area
of the chromatograms which contained "P-labeled adducts consisted
of a diagonal zone from the origin to the upper right region of the
chromatograms. This area was excised and the levels of radioactivity
were determined by Cerenkov counting. Background levels, obtained
by counting areas of the chromatograms not containing adducts, were
subtracted. The levels of adducts present in a DNA sample were then
calculated from the radioactivity present in the adduci spots, and the
specific activity of the [-y--"P]ATP was used and expressed as fmol
adducts/Vg DNA (1 fmol,'//n DNA is equivalent to 33 adducts/ 10*
nucleotides). The limit of detection was estimated to be approximately
3 attomoles/Mg DNA (1 adduci/ 10' nucleotides).
Calculation and Statistics. For statistical evaluations, Kendall rank
correlation, Spearman rank correlation, and Pearson producl-momeni
correlation were used. Kendall rank corrélationgave ihe lowesl signif
icance level and is shown in ihe tables.
RESULTS
C
where a and c are coefficients for high- and low-modified DNA,
respectively, and b and d are the intercept with the x axis for high- and
low-modified DNA, respectively. All values were calculated with linear
regression of logio of modified BPDE-DNA.
"P-Postlabeling Assay. Samples of DNA (4 ^g) were digested with
micrococcal nuclease and spleen phosphodiesterase and then with nuclease PI essentially as described previously (30, 31) with minor mod
ifications (32). The DNA digest was then "P labeled with carrier-free
[v"P]ATP (50 /iCi, 1.85 MBq; purchased from Amersham Interna
tional, Amersham, Buckinghamshire, United Kingdom) or synthesized
in the laboratory (33) using [':P]P, (also obtained from Amersham
International) and polynucleotide kinase; the reaction was terminated
by the addition of apyrase (30). Resolution of ' 'I'-labeled adducts was
then carried out by chromatography on 10- x 10 cm polyethyleneiminecellulose thin layer chromatography sheets using the solvents described
previously (25), namely: direction I, l M sodium phosphate, pH 6.0
(overnight, onto a filter paper wick); direction 2 (opposite to direction
1), 3.5 M lithium formate-8.5 M urea, pH 3.5; direction 3 (at 90'C to
direction 2), 0.8 M LiCl-0.5 M Tris-HCl-8.5 M urea, pH 8.0; direction
4 (same as direction 3), 1.7 M sodium phosphate, pH 6.0 (onto a filter
paper wick). The presence of radiolabeled adducts on the chromatograms was detected by autoradiography at -70°C using intensifying
screens.
For each batch of [32P]P¡,
the specific activity of the laboratory-
100
80
60
40
20
0.1
1
fmol PAH odducts/Vg DNA
Study Population. DNA was isolated from WBCs obtained
from 68 coke oven workers and 13 local controls. All samples
were analyzed for PAH-DNA adducts with immunoassay
(USERIA) and the 32P-postlabeling technique. There were only
small variations in age and smoking habits between the groups;
however, mask use was most common in the high-exposure
groups as expected (Table 1). There was a higher representation
of young workers in the high-exposure group. Most of the
individuals sampled in January were also sampled 6 months
later. In general, similar levels of adducts were detected in these
two groups of samples. However, a consistent increase in adduci
levels from January to June was found in the exposed groups
by 32P postlabeling.
PAH-DNA Adduct Levels Quantitated by USERIA. Mean
adduci levels (fmol/^g) increased with the exlenl of exposure.
The levels were 0.316, 0.340, 0.398, and 0.440 for Ihe control,
low-, medium-, and high-exposure groups, respeclively. A box
plol of adduci values versus exposure groups is shown in Fig.
2. A dose-response relationship is evidenl because Ihe median
increases wilh exposure.
Of Ihe samples analyzed, 44 (37%) were positive, i.e. above
Ihe detection limit, by USERIA. The mean adduci level for the
coke oven workers was 0.390 fmol//ig (12.9 adducts/ IO8 nu
cleotides) and 0.316 fmol/Mg (10.4 adducts/ IO8nucleotides) for
the control group. The percentage of positive samples did noi
correlale wilh exposure. Smoking habits did not influence the
adduci levels as measured by USERIA in Ihe eilher exposed or
ihe unexposed group.
Since Ihe exposure levels were divided inlo groups, a nonparametric rank correlalion lest was used. The resulls from a
Kendall rank correlalion lesi are shown in Table 2. Adduci
levels are significantly correlated lo exposure groups. This
correlalion (P < 0.05) was also found wilh Spearman rank
correlation and Pearson product-momenl correlations (data not
shown). No significant correlation was found between adduci
levels and use of proleclive mask, age, or number of years
employed al Ihe plani.
PAH-DNA Adducls Quantitated by "I' Posllabeling. Chro
matograms of 32P-labeled digests of WBC DNA revealed a
Fig. 1. Competitive inhibition of polyclonal antibody binding to plates coated
with BPDE-modified DNA. Standard inhibition curves obtained by I'M RIA.
The inhibitors were serially diluted (•)highly modified BPDE-DNA (36 pinol
BPDE/Mg DNA and (•)low-modified BPDE-DNA (3.35 fmol BPDE/Mg DNA).
Error bars, SD from 25 standard curves. In the calculation of the correction
factor, all values from low-modified BPDE-DNA and only the 4 middle values
from the highly modified curve were used.
diffuse diagonal zone of radioactivity (not shown). These pro
files, indicative of adduci format ion by a complex mixture of
compounds, were qualilalively similar to those previously seen
in sludies of workers in iron foundries (15), coke ovens (11),
and aluminum plañÃ-s
(17).
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PAH-DNA ADDUCTS IN HUMANS
Table 1 Coke oven workers summary data, by estimated PAH exposure group
group1313
exposure44261861.4
exposure40231756.1
exposure3118
samples12880
samplesJanuary
No. of
June%
1350.0
046.1
smokers
(SD)%
(51.9)0
mask use
(SD)Mean
(0)48.8
age (yr)
(SD)ExposureControl
(9.9)NoneLow
(50.2)68.3(47.1)38.3
(9.5)Maintenance
workersMedium
•¿T
1
0.6
•¿oo
0<.4Q
°-2EO-
AContro
(50.9)93.3
(49.9)68.0
(46.2)36.7
(25.4)33.4
(46.8)37.7
(11.6)Side-oven
(10.7)Topside
workersHigh
oven
workersAll
(11.3)
cant correlation was found between adduct levels measured by
USERIA and 32P postlabeling.
i•
1I
(49.3)70.5
Comparison between USERIA and 32PPostlabeling. The mean
adduct level (adducts/IO8 nucleotides) using the USERIA tech
nique was 7-fold higher than with 32P post labeling. No signifi
zoo»^
u•o
4855.5
DISCUSSION
.Medium'
exposureTîexposure
groupoI1OLow-
There have been several studies in which 32P-postIabeling
analysis and/or immunological techniques have been used to
determine the levels of PAH-DNA adducts in WBCs from
occupational!)' exposed workers. With both of these methods,
differences between the occupational!}' exposed groups and
unexposed controls have been reported (6, 7, 10-15).
However, with neither of these techniques is it possible to
regard the levels of adducts determined as absolute levels when,
as here, the DNA damage is thought to be due to many
components. 32P-postlabeling efficiency and degree of antigenic
Highexposure
Fig. 2. Box plot of PAH-DNA adduct levels in DNA isolated from WBCs
from coke oven workers and a local control group measured by USERIA. For
each box, the middle line represents the median, the box encompasses the 75th
through 25th percentiles, and the caps are at the 10th and 90th percent ¡les.Values
outside 10th and 90th percentiles are plotted individually. Dotted lines, mean
values for each group.
Table 2 Statistical evaluation (Kendall rank correlations) of DNA adduct levels
determined by USERIA and "Ppostlabelling and confounding factors
PAH-DNA
Exposure
groupsUSERIA"
adducts
mask
use0.1294
employed-0.1572
0.2403*
0.0145C
0.1714
0.9564
0.1155
0.1081
"P postlabel0.1122*
-0.0631
0.0098
0.0223
0.0299
ling'
0.0594CSmokers0.1533
0.2892"
0.6152Protective
0.8692Age0.0055
0.7088Year
Sample size, 44.
* Coefficient.
' Significance level.
' Sample size, 125.
response cannot be precisely determined in such circumstances.
Therefore, the values obtained with each method indicate rela
tive levels between individuals and exposure groups, and differ
ences in the total levels determined by the two methods are not
unexpected.
In this investigation, we analyzed DNA from WBCs from
115 coke oven workers and 13 local controls. The present study
is a follow-up of a similar investigation at the same coke oven
plant by Haugen et al. (10), with the inclusion of side-oven and
maintenance workers in addition to the highly exposed top20
."5 10
The results of the determination of the PAH-DNA adduct
levels in the exposure groups are shown in Fig. 3. All samples
showed adduct levels above the detection limit of 1 adduct/IO9
nucleotides. The mean PAH-DNA adduct level in the exposed
group was 1.67 adducts/IO8 nucleotides compared to 1.54
adducts/IO8 nucleotides in the control group. The correlation
coefficients are summarized in Table 2. The correlation between
adduct levels and estimated exposure (work categories) is of
borderline significance.
Comparisons between smokers and nonsmokers are shown
in Fig. 4. In the control group, smokers have a higher median
than nonsmokers, and in this group smoking is significantly
correlated with the adduct level (P = 0.02, Kendall rank
correlation)
Control
group
Lowexposure
Mediumexposure
Highexposure
Fig. 3. Box plot of PAH-DNA adduct levels quantitated with the 32P-postlabeling assay. DNA was isolated from WBCs from coke oven workers and a local
control group. For each box, the middle line represents the median, the box
encompasses the 75th through 25th percentiles, and the caps are at the 10th and
90th percentiles. Values outside the 10th and 90th percentiles are plotted individ
ually. Dotted lines, mean values for each group.
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PAH-DNA ADDUCrS
IN HUMANS
Seasonal variations in PAH-DNA adduct levels in WBCs
from lung cancer patients and controls were described by Perera
et al. (21). They found the lowest levels in the coldest seasons.
This is in accordance with our "P-postlabeling results which
indicated higher levels in June samples than in January samples.
Generally, the exposure to PAH in coke oven plants and
aluminum plants is higher than found, for example, among
foundry workers (38). High adduct levels have been reported in
samples from coke oven workers (7, 10). But in DNA from
workers at aluminum plants, mostly low adduct levels have
been found. One study used SFS, and the authors found one
positive sample out of 30 for adducts (8). In a more recent
study, van Schonten (16) reported no detectable level adducts
by ELISA and 91% positive samples by 32P post labeling.
20
10
O
O
Control
group
Exposed
Fig. 4. Box plot of PAH-DNA adduci levels quantitated by the "P-postlabeling
assay. Samples are divided into a control group and a group of exposed workers,
and each of these is divided into smokers and nonsmokers. For each box, the
middle line represents the median, the box encompasses the 75th through 25th
percent ¡Ics,and the caps are at the 10th and 90th percentiles. Values outside the
10th and 90th percentiles are plotted individually. Dotted lines, mean values for
each group.
oven workers. We found a dose response between adduci levels
and exposure groups, defined as work categories. The mean
adduci level by USERIA in the 1986 study was 1.7 compared
to 0.44 fmol/^g DNA in the present study. This difference is
greater if we consider our use of low-modified DNA as the
standard, which increased our mean approximately 3-fold. In
both studies, no influence of smoking was found. Several studies
have been performed at this plant in the last 10 years, resulting
in an increased awareness of the benefits of a healthy working
environment at the plant. In the high-exposure group, 93% now
use protective masks compared to 80% earlier. It is also possible
that there has been an improvement in work practice during
this period, resulting in reduced exposure at work. These factors
may explain the lower adduct levels in this study.
When comparing our results with earlier coke oven plant
studies (7, 10), we found low adduct levels. But compared to 2
recent coke oven plant studies (11, 12), similar adduct levels,
as determined by immunoassay, were found. Reported mean
values for exposed groups were 0.155 (12) and 0.460 fmol
adducts/Vg DNA (11) compared to our values (0.390 fmol
adducts/Mg DNA). An interlaboratory comparison of immu
noassay for benzo(a)pyrene-modified
DNA has shown that
adduct levels differ as a result of different antibodies and assay
methods. The assay methods tested were USERIA, color
ELISA, and fluorescence ELISA (29). Therefore, one should
be cautious when directly comparing adduct levels between
different studies. Adducts increase the depurination of bases in
DNA, resulting in a loss of adducts (34). Although the DNA
isolation methods used in most studies of adduct levels are
based on standard procedures involving phenol/chloroform ex
traction and precipitation with ethanol, the possible partial loss
of adducts during such procedures cannot be discounted.
The influence of smoking on adduct levels has been deter
mined quantitatively with immunoassay and the 32P-postlabeling technique. In tissue samples from smokers, increased adduct
levels have been found with all of these methods. The tissue
analyzed was from placenta (35) and lung (23, 36, 37). Several
studies have been published concerning the influence of smok
ing on DNA adduct levels in DNA from WBCs, but a less clear
relation has been found (18-22, 25, 37).
Schoket et al. (17) studied two plants but found a higher mean
adduct level, compared to a nonlocal control group, in samples
from only one of the factories. This is in contrast to foundries,
which normally have low benzo(a)pyrene levels (14, 38), but
several reports have been published demonstrating relatively
high DNA adduct levels in workers' blood cells (13-15). These
observations suggest that uptake may vary among coke ovens,
aluminum plants, and foundries, and may also vary between
different coke oven plants. The nature of such a putative differ
ence in bioavailability is unknown, but absorption of PAH to
particulate matter and size of these particles can influence
uptake. In a study of lung cancer in China, increased lung
cancer incidence was associated not only with PAH air concen
trations but also particle size (39).
The adduct levels determined by ' P post labeling in our study
are lower than those obtained in a recent study of coke oven
workers (11). We found a ratio of adduct levels determined by
USERIA and adduct levels determined by 32P postlabeling
varying from 5 to 10 in the different work categories. This
USERIA to 32P-postlabeling ratio can be calculated from the
results in a few reports in which the samples were analyzed by
both immunoassay and the "P-postlabeling assay. Everson et
al. (35) quantitated adducts in smokers and nonsmokers and
used both monoclonal and polyclonal antibodies in their im
munoassay. We calculated a ratio of 5.3 and 0.35 with the
monoclonal antibody for nonsmokers and smokers, respec
tively. The similar numbers for the polyclonal antibody were
2.1 and 0.1. In two more resent publications, this USERIA to
32P-postlabeling ratio can be calculated to be 3.1 (Ref. 36) and
0.6 and 1.2 (Ref. 11). This ratio is likely to vary with type of
'••P-post
labeling method, antibody in the immunoassay, stand
ard modified DNA, and samples.
In summary, we have found a dose-response relationship
between estimated exposure defined by job categories and ad
duct levels with USERIA. In the control group, smokers had
higher adduct levels than nonsmokers when determined with
the 32P-postlabeling technique.
ACKNOWLEDGEMENTS
We wish to thank Dr. H. Hofstad Andreasen for sampling and
administration of questionnaires at the plant. The technical assistance
of Ingrid V. Botnen is gratefully acknowledged. We also thank Dr.
Glenwood E. Trivers for advice concerning the USERIA assay.
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PAH-DNA ADDUCTS IN HUMANS
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1992 American Association for Cancer Research.
Detection of Polycyclic Aromatic Hydrocarbon-DNA Adducts in
White Blood Cells from Coke Oven Workers: Correlation with
Job Categories
Steinar Øvrebø, Aage Haugen, David H. Phillips, et al.
Cancer Res 1992;52:1510-1514.
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