AATEX 11(2), 2005
ORIGINAL ARTICLE
Development of a Modified Local Lymph Node
Assay using ATP Measurement as an Endpoint
Kunihiko Yamashita, Kenji Idehara, Norio Fukuda,
Gaku Yamagishi and Naoki Kawada
Daicel Chemical Industries, Ltd.
1239, Shinzaike, Aboshiku, Himeji, Hyogo 671-1283, Japan
Abstract
Several proposed modified local lymph node assays (LLNAs) use a non-RI method, but such methods
tend to yield lower stimulation indices (SIs) than those obtained with the standard LLNA when used to
assess moderate sensitizers. To address this issue, we propose a modified LLNA in which mice are exposed to the test material four times, instead of three times, and adenosine triphosphate (ATP) content is
used as a measure of proliferation of lymph node cells (LNCs). The chemicals tested were 2.4- dinitrochlorobenzene (DNCB), eugenol, and -hexyl cinnamic aldehyde (HCA), which are classified as
strong-to-moderate sensitizers in the LLNA. Methyl salicylate (MS) was used as an example of a
non-sensitizer. Using an SI of 3 as a cut-off, as recommended in the standard LLNA, DNCB, eugenol
and HCA were classified as positive, while MS was classified as negative using the ATP-based non-RI
method. Furthermore, there were no marked differences in SI levels for 10% eugenol and 10% HCA (a
moderate sensitizer) between the non-RI method and the standard LLNA. Thus, it is reasonable to conclude that the non-RI method proposed in the present study could be a valid alternative to the standard
LLNA.
Key words: local lymph node assay, non-RI method, stimulation index, adenosine triphosphate (ATP),
eugenol
No. 429 (OECD 2002)). Although the LLNA is not
quite as sensitive as the GPMT, it has several advantages over the GPMT, including generation of
quantitative data, a reduced test time (Hanake et al.,
2001; Basketter et al., 1996), a reduction in the
required number of animals, and improved animal
welfare.
The LLNA uses [3H]thymidine to measure
lymphocyte proliferation, and therefore requires
special facilities that enable safe handling of radioactive substances (Takeyoshi et al., 2001).
Thus, efforts are being made to develop a modified
LLNA that uses a non-radioisotope (non-RI)
method, such as histological assessment of 5bromo-2’-deoxyuridine (BrdU) incorporation into
LNCs or assessment of the amount of interleukin-2
Introduction
Exposure to sensitizing chemicals may cause serious health problems, such as glutaraldehyde-induced allergic reactions among healthcare
workers and metal allergy associated with ear
piercing (Katsumura et al., 1996). The Guinea Pig
Maximization Test (GPMT) has long been used to
assess the sensitizing potential of chemicals
(Magnusson et al., 1969). In recent years, the murine LLNA has been developed, and after thorough
evaluation and reviews, this test was adopted by
the OECD as a stand-alone alternative for contact-sensitization hazard assessment (OECD Test
Guideline No.429 ) (Kimber et al., 1986; Kimber
et al., 1989; Basketter et al., 1996; Dean et al.,
2001; Gerberich et al., 2000; Haneke et al., 2001;
136
AATEX 11(2), 2005
(IL-2) produced by stimulated lymphocytes (Suda
et al., 2002). Assessment of both the CD4/CD8
ratio in lymph nodes and IL-2 production can be
useful for differentiation of irritant and allergic
contact sensitizers (Hariya et al., 1999). A recent
study using BrdU incorporation instead of isotope
incorporation by LNCs as a measure of LNC proliferation was, in principle, conducted according to
the standard LLNA protocol (Takeyoshi et al.,
2001). However, when used to assess certain types
of allergens, including eugenol and HCA, non-RI
methods tend to yield lower SIs than those obtained with the standard LLNA, and may be subject to false-negative results. Therefore, the purpose of the present study is to develop a non-RI
method with greater sensitivity and accuracy than
existing non-RI methods.
per hour. All procedures were performed according
to the guidelines of the Japanese Association for
Laboratory Animal Science.
Chemicals and Instruments
The chemical agents tested in the study included
(i) 2.4-dinitrochlorobenzene (DNCB) (Wako Pure
Chemical Industries, Ltd.), which was used as an
example of a strong sensitizer, (ii) eugenol (Wako
Pure Chemical Industries, Ltd.) and -hexyl cinnamic aldehyde (HCA) (Wako Pure Chemical Industries, Ltd.), which were included as moderate
sensitizers, and (iii) methyl salicylate (MS) (Wako
Pure Chemical Industries, Ltd.), which was used as
an example of a non-sensitizer. Sodium lauryl sulfate (SLS) was used to pretreat the test materials
prior to application of the agents (or vehicle) to
mice. Suitable vehicles and concentrations for
testing were determined based on previous reports
(Hariya et al., 1999; Van Och et al., 2000).
Eugenol, DNCB, HCA and MS were dissolved in
4:1 v/v acetone and olive oil (Yoshida Pharmaceutical Co. Ltd.) (AOO) solvent vehicle, and SLS
was dissolved in distilled water. Phosphate-buffered saline (PBS pH7.2) was purchased
from Invitron Corporation.
A ViaLightTM HS (Cambrex BioScience Walkersville Inc., formerly BioWhittaker Inc.) cytotoxicity detection kit was used to measure adenosine
triphosphate (ATP) content in the diluted LNC
suspension and to assess ATP-dependent enzyme
reactions. Bioluminescent measurement of ATP
was performed using a LumitesterTM C-100 (Kikkoman Corporation, Japan). A cell strainer (Falcon
35-2340, Becton Dickinson) was used to homogenize the LNCs.
Materials and Methods
Animals
Female CBA/JN mice (Charles River Japan Inc.,
Kanagawa) of 7 to 11 weeks old were used in the
experiments, and each test group or control group
contained three mice. Temperature and humidity
inside the animal holding rooms were maintained
at 232ºC and 55%15%, respectively, and each
room was ventilated at a frequency of 8-10 cycles
Fig.1 Experimental schedules for improving the
stimulation index of lymph node weight.
Pre treatment of 1% SLS solution
Sample application
Excision of lymph node
137
Experiment 1
Determination of an Optimal Dosing Schedule
Two experiments were carried out to determine an
optimal dosing schedule. In the first experiment,
0.25% DNCB, 10% eugenol and AOO vehicle
were tested, with three groups of mice (Group
1-3) assigned to each test material, giving a total
of nine groups of mice. The mice were treated by
topical application of the agent to the dorsum of
both ears once per day over three consecutive
days. The draining auricular lymph nodes were
collected from the mice four days (Group 1), five
days (Group 2), and seven days (Group 3) postapplication to calculate the lymph node weight
(LNW) in individual mice. The specific dosing
schedule is shown in Figure 1(A). In the second
AATEX 11(2), 2005
experiment, four groups of mice were treated with
10% eugenol, according to the schedule specified
in Figure 1(B), to evaluate whether application of
the agent four times instead of three times is useful
for increasing lymph node proliferation in the proposed non-RI method. Mice were divided into two
groups: those that did and those that did not receive the fourth treatment six or seven days after
the initiation of treatment. Nodes were collected on
either the seventh or the eighth day, after euthanasia of mice by an overdose of ether. In both experiments, mice were pretreated an hour prior to
ear challenge with application of a small amount of
1% SLS to each ear, which is known to help improve the detection sensitivity of the test (Van Och
et al., 2000).
Experiment 4
Assessment of Sensitivity and Specificity
HCA and MS were both tested at concentrations of
5%, 10% and 25% to assess the sensitivity and
specificity of the proposed non-RI method. HCA is
recommended as a positive control for sensitization in the LLNA (Basketter et al., 2002), whereas
MS is known to be a non-sensitizer (Kimber et al.,
1995). Mice were treated with these agents at four
times, and the LNC proliferation was measured
based on the LNW and the ATP content, and expressed as the SI.
Statistical analysis
Results are expressed as means  SD. An F-test
indicated that the LNW results were normally distributed, and therefore the Student t test was used
to compare data. P <0.05 was considered to be
significant.
Experiment 2
Measurement of ATP
To calculate the LNW for each mouse, both
auricular lymph nodes were collected after euthaResults
nasia of mice by an overdose of ether. LNCs were
Determination of the Experimental Schedule
filtered through a cell strainer and suspended in
The LNW-based SI, or the ratio of the LNW of the
500 l of PBS. An aliquot of this suspension was
test group to that of the control group, was calcudiluted 1:100 in PBS, and the ATP content in the
lated for each concentration of DNCB and eugenol
diluted LNC suspension was measured using bio(Table 1, Figure 2). The LNW increased with time
TM
HS,
luminescent
technology
(ViaLight
in all the DNCB-challenged groups. The eugenolTM
Lumitester C-100). A mixture of 90l of diluted
challenged groups that received three applications
cell suspension and 90l of ATP extract was preof the agent ceased to show a statistically signifipared and left to stand for five minutes at room
cant increase in LNW after four or five days.
temperature (232C). ATP content (or
Table 1
intensity of light emitted) was measured
TM
Results of non-RI LLNA on different experimental schedules.
with a luminometer (Lumitester
C-100) immediately after addition of
20l of luminescent reagent to the above
Group
Day
Lymph node weight (g)
mixture. Lymph nodes were excised from
(n=3)
Mean
SD
mice treated with 0.25% DNCB, 10%
control
4
0.0039
0.0006
eugenol and AOO vehicle, respectively,
5
0.0047
0.0011
according to protocol No. 3 in Figure
7
0.0045
0.0001
1(A).
DNCB
0.25%
4
0.0072
0.0013
5
0.0114
0.0022
Experiment 3
Assessment of Intermediate Precision
7
0.0180
0.0011
Six independent experiments were perEugenol
10%
4
0.0047
0.0002
formed, each of which used a group of
5
0.0036
0.0017
three mice treated with 10% eugenol,
7
0.0075
0.0012
according to protocol No. 3 in Figure
1(A). LNC proliferation in the control
Results represent mean values and standard deviation
group was measured based on the LNW
(SD) in three mice. The stimulation index (SI) was
and the ATP content, and expressed as the
calculated by the mean value obtained in each experimental schedule group of agent by that of the vestimulation index (SI).
hicle control group.
138
AATEX 11(2), 2005
Lymph Node Weight(g)
0.025
0.020
day4
day5
day7
0.015
0.010
0.005
0.000
AOO
DNCB(0.25%)
Eugenol(10%)
Fig. 2 Comparison of lymph node weight (LNW) on different experimental schedules.
The data are expressed as LNW obtained from individual mice (n=3) exposed to each agent by different
experimental schedules.
: The LNW of the mice that were excised on day 4, which were exposed on day 1,2 and day 3.
: The LNW of the mice that were excised on day 5, which were exposed on day 1,2 and day 3.
: The LNW of the mice that were excised on day 7, which were exposed on day 1,2,3 and day 6.
* P <0.05 as change compared to different protocol groups.
0.0160
Lymph Node Weight.(g)
0.0140
0.0120
0.0100
0.0080
0.0060
0.0040
0.0020
0.0000
day 7
E(-)
day 7
E(+)
day 8
E(-)
day 8
E(+)
day 7
E(+)
day 8
E(+)
Fig. 3 Evaluation of additional application.
day 7 (or day 8) E(-):The LNW of the mice that were excised on day 7 (or day 8), which were exposed
on day 1,2 and day 3. day 7 (or day 8) E(+): The LNW of the mice that were excised on day 7 (or day
8), which were exposed on day 1,2,3 and day 6 (or day7). ＊: Values significantly different between
day 7 E(+) and day 7E(-). （P <0.05）
Whereas those groups that received a fourth application of the agent on the sixth day of the experiment continued to show a statistically significant
increase in LNW. The LNW-based SIs for DNCB
and eugenol on the seventh day of the experiment
were 3.97 and 1.75, respectively (Table 1). The
LNW was larger for mice that received the fourth
application of eugenol on the sixth day, compared
to mice that only received three applications of the
agent. However, there was no statistically signifi-
cant difference in the LNW between groups that
received the fourth application of eugenol on the
sixth day and those that received it on the seventh
day (Figure 3).
Assessment of ATP Content
The ATP content in the diluted LNC suspension
obtained from auricular lymph nodes of mice exposed to DNCB or eugenol was measured to calculate the ATP-based SIs. The ratio of the ATP
139
AATEX 11(2), 2005
Table 2
The SI values of lymph node weight (LNW) and ATP content (ATP) with DNCB and eugenol were used.
Animal LNW(g)
ATP as bioruminescent (RLU)
No.
weight
mean
SD
SI
RLU
mean
SD
control
1
0.0044
0.0049
0.0011
1.00
2921
4544
1619
2
0.0042
4552
3
0.0062
6158
DNCB 0.25%
1
0.0218
0.0208
0.0035
4.21
51036
51599
15717
2
0.0236
67590
3
0.0169
36172
Eugenol 10%
1
0.0106
0.0100
0.0013
2.03
18245
20054
4544
2
0.0110
25223
3
0.0085
16693
Results represent mean values and standard deviations (SD) of LNW or ATP content in three mice.
The SI values were calculated by the mean value obtained in agent by that of the vehicle control group.
Stimulation Index (SI)
Group
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
SI
1.00
11.36
4.41
SI value(LNW)
SI value(ATP)
control (AOO)
DNCB (0.25%)
Eugenol (10%)
Fig. 4 SI values of lymph node weight (LNW) and ATP content (ATP).
The data are expressed as mean SI ± SD of LNW and the mean SI ± SD of ATP for each agent.
content in the LNC suspension obtained from test
groups to that in the LNC suspension obtained
from control groups was also calculated. The
ATP-based SI was 11.36 for DNCB-challenged
mice and 4.41 for eugenol-challenged mice,
whereas the LNW-based SI was 4.21 for
DNCB-challenged mice and 2.03 for eugenolchallenged mice (Table 2, Figure 4).
and 2.54, respectively. The mean ATP-based SI,
standard deviation (SD), and coefficient of variation (CV) were calculated as 4.0, 0.7 and 17.3%,
respectively.
Assessment of Sensitivity and Specificity
While a concentration-dependent increase was observed in the SIs for the HCA-exposed groups, no
increase was observed in the SIs for the
MS-exposed groups (Figures 6 and 7, respectively).
The ATP-based SIs for the groups exposed to HCA
were 1.29, 2.58, and 6.47, respectively, and these
values for groups exposed to MS were 0.72, 0.96
and 1.20, respectively.
Assessment of Intermediate Precision
The results obtained from six independent experiments conducted in mice treated with eugenol are
summarized in Figure 5. The ATP-based SIs for
each experimental group were 3.39, 3.55, 3.72,
5.22, 4.25 and 3.60, respectively, and the
LNW-based SIs were 2.26, 2.17, 2.56, 3.07, 2.16
140
Stimulation Index (SI)
AATEX 11(2), 2005
8
7
6
5
4
3
2
1
0
SI value(ATP)
SI value(LNW)
1
2
3
4
Experimental No.
5
6
Fig. 5 SI values of ATP content (ATP) and lymph node weight (LNW).
The local lymph nodes were excised on day 7 according to the Fig. 1(A) No. 3 procedure, and the ATP
and LNW were measured. The data showed the mean SI ± SD of ATP and the mean SI ± SD of LNW in
the six independent experiments.
9
Stimulation Index (SI)
8
7
6
5
4
3
2
1
0
control(AOO)
5%
10%
25%
HCA
Fig. 6 Proliferation of local lymph node in response to α-hexyl cinnacmic aldehyde (HCA).
The lymph nodes of the mice that were excised on day 8, which were exposed on day 1,2 and day 3.
day 7. The data are expressed as mean SI ± SD of LNW and the mean SI ± SD of ATP for each
concentration of HCA.
method, including (i) measurement of cytokine
production by LNCs (Derman et al., 1999), (ii)
flow cytometric analysis of LNCs (Hatao et al.,
1995), and (iii) assessment of BrdU incorporation
into LNCs as a measure of lymphocyte proliferation (Takeyoshi et al., 2001). However, when used
to assess moderate sensitizers (such as eugenol and
HCA), non-RI methods tend to yield lower SIs
than those obtained with the standard LLNA
(Hatao et al., 1995, Takeyoshi et al., 2001). Thus,
using several dosing schedules, we examined
whether LNW-based SIs and ATP-based SIs are
Discussion
The LLNA has been developed as a screening test
to detect the effects of skin sensitizers, and the
OECD guideline (No. 429) for conducting the
LLNA was adopted in 2002 after thorough evaluation and reviews of the test. Because the standard
LLNA uses a radioisotope to measure lymphocyte
proliferation, it requires special facilities for the
safe handling of radioactive substances (Takeyoshi
et al., 2001). Several attempts have been made to
develop a modified LLNA that uses a non-RI
141
AATEX 11(2), 2005
8
Stimulation Index
7
6
5
4
3
2
1
0
control(AOO)
P. C.
5%
10%
25%
MS
Fig. 7 Proliferation of local lymph node in response to methyl salicylate (MS).
The lymph nodes of the mice that were excised on day 8, which were exposed on day 1,2 and
day 3. day 7. The data are expressed as mean SI ± SD of LNW and the mean SI ± SD of ATP for
each concentration of MS and 15% HCA as positive controls.
valid measures of LNC proliferation, with the ultimate goal of developing a non-RI method that
can yield SIs comparable to those obtained with
the standard LLNA, without the need to use higher
sample concentrations.
The results obtained from an experiment conducted in mice treated with 10% eugenol indicated
that applying the agent four times was effective for
improving the LNW-based SI (the standard LLNA
protocol recommends that mice are exposed to the
agent three times). However, the LNW-based SI
for eugenol was only 1.75. The SI for eugenol is
usually higher in the standard LLNA; in fact, SIs
of 2.4 and 3.2 have been reported for eugenol
tested in two different laboratories, according to
the standard LLNA protocol (Loveless et al., 1996).
To develop a better measure of LNC proliferation,
the ATP content in the LNC suspension was measured. ATP bioluminescence has long been used as a
measure of cell proliferation and cytotoxicity in
studies of bacteria and cultured cells (Crouch et al.,
1993; Dexter et al., 2003). Assuming that LNC
proliferation is mostly due to lymphocyte proliferation, and that ATP content per cell is similar
across LNCs, ATP content can be a useful measure
of LNC proliferation. In the present study, the
ATP-based SI was 4.41 for 10% eugenol and 11.36
for 0.25% DNCB, whereas the LNW-based SI was
2.03 and 4.21 respectively (Figure 4), suggesting
142
that the ATP-based SI provides a more sensitive
index of LNC proliferation than the LNW-based SI.
The minimum concentration of eugenol required to
yield a SI of 3 or higher in the standard LLNA is
10% (Basketter et al., 1996); thus, it is justifiable
to conclude that, as far as eugenol is concerned, the
sensitivity of the proposed non-RI method is comparable to that of the standard LLNA. On the other
hand, while 0.25% DNCB induced an ATP-based
SI of 11.36 in the present study, the standard
LLNA yields an SI of 14.2 for DNCB tested at the
same concentration (Kimber et al., 1989). To determine if this difference may affect the sensitivity
of the proposed non-RI method, further studies are
required using lower concentrations of DNCB.
Six independent experiments in mice treated
with 10% eugenol were performed to assess the
intermediate precision of the results obtained from
the proposed non-RI method. The mean ATP-based
SI, SD and CV were calculated as 4.0, 0.7 and
17.3%, respectively. In a previous study conducted
in mice treated with HCA (Haneke et al., 2001),
six experiments were carried out to assess the repeatability of the effective concentrations for elicitation of a 3-fold increase in [3H]thymidine incorporation (EC3). The EC3 values obtained from the
six experiments were 7.9, 6.9, 9.6, 8.7, 4.0 and
9.2% and the mean EC3, SD and CV were calculated as 7.7, 2.1 and 26.7%, respectively.
AATEX 11(2), 2005
We also performed an experiment in mice
treated with HCA and MS to assess the sensitivity
and specificity of the proposed non-RI method.
HCA is known to induce stable responses over
time in the LLNA, and has been tested in several
modified LLNAs using non-RI methods, while MS
is known to be a non-sensitizer. The two substances were tested at concentrations of 5%, 10%
and 25%, and the ATP-based SIs for each concentration of HCA were 1.29, 2.58 and 6.47, respectively. A similar test of HCA at the same concentrations using the standard LLNA protocol yielded
SIs of 1.1, 2.5 and 10.4, respectively (Gerberick et
al., 1999). On the other hand, a modified LLNA in
which HCA was tested at concentrations of 12.5%,
25% and 50%, using BrdU incorporation as a
measure of LNC proliferation, yielded SIs of 1.58,
2.40 and 3.63, respectively (Takeyoshi et al., 2003),
and a second modified LLNA protocol using IL-2
production as a measure of LNC proliferation
yielded a SI of approximately 6 for an HCA concentration of 50% (Hariya et al., 1999). Thus, it
can be concluded that the sensitivity of the proposed non-RI method is comparable, if not superior, to that of existing non-RI methods, with respect to assessment of the sensitizing potential of
HCA. The ATP-based SIs for MS, a non-sensitizer,
were all lower than 3, which is the usual cut-off
value in the standard LLNA. Based on this criterion, the results obtained from the non-RI method
proposed herein indicate that HCA is a sensitizer
and MS is a non-sensitizer.
To summarize, the modified LLNA proposed
in the present study has the potential to serve as an
alternative to the standard LLNA. The modified
approach is characterized by exposure of mice to
the test agent four times, instead of three times,
and by the use of an SI based on ATP content as a
measure of LNC proliferation. Regarding the
chemicals tested in the present study, the
ATP-based SIs were close to those calculated
based on [3H]thymidine incorporation in the
LLNA. Furthermore, the proposed non-RI method
was proven to be as effective as the standard
LLNA in detecting moderate sensitizers, suggesting that it is less likely to yield false negative results, compared to existing non-RI methods. To
demonstrate the overall validity of the proposed
method further experiments will be required, using
other chemicals that have been tested in the standard LLNA.
143
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(Received: February 9, 2004 /
Accepted: November 4, 2005)
Corresponding author:
Kunihiko Yamashita, Ph.D.
Daicel Chemical Industries, Ltd.
1239, Shinzaike, Aboshiku, Himeji, Hyogo 671-1283,
Japan
Tel, +81-792-74-4096 Fax, +81-792-74-5831
E-mail: [email protected]
Loveless, S. E., Ladics, E. W., Gerberich, G. F.,
Ryan, C. A., Basketter, D. A., Scholes, G. S.,
House, R. V., Hilton, J., Dearman, R. J. and
Kimber, I.(1996) Futher evaluation of the local
lymph node assay in the final phase of an international collaborative traial, Toxicol, 108, 141152
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