United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 2771 1
EPA-600/1-79-039
September 1979
Research and Development
Effects of
Pesticides on the
Immune Response
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
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The nine series are:
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ances of man for unhealthful substances or conditions. This work is generally
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clude biomedical instrumentation and health research techniques utilizing ani-
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This document is available to the public through the National Technical Informa-
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EPA-600/1-79-039
September 1979
EFFECTS OF PESTICIDES ON
THE IMMUNE RESPONSE
by
Walter B. Dandliker, Arthur N. Hicks,
Stuart A. Levison, Kris Stewart,
and R. James Brawn
Department of Biochemistry
Scripps Clinic and Research Foundation
La Jolla, CA 92037
Grant No. R803885
Project Officer
August Curley
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, NC 27711
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NC 27711
-------
DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation for use.
11
-------
FOREWORD
The many benefits of our modern, developing, Industrial society are
accompanied by certain hazards. Careful assessment of the relative risk of
existing and new man-made environmental hazards Is necessary for the estab-
lishment of sound regulatory policy. These regulations serve to enhance the
quality of our environment 1n order to promote the public health and welfare
and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park, conducts
a coordinated environmental health research program 1n toxicology, epidemi-
ology, and clinical studies using volunteer subjects. These studies address
problems 1n air pollution, non-1on1z1ng radiation, environmental cardno-
genesls and the toxicology of pesticides as well as other chemical pollutants.
The Laboratory participates 1n the development and revision of air quality
criteria documents on pollutants for which national ambient air quality
standards exist or are proposed, provides the data for registration of new
pesticides or proposed suspension of those already 1n use, conducts research
on hazardsous and toxic materials, and 1s primarily responsible for providing
the health basis for non-1on1z1ng radiation standards. Direct support to the
regulatory function of the Agency 1s provided 1n the form of expert testimony
and preparation of affidavits as well as expert advice to the Administrator
to assure the adequacy of health care and surveillance of persons having
suffered Imminent and substantial endangerment of their health.
This report summarizes the results of a study to determine the effects
of pesticides on Immune responses. Immediate toxic effects are relatively
readily assessed but slow or delayed effects are more difficult to detect
and yet may be more Important -- possibly leading to altered susceptibility
to disease, damage In utero, accelerated aging, tumorgenesls, etc. Immune
responses, although very little studied, offers one parameter which Is a
sensitive Indicator of a variety of physiological functions and which may be
quantltated. The results of this study offers methodology useful 1n assessing
the adverse effects of pesticides exposure on Immune response.
F. G. Hueter, Ph.D.
D1rector
Health Effects Research Laboratory
111
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Introduction
The world-wide use of pesticides makes it urgent to know as
much as possible about the effects of pesticides and their degrada-
tion products on humans and animals. Immediate toxic effects are
relatively readily assessed but slow or delayed effects are more
difficult to detect and yet may be the more important -- possibly
leading to altered susceptibility to disease, damage in utero,
accelerated aging, etc.
The immune response offers one parameter which is a sensitive
indicator of a variety of physiological functions and which is
readily quantified in a number of ways. The early work in this
area was reviewed by Ercegovich (1). Since that time there has
been a growing interest in evaluating pesticide effects on a number
of different aspects of the immune response. These include effects
on antibody production, dermal reactions to specific immunogens,
immunoglobulin levels, resistance to infection, complement levels,
lymphoid cell counts and effects on lymphoid organs detected
histologically.
The parameters most easily quantified are those connected with
the humoral response since serum antibody can be easily obtained
and can be characterized in a number of ways. The production of
anti-human serum albumin was found to be inhibited in rats injected
with Lindane, 60 or 120 mg/kg (2), while the titer of anti-bovine
serum albumin was not consistently altered in chicks fed mash
containing up to 625 parts per million (ppm) of DDT (3). Hemag-
glutinin levels of rats immunized against sheep red blood cells
were suppressed by an oral dose of Methylnitrophos or Chlorophos,
1
-------
5 or 7 mg/kg/day, especially in rats fed a protein-deficient diet
(4). A second class of specific immunogen used to monitor the
humoral immune response in pesticide-treated animals includes
bacteria and viruses. Significantly lowered titers of anti-
Salmonella typhii were found in rabbits given drinking water
containing 200 ppm DDT (5-7). Also the expected increase in the
y-globulin 7S fraction in response to Salmonella inoculation was
•
inhibited by Dieldrin and benzene hexachloride (8). On the other
hand, no consistent effect was found in the anti-Salmonella
pullorum titer of DDT-fed chicks (3). Similarly, no differences
in bacterial agglutination, indirect hemagglutination, indirect
hemolysis, or precipitation were noted between Warfarin-treated
and untreated rabbits immunized with purified Salmonella typhii
endotoxin (9). Lower titers of tetanus antitoxin were consistently
found when animals immunized with tetanus toxoid were given dietary
Aroclor 1260 or Clophen A60 (50 ppm) (10) or 0.1 - 0.2 LD5Q of
Carbaryl orally (11) . Effects on antibody-mediated immunity
have been investigated for Anthio and Milbex in goats (12,13) and
for Minex and DDT in chickens (14). Examination of lymphoid organs
proved to be a sensitive indicator of immunosuppression in a
study of the effects of DDT, Aroclor 1254, Carbaryl, Carbofuran
and Methylparathion in rabbits (15).
The present study was designed to simultaneously analyze the
influence of various pesticides on the humoral and cellular immune
responses to a well defined immunogen (fluorescein labeled ovalbumin)
The pesticides were administered in one oral dose preceding primary
immunization. Booster immunizations were then given periodically
-------
after sampling the serum and performing in vivo tests of cellular
immunity. These included visual evaluation of redness and swelling
of a challenged footpad and measurement of the temperature difference
between challenged and control footpads. Serum antibody was
characterized by fluorescence polarization. In addition, body
weight was followed as an indicator of gross physiological
status throughout the experiments.
Materials and Methods
Pesticides. Dinoseb, Parathion and Pentachloronitrobenzene
were analytical standards (16) from the Environmental Protection
Agency, Triangle Park, N.C. Resmethrin, piperonyl butoxide and
mixed natural pyrethrins were generously donated by FMC Corporation,
Agricultural Chemicals Group, Richmond, CA, and Aroclor 1260 was
purchased from Chem Service (West Chester, PA). Methotrexate was
the pharmaceutical product from Lederle Laboratories (Pearl River,
N.Y.).
Animals. Inbred male hamsters, Strain LHC/LAK (Lakeview
Hamster Colony, Newfield, NJ) 5 to 8 weeks old and weighing about
100 g each were used for all experiments.
Preparation of Immunogen. To 5 g of chicken ovalbumin (Mann
Research Laboratories, New York, NY, 5X crystallized) dissolved in
15 ml of 0.5 M carbonate buffer (0.4M NaHCOj, 0.1 M Na2CO_, pH 9.3),
43 mg fluorescein isothiocyanate (Isomer I, Sigma Chemical Co., St.
Louis, MO) was added. The mixture was held at 4° for 16 hr and
passed through a column (3.7 cm in diameter X 58 cm long) of
Sephadex G-25 medium (Pharmacia Inc., Piscataway, NJ) previously
equilibrated with 0.15 M NaCl. The void volume as determined by
-------
blue dextran was 250 ml. The fluorescein labeled ovalbumin (FO)
was collected in a volume of 143 ml beginning at 246 ml of effluent.
Optical density measurements at 280 and 490 nm indicated a labeling
ratio of 1.0 utilizing the molar extinction coefficients for fluoro-
scein previously determined (17) and assuming values for ovalbumin
i *
of 7.34 for E2go and of 46,000 for the molecular weight. The FO
solution was stored at -20° until used.
To prepare FO for injection, a solution containing 20, 2 or
0.4 rag/ml (in 0.15 M NaCl) was homogenized with an equal volume of
Complete Freund's Adjuvant (CFA) (DJFCO Laboratories, Detroit,
MI) by means of two syringes connected together by a plastic 3-way
stopcock (Type K-75, Pharmaseal, Toa Alta, Puerto Rico) .
Pesticide Administration. Each animal received one dose of
pesticide or other compound equal to one-half of the LDgg (16)
dissolved in 1 ml of corn oil. The pesticide solution was. administered
as a bolus by intragastric feeding tube 24 hr after the first injection
of FO. The animals were fasted during this 24 hr period with water
ad lib.
Immunization. For all immunizations, 0.2 ml of a mixture
consisting of equal volumes of FO and CFA were injected subcutan-
eous ly into each flank. For primary immunizations FO at either
10 mg/ml or 1 mg/ml (final concentration in homogenized mixture)
was used resulting in a dose of either 4 mg or 0.4 mg per hamster.
Booster immunizations consisted of 0.2 ml of FO-CFA mixture
containing 200 yg FO/ml into each flank. The primary immunization
was given 24 hr after the pesticide; boosters were administered at
7 day intervals after the primary immunization.
-------
Serum preparation. All blood was drawn by cardiac puncture,
allowed to clot 2 hr at room temperature, overnight at +4° and
the serum drawn off after centrifugation. A preimmunization bleeding
was obtained before pesticide administration and thereafter before
each booster immunization.
Immunoglobulin preparation. Serum pooled from several
individual bleedings was fractionated by ammonium sulfate precipita-
tion to give an immunoglobulin preparation substantially free of
serum albumin (which if present in sufficient concentration, would
interfere in the titrations by binding fluorescein non-specifically).
To one volume of serum, 0.58 volumes of saturated ammonium
sulfate (adjusted to pH 8.1 to 8.2 by the addition of concentrated
NH^OH) was added rapidly while mixing, at room temperature. The
precipitate was immediately centrifuged at 16,000 X g for 30 min
at 20°C. The supernatant fluid was decanted; the centrifuge tube
vras drained for about 5 min to remove as much fluid from the
precipitate as possible and the precipitate was dissolved in 1
serum volume of 0.15 M NaCl containing 0.001 M NaN,.
Buffers. 0.15 M NaCl, 0.01 M K2HP04, 0.005 M KH2P04, 0.001 M
NaN- and 0.1 mg/ml, rabbit yglobin (Schwarz/Mann, Orangeburg, NY)
Hepes buffered Hanks (Flow Laboratories, Rockville, MD).
Characterization of the humoral immune response by means of
fluorescence polarization. Immunoglobulin (Ig) equivalent to 10,
30 or 100 yl of serum was diluted in a fluorescence cuvette to 3 ml
with buffer and the blank fluorescence measured by a fluorescence
polarometer (18). Fluorescein (3, 10 or 30 yl of 10" M) was added to thr
diluted Ig and the solution was mixed with a Pasteur pipette.
After 30 min at room temperature the fluorescence intensities and
5
-------
polarizations were measured. Fluorescence parameters for free,
unbounded fluorescein were measured in the presence of normal Ig
only. The data were treated as described in the Appendix to give
the serum antibody site concentration, the antibody-hapten binding
affinity and the antibody heterogeneity index. Alternatively,
the polarizations themselves can be viewed as a kind of titer
assessing the immune response.
Quantification of the cellular immune response. To assess
the magnitude of the cellular immune response against FO the test
animals were challenged with 0.1 ml of FO (400 ug/ml in Hepes
buffered Hanks) in one footpad and with the buffer alone in the
contralateral footpad. Twenty-four hours later the response was
quantified in two ways. In the first the immune response was
graded subjectively on the basis of color and swelling on a
scale of 1 through 4 and plotted as an average difference between
experimental and control feet for all animals in the group (usually
5 animals). In the second method (thermometric footpad assay)
thermocouples were attached with adhesive to the test and control
feet and the difference in footpad temperature was measured with the
circuit shown in Figure 8. In some cases the footpads were dissected
after temperature measurement in order to determine the degree of
correlation between temperature and histological findings.
RESULTS
The effect of pesticides on the cellular immune response was
measured by two methods, the first being a visual evaluation of the
intensity of inflammation and swelling of an antigen challenged
footpad as compared to the contralateral pad treated with buffer
alone. Figure 1 shows that by this measurement Dinoseb and
6
-------
Parathion markedly depress the response while Resmethrin gives
a large stimulation appearing very early after primary immuniza-
tion. Methotrexate and PCNB appear also to stimulate cellular
immunity but only late in the immune respone. Aroclor, piperonyl
butoxide and mixed pyrethrins have little if any effect. The
second method for evaluating cellular immunity involved a
differential temperature measurement between the antigen-
challenged and control footpads. The results of this thermo-
metric footpad assay were found to correlate positively with
visual evaluation of the degree of inflammation and with patho-
logical findings in the antigen-challenged footpad tissue.
Results of the thermometric footpad assay are shown in Fig. 2.
Resmethrin as before shows an early, sometimes very large,
stimulation while Methotrexate, PCNB and possibly pyrethrins
give a late stimulation. Both Dinoseb and Parathion are
depressive while the other pesticides show no detectable
effects. The effect of size of the first dose of immunogen is
obviously quite important as shown by Figs. 1 and 2. The smaller
dose (0.4 mg per hamster) resulted in a much larger response than
did the larger dose (4 mg) .
Pesticide effects on the humoral response were assessed by
fluorescence polarization measurements after adding fluorescein
to an Ig preparation from serum.' The polarization itself can be
thought of as a titer dependent upon both antibody concentration
and antibody binding affinity. The polarization titers of Fig. 3
show depression by Dinoseb and Parathion and not much effect of
the other pesticides. The effect of size of immunizing dose is
again evident, the smaller dose giving the larger response.
7
-------
All examined preimmunization bleedings gave only background polari-
zation vlaues (0.027, the same as in buffer alone).
As mentioned above, polarization is dependent upon both the
antibody concentration and the binding affinity. These two
variables together with a third variable the heterogeneity index
can be segregated by an analysis of the complete titration curve.
The results of these computations are shown in Figs. 4 and 5. In
Fig. 4 the quantity F, __ , which is equal to the molar concentration
D | luclX
of antibody combining sites in a 300 fold dilution of serum, is
shown for different pesticides during progression of the immune
response. A marked depression can be seen for Dinoseb and
Parathion with only minor differences from controls for the other
pesticides. In Fig. 5 the largest effect on the binding affinity
as measured by the average association constant seems to be pro-
duced by varying the size of immunizing dose. All values of K
8 9 -1
lay between 10 and 10 Imole which is a rather small variation.
The typical appearance of fluorescence polarization titration
curves can be seen in Fig. 6 for piperonyl butoxide at 36 days.
2
The agreement between experiment and theory is very close (chi
for these data is 1.19). For the other data shown in Figs. 4 and
2
5, the values of chi varied from 0.23 to 2.2. A third variable,
a, the heterogeneity index was also obtained from the titration
data. The values ranged from 0.7 to 1 and are shown in Table 1
with results of all the computations.
The general physiological state of the animals was monitored
by the change in body weight during the experiments (Fig. 7). Both
Dinoseb and Parathion show a prolonged effect in depressing growth
8
-------
while Methotrexate and Aroclor give transient depressions. The
high dose of immunogen itself shows a marked depression when com-
pared to the low dose control.
The schematic for the circuit used in the thermometric
footpad assay is shown in Fig. 8.
DISCUSSION
The experimental data obtained shows that a single dose of
orally administered pesticide may exert large, long-lasting
effects on the immune response. The effects observed may be
either stimulation or depression and may be directed selectively
towards either the cellular or hurmoral immune response depending
upon the pesticide.
In this study these effects were monitored by measurement
of several parameters related to various aspects of the immune
response to a single well-defined immunogen, fluorescein labeled
ovalbumin (FO). The humoral response measured was directed
against fluorescein itself while the cellular response was that
directed against the entire immunogen. The effects observed as
summarized in Table 2 show that the most significant findings
are a marked depression of both the cellular and humoral immune
response by Dinoseb and Parathion and an early and sometimes
very pronounced stimulation of the cellular response by Resmethrin.
The latter effect is of considerable interest in two contexts. First,
Resmethrin when used with another pesticide may exaggerate any
antipesticide reactions in humans or animals exposed to the two
substances together. Secondly, and perhaps more important, the
behavior of Resmethrin may provide an important clue towards
-------
designing potent stimulators of the cellular immune response.
Such materials could be of great value in treating bacterial,
viral and certain neoplastic diseases. The action of Parathion
and Dinoseb are remarkably long lasting and the depression of
the immune response which they evoke could lower resistance to
a variety of infectious diseases. The nature of the effect on
the humoral response is seen to be chiefly on the amount of
antibody produced and not upon its binding affinity. Probably
the type of antibody is unchanged by these pesticides but the
amount is decreased.
The results of this study provide important leads which
should be followed up. First, the immunological effects of
many other pesticides and organics generally should be investi-
gated and secondly, the effects should be studied not only in
the whole animal but also at the level of T-cell and B-cell
activation.
10
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TABLE 1. Effects of Pesticides on the Immune Kespons- as shown by Parameters Pertaining to
Serum Antibody
Pesticide Dose of
Imiuunogen
Control
"
"
Control
ii
Methotrexate
"
Aroclor
"
Dinoseb
Parathion
"
PCNB
"
Pip. Butox
H
Pyrethrins
11
Resmethrin
11
L
tl
II
H
II
Ii
II
1.
II
I
L
II
H
II
L
II
H
II
L
11
Days
31
46
52
4b
52
46
52
29
36
49
42
49
46
52
29
36
46
52
29
36
Pb
0.48
0.45
0.42
0.40
0.40
0.36
0.42
0.43
0.44
0.42
0.45
0.45
0.42
0.40
0.45
0.45
0.40
0.42
0.46
0.47
Qf/Qb
7.1
5.0
3.1
4.0
4.0
6.2
5.9
6.4
4.5
5.0
5.8
4.4
5.8
8.6
6.4
6.6
5.9
4.7
5.8
5.0
10'
5
0
3
2
0
0
0
3
3
2
6
1
1
1
2
5
0
0
5
10
'«.
.0
.9
.8
.0
.20
.77
.76
.1
.3
.3
.6
.4
.00
.15
.2
.1
.67
.35
.5
.1
0.
0.
0.
1
0.
0.
0.
0.
0.
0.
1.
0.
0.
0.
0.
0.
0.
0.
0.
0.
a
75
77
77
85
85
77
70
72
93
0
77
87
83
76
77
91
89
80
83
,.',
0
4
6
0
4
9
5
5
6
1
0
1
4
4
5
6
2
3
4
9
'b.max
.90
.5
.5
.86
.7
.9
.1
.4
.3
. 11
.41
.3
.1
.0
.0
.7
.0
.5
.7
.0
X2
1 .38
1 .49
1.78
1.06
0.37
0.47
1.86
1.11
0.76
1.02
1.41
0.69
2.40
0.42
1 .23
1.19
0.35
0.76
1.17
1.24
L « 0.4 mg iramunogen/hamster
II " 4 rag immunogen/liamster
P^, the polarization of bound fluorescein
the fluorescence ratio of free to bound
KO> the association constant, Iroole
a, the heterogenecity index
FH m=,v> tne antibody site concentration in 300 X diluted serum
o j ma A
X2 • 100 t /Pobs pculc)2. statistical measure of fit between observed and calculated
2
polarization
11
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TABLE 2. Summary of Effects of Pesticides on the Immune Response
ro
Pesticide
Methotrexate
Aroclov
Dinoseb
Parathion
PCNB
Pip. Butox.
Pyrethrins
Kusmethrin
Cellular
Visual
Evaluation
+ (late)
0
-
-
+ (late)
0
0
..+ (early)
Thermometric
Footpad
+ (late)
0
-
-
+ (late)
0
+. (late)
+ (early)
Humoral
Polarization
liter
0
0
-
-
0
0
0
0
Antibody
Concentration
0
0
-
-
0
0
0
0
Binding
Affinity
0
0
0 to -
0
0
0
0 to -
0 to +
(-): Depression
(+): Stimulation
(0): Little or no effect
-------
References
1. Ercegovich, C. D., Fed. Proc. .32(9), 2010-2016 (1973).
2. Rosival, L., Barlogova, S. and Grunt, J., Gig. Tr. Prof. Zabol.
(6), 53-55 (1974). (Russ), -
3. Latimer, J.W. and Siegel, H. S., Poult. Sci., 5^(3), 1078-
1083 (1974).
4. Shtenberg, A. I., Khovaeva, L. and Zavarzin, M. V-, Vopr.
Pitan. (4), 35-42 (1974).(Russ).
5. Wassermann, M., Wassermann, D., Kedar, E. and Djavaherian, M.,
Bull, Environ. Contam. Toxicol.,
-------
16. Analytical Reference Standards and Supplemental Data for
Pesticides and Other Organic Compounds, Thompson, J. F.,
ed. Publication No. EPA - 600/9-76-012, Environmental
Protection Agency, Technical Publications Branch, Office
of Administration, Research Triangle Park, N.C. 27711 (1976).
17. Dandliker, W. B. and Alonso, R., Immunochem. 4_, 191-196 (1967).
18. Kelly, R. J., Dandliker, W. B. and Williamson, D.E., Anal.
Chem. 48 ( ), 846-856 (1976).
19. Dandliker, W. B., Schapiro, H. C., Meduski, J. W., Alonso , R.,
Feigen, G. A. and Hamuck, J. R., Immunochem. 1^ (3), 165-191
(1964).
20. Nisonoff, A. and Pressman, D., J. Immunol., 80 ( ), 417-428
(1958).
14
-------
0
2
0
2
0
2
0
2
Control (L)
Control (L)
Control (H)
Methotrexate (H)
Aroclor (L)
Dinoseb (L)
Parathion (L)
PCNB (H)
Piperonyl Butoxide (L)
Pyrethrins (H)
Resmethrin (L)
Resmethrin (L)
10 20 30 40 10 20 30 40
Days
Figure 1. Effect of pesticides on the cellular immune response as measured by
visual evaluation on a 1 to 4 scale of the inflammation and swelling of the
footpad challenged with antigen as compared to the contralateral pad chal-
lenged with buffer alone. Animals were treated on day zero with pesticide in
corn oil or with corn oil alone (control) and immunized with either a low
dose (L) or a high dose (H) of FO as described in Materials and Methods. The
curve of each pesticide must be compared with the appropriate control.
15
-------
\
0
1
0
1
0
1
0
1
0
1
0
Control (L) r,
Rl ^
^ S
Control (L) E
s
s
s
s
Control (H)
*
~ ?3 — _
Methotrexatelj
(H) S
• s
s
Aroclor
- (L) 1
s
s
s
s
^
£3
Dinoseb (L)
ra
E3~
i
R R
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i
s
s
s
5 S r.
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N
V
S
S
S
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Parathion(L)
PCNBO
1
H
S
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WSS//SSSS/SSA
s
s
N
S
s
s
s
^
s
s
s
s
V
s
s
s
s
s
s
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Pip. But
Pyrethrins (
S
Resmeth
Ii
ri
i
V////////7/77K
H
1
)
n(L)
L
ox.(L)
«
s
s
s
J
s
s
;
s
Resmethrin
(L)
S i S
i i i i
0
0
0
0
0
10 20 30 40
10 20 30 40
Days
Figure 2. Effect of pesticides on the cellular irrmune response as measured by
average values of temperature differences (AT) between the footpad challenged
with antigen and the contralateral pad challenged with buffer alone. Animals
were treated with pesticide in corn oil or with corn oil alone (control) and
immunized with either a low dose (L) or a high dose (H) of FO as described
under Materials and Methods. Temperature differences were measured with the
circuit of Figure 8.
16
-------
0.3
0
0.3
o. 0
0.3
0
0.3
0
0.3
n
Control (L)
S S
. Ill
1
*
Control (H)
Methotrexate (H)
^
ra o
_ N X
^
I
Aroclor (L) ra ra
- 1 \\
SI ^> ^ S
Dinoseb (L)
Parathion (L)
PCNB (H)
Pip. Butox
iuil
1
1
Pyrethrins (H)
Resmethrin
(L) i
1
Resmethrin
- I
. .' S I
Y7/Z7//&m
\
V
(L
Y/S/Z7/&/ZA
i
20
40
20
40
Days
Figure 3. Effect of pesticides on the humoral immune response as measured by
polarization titers. The polarization of fluorescence was measured 30 min
after adding 10~9 M fluorescein to a solution of Ig (equivalent to 100 ]i\ of
serum) in 3 ml of buffer. The polarization is a function of both antibody
concentration and antibody binding affinity and is synbatic with both of these
variables. Animals were treated on day zero with pesticide in corn oil or
with corn oil alone (control) and immunized with either a low dose (L) or a
high dose (H) or FO as described in Materials and Methods.
17
-------
X
a
.a"
U_
a>
O
5
0
5
0
5
0
5
0
5
0
5
Control (L)
im
Ri fcsi v
•
Control (H)
ra R
Methotrexate (H) fc
h
Aroclor (L
)
ii
Dinoseb (L)
•
ra
Parathion (L)
PCNB(H)
Pip.Butox. (L]
i
i
Pyrethrins (H)
s i
Resmethrin (L
s
i
20
40 20 40
Days
Figure 4. Effect of pesticides on the humoral immune response as measured by
the concentration of serum antibody against fluorescein as a function of time
after primary immunization. The quantity, Fj>>max is the molar concentration
of antibody combining sites specific for fluorescein present 1n a 300 fold
dilution of serum. Values of Fb>max were computed from fluorescence polari-
zation measurements, cf. Appendix.
18
-------
00
I
5
0
5
0
o 5
^ ^^
5
0
5
0
5
n
Control (L)
1
-
Control (H)
Methotrexdte (H)
ra ra
Aroclor (L)
Dinoseb (L)
i
Parathion (L)
I.
PCNB (H)
(S3 S
Pip. Butox. (L)
1
Pyrethrins (H)
en Rn
1
Resmethrin(L) ^
I ^
20 40 20 40
Days
Figure 5. Effect of pesticides on the humoral immune response as measured by
the average association constant (KQ) of the serum antibody present against
fluorescein at different times after primary immunization. The values of KQ
were computed from fluorescence polarization measurements, cf. Appendix.
19
-------
I09M
Figure 6. Typical appearance of fluorescence polarization titration curves
showing polarization, p, as a function of M, the final molar concentration
of fiuorescein in the titration cuvette. The data points are for piperonyl
butoxide at 36 days and the smooth curves are theoretical for n>,max ~ _,
6 74 x 10-* M, C* 5.13 x 10* r", a - 0.77, pf • 0.0272, Pb - 0.45 and
Qf/Qb s 6-6- Se* Appendix for a Discussion of these quantities.
20
-------
16
0
16
0
16
0
16
0
16
0
16
0
Control
R ^
(L)
I
Control (L)
Control (H)
R £3 m
Y//S////////S.
I
1
Es is R!
Methotrexate (H)
• Aroclor (L)
S 1
Dinoseb (L)
Parathion (L)
PCNB (H)
Pip. Butox. ^
(L) 1 ^
SI
^
s
^
Y///////////S.
i
Pyrethrins(H)
Resmethrin (L)
Resmethrin H
V///////S.
i i i i i t
6
Weeks
Figure 7. Effect of pesticides on body weight as a function of time after
pesticide administration expressed as a percentage change of the initial
weight. Animals were treated with pesticide in corn oil as indicated or with
com oil alone (control) and immunized with either a low dose (L) or a high
dose (H) of FO as described in Materials and Methods.
21
-------
IC2
l//f Polycarbonate
ro
Output = K T
15V DC
^p I0//f Tantalum
m
-I5VDC
•y 10//f Tantalum
m
Figure 8. Schematic of the temperature measurement circuit for the thermo-
metric footpad assay. The amplifiers AD522AD and AD504MH (Analog Devices,
Norwood, Mass) give voltage gains of 100 and 201, respectively. The thermo-
couples Tc-i and Tco were copper constantan (Thermometrics, Northridge, CA).
The output if maintained between -10 and +10 volts is proportional to AT and
is conveniently read on a digital voltmeter. After a warmup of 15 to 30 min,
the offsets were adjusted by shorting the input and adjusting the 2K potentio-
meter of 1C] to give zero volts at the output. The 10K potentiometer of IC2
does not need routine adjustment but can be set initially to give zero output
after shorting the input to Ify* i-6" tne output of IC-j.
-------
APPENDIX
In this appendix a brief review of fluorescence polarization
is given together with the necessary equations for interpreting
polarization data and procedures for computing several derived
parameters.
The light emitted from fluorescent solutions is partially
polarized; it consists of a mixture of linearly polarized and
unpolarized light. The origin of this partial polarization and
its implications concerning the kinetic unit carrying the fluor-
escent moiety can be seen from the following considerations.
Classically, the emission from a single molecule may be regarded
as radiation from a single oscillating dipole. This radiation
has an oscillating electric field parallel to the direction of
oscillation of the dipole and is said to be polarized in the
same direction. If a randomly oriented assembly of molecules
is excited by fully polarized light, their fluorescence is only
"partially" polarized, even if the molecules are prevented from
rotary Brownian motion in solution. For simplicity, assume that
the direction of the absorption and emission oscillators in a
single molecule are the same and that they are rigidly fixed
with respect to the geometric axis of the molecule. Furthermore,
assume the molecule rigidly fixed in position during the interval
between absorption and emission (typically a few nanosec). The
probability of absorption of light is proportional to the square
of the magnitude of the component of the electric vector of the
exciting light in the direction of the oscillator. If the
absorption and emission oscillators are parallel the emitted light
23
-------
will be partially polarized with a degree of polarization, p. This
quantity is defined in terms of intensities, I, polarized either
parallel of perpendicular to the incident electric field.
For randomly oriented molecules in a rigid medium, the maximum
value of p observed with linearly polarized light is one-half.
If, instead of being rigidly fixed, the molecules are subject to
rotary Brownian motion, the molecular rotation taking place
between the times of absorption and emission may be expected
to result in values of p lying between one-half and zero. The
extent of this rotation is a function of molecular dimensions and
structure, solvent and temperature. Low molecular weight compounds
such as fluorescein will give rise to virtually completely de-
polarized fluorescence; some polarization will be retained as
molecular size increases. Considering two molecules of equal
size, the fluorescence of the more asymmetric rigid structure
will be more highly polarized.
The essential feature of applying these phenomena to anti-
body hapten binding consists in observing the degree of polariza-
tion and intensity of the fluorescent light when measured
quantities of the hapten and antibody are allowed to interact.
Reaction results in an increase in size of the fluorescent kinetic
unit and in a retardation of the rotary Brownian motion manifested
as an increase in the polarization of fluorescence. From
measurements of polarization the final, derived parameters which
*
can be obtained are 1) binding site concentration, 2) an average
24
-------
association constant and 3) an index of the heterogeneity of the
binding sites. The general type of reaction assumed is that in
which a fluorescent ligand^tbinds to a receptor vb, to
reversibly form a complex ^fa Q\J :
Symbols
a, heterogeneity index
b, subscript indicating "bound"
F, molar concentration of W-r
f, subscript indicating "free"
F. _ax The maximum value of F, ; taken to be equal to the
total molar concentration of receptor sites
M, the total molar concentration of yt in both free
and bound forms (AM in computer program)
p, the polarization of the excess fluorescence, i.e.,
p = (Av - Ah)/(Av + Ah), where AV and Ah are the
intensities in arbitrary units of the components
in the excess fluorescence (above that of the
blank) polarized in the vertical and horizontal
directions, respectively
Q, molar fluorescence of a mixture of free and bound
forms of v* as they exist in a solution under
observation, i.e., Q = (AV + Ah)/M
Fluorescence polarization and intensity measurements
provide a direct and rapid measure of the bound/free ratio
25
-------
and
F, Qf - Q
*f~ = Q - Qb (4a)
In order to utilize these equations, the constants Qf, Q,,
and p^ must be determined for a particular system under study.
No problem is posed in finding Q^ and P£, since these come
directly from a measurement on the labeled component alone.
The determination of Q, and p, , however, implies measurements
on a state in which the fluorescent labeled material is completely
bound to its complementary partner. Since complete binding
cannot be realized physically, an extrapolation is involved.
If equilibrium values ofp plotted against M are extrapolated
to M = 0, p approaches a limit, p'. Values of p' for different
antibody concentrations plotted against (p' - pf) divided by
antibody concentration in any arbitrary convenient units give
p, as the intercept of a straight line, for classical mass law
(18).
Qf(P' - Pf)
^ = pb - ofm .,. ^
This procedure makes it unnecessary to know absolute values of
KF, beforehand. A similar relationship facilitates the
b , max
determination of Q, :
Q - Q'
KF
Khb,max
(6a)
In the program given below the measured values of M, P. Q, P
Qf and relative antibody concentrations (AB) with or without
tentative estimates of P^ and Q^ and used in an iterative
computation to derive the final best values of P^, Q^, KQ, a
26
-------
Fu m*v- These computations are based upon achieving a chi
D 9 Ju3X
square fit of the data to the Sips equation (19,20) which defines
Ff as:
i r_
Ko jV
max - Fb
Substituting M = F, + Ff into eq. (3a) and rearranging gives:
_ (Pfqf - PbQb) Pf - PbQbM
- CQf - V F£ * V -
Inspection of eq. (7a) and (8a) shows that there are five
unknowns, F, , a, K , P, and 0, to be evaluated from measured
b,max o D D
individual values of P, M and Q, viz. P., M. and Q. and from the
measured values of Pf and Q .
In the procedure given below the user may either specify
initial estimates for these unknowns along with measured molarity
and polarization data, or the user can allow the program POLAR
to make the initial estimates. Once initial estimates have been
made for the five parameters , the program proceeds to improve
these estimates in an iterative fashion until a stopping
criterion specified by the user is met. The measure of goodness
to fit to eq. (8a) is a modified chi-square defined by:
/p - P .} *
* i 1
CHISQ = sum .
where P is the measured polarization and Pcaic is the value com-
puted from eq. (8a) given the current estimates for the five
parameters. The iterative improvement performed by POLAR con-
sists of repeatedly moving away from the current best estimate
of each of the parameters either by a user specified value (if
27
-------
Q = molar fluorescence
Using these data, the program proceeds to:
1. Extrapolate each set of P and Q data to a zero molarity
value (called P1 and Q') using the subroutine FLAGR which per-
forms Lagrange interpolation. Therefore, for each antibody,
there is P' = P(0) and Q1 = Q(0) which are put in the arrays
APE and QPE.
2. Obtain a least squares fit to a straight line by SQRL
which assembles and solves the normal equations to obtain
initial estimates of P, and Q, :
P' = slope
Q1 = slope
F'Qf - Q')1
I AB J
V
3. Iteratively find initial estimates for F, __„, K , and
D y III 3.X O
a (renamed in the program: FBMAX, OK,A).
4. Use the chi-square fit to improve these initial
estimates.
Option 2. The program reads in P£, Q£, N (= number of antibody
concentrations) and RANGE.
Then for each antibody the following data are read:
AB = antibody (scaled)
NP = number of molarities at which P has been measured
followed by NP sets of:
AM = M = molarity
P = polarization
28
-------
the user provided the initial estimates) or by 10% of the current
estimate (if POLAR computed the initial estimates) , computing
the chi- square value that results and keeping track of the para-
meters that give the smallest chi- square value. As the iteration
proceeds, the amount that is being added or subtracted from the
current best estimate is halved as the value of the parameter
nears an optimal value. The iteration continues until all the
parameters satisfy the following test, where RANGE is a value
entered by the user at the onset:
cur?entmvalue ^ - ***** £or a11 five Parameters (10a)
- A frequently occurring computation in this search for optimal
parameters is the value of F£. . This is accomplished by a root-
finding routine ZEROIN which will find the zero of a function,
given an initial interval in which that root must lie. Equation
(7a) for F£. is written as follows to avoid the singularity present
when Fbi approaches Fb
CVfi (Fb,max :.**'* Ffi> - AM + Ff. = 0 (lla)
_,**"-- •'•-?" ^
using the fact that molat^ty, AM = M = Ffei + Ff . .
The program POLAR operates in two modes dajJending on the
f ^
amount of data the user can supply. ^ ~
Option 1. The program reads in Pi, Qf» N (= number of
9
antibody concentrations) and RANGE. Then, for each antibody/
v" - '
concentration the following data are read:
AB = antibody concentration (scaled so smallest is 1)
NP = number of molarities at which p and Q have been measured,
followed by NP sets of:
AM = M = molar ity
P = polarization
29
-------
The program then reads in initial estimates provided by the
user for the five parameters,
FBMAX, QF/QB (only ratio is important), A, OK, PB,
followed by five values specifying how much the current values
of the parameters are to be varied in optimizing the chi square
fit. If the parameter is not to be changed by the program,
then a zero should be entered for the corresponding variance.
Otherwise a good value to use is 10% of the original estimate.
The program proceeds then to improve these initial estimates
using the chi-square criterion. As an aid to the user, a
print out of the entire program with subroutines as well as
of the runs for piperonyl butoxide at 36 days using first
option 1 and then option 2 are given below.
30
-------
1 PROGRAM POLAR (INPUT»LIST,TESTER*TAPE 1-TESTER*TAPE3-L1ST)
C N-NUMBER OF SETS OF DATA POINTS
5 DIMENSION OPE(15)»OPA(15)(NP(15)*P(30tlS)*AM(30flll*
1 APEn5){APA,ABtl5f,a{30tl5),FFT30,15|,Fa]30.15>,X<15),
2 OKS<450)*Plf6)*PVARf6>.FlX(6!*PT(6*6)*PCt30*is)*PFIT(6)*TITL{4)
COMMON A»AMOL»FBM»OK
C READ HEADING AND INCUT. HEADING IS 40 SPACES.
REWIND 1
99 READ «1.59)TITL,[NDIC
19 59 FOR1AT(4A10*m
IF(INDIC)800»64»314
64 WRJTE(3,88I
88 FORMAT! 1HH
W AND ONE A9 VALUE PRECEED EACH SET OF POINTS
DO 4 KK-l.N
REAtm.101 )NPIKK),A8(KK)
101 FORMAT(IZ,F10.5I
35 C ONE VALUE OF MOLARITV AND THE CORRESPONDING P AND 0 VALUES ARE TO IE PUNCHED
C riN EACH CARD. AM»HOLAR lTY*P-POLARllATION» AND Q'OUENCHING.
NB-NPIKKI
4 READ jl.102) (AM(I*KK),P(I*KKI*0(I*KK),I>l*NBt
.>_!
-------
T
j,
'V,
C FORH INDEPENDENT VARIABLES, APA AND QPA. FOR LEAST SQUARES FIT
60 C
DO 21 1-1, N
APA(l) - (APEU I - PF)/AB(I)
OPA(I) - (OFOPEmiSABd) >•
21 CONTINUE ••'.
C LEAST SQUARES FIT FOR PB AND OB
CALL SORL ( APAf APE • N, SLOPE, PB, STDF.RR ) \
CALL SORL (QPA,aPE,N.SLOO,OB,STDORR)
70 C
DO 22 IMO-1,N
IR-NPUMO)
DO 22 IZZZ-l.lR \
PBIG"P(IZZZ,IMO) . N.
75 IF(?B-PBIG)20,20.22
20 PB*1.2+P8IG
22 CONTINUE -,,,/.»
C PRINT THE OUTPUT
80 C
WRITE (3.107) PB, OB, SLOPE, SLOQ
107 FORMAT(//6H PB » ,FB . 5, 3<.X, 6H OB - »F8.4,/,SH SLOPE- ,F1
* 7HSLOPE- ,Fe.5l
WRITE (3,10R) STDERR,STD3RR
8t> 108 FORHAT (IBM STANDARD ERROR • , F7.5,23X,18H STANDARD ERROR • ,
1 F 7 • 5 I
C
CALCULATE FF AND FB.
90 DO 121 J-1,N
NB-NP(J)
00 121 I«1,N3
FF( I,JI-QB*AM( f ,J)»(PB-P( I,J) )/IP( l,J)*(OF-QB)-PF*QFtPB»QB)
121 FBI I,J)-iH(I,J)-FF(I,J)
812 FORHATl/'/^OHlCALCULATEO VALUES Of FF AND FB, BASED ON FIRST ESTIM
1ATES OF PB AND QB,//,7lH ANTIBODY HOLARITY
2 FF FB )
100 00 814 JO-l.N
NPOL-NP(JO)
WRITE (3,813) AB(JO)
613 FORMAT! 3H ,El
WRITE (3,816)
816 FORHAT(IHO)
814 CONTINUE
110 C DETERMINE APPROXIMATE VALUES FOR A, KQ. AND FBMAX .....
817 SIGMA - 1000.
A=. 9
SIZE-.l
-------
115 123 NKl-0
00 122 J-l,N
NR-NPm-l
DO 122 I-l.NB
NK1-NK1+1
120 TOK.((FBU,J»/FFC I,J)**A-FBU»l.J)/FF(I*l,J)**AmFB(m« J)-FBCI,J
124 OKS(NK1)-0.0
GO TO 122
12!) 125 QKS(NK1)-TOK**(1./AI
122 CONTINUE
ENK-NKl-1
NZIP-NK1-1
SUM - 0.0
130 00 126 I'liNZIP
RASMA • OKSdf » nKSII+1)
IF(RASMA) 822,822*821
821 POS-SQPT(4.*UOKS(I )-OKSII»l HMOKSII J+OKSU + 1
SUM • SUM + ROS
139 GO TO 126
R22 SUM • SUM * 2.0
126 CONTINUE
SIG-SUM/ENK
TEMPK-0.0
0-0.0
00 127 I-UNK1
IFtnKSIllJ 127*127,128
128 TEMPK-TEMPK+QKSII)
0-0+1.0
127 CONTINUE
IF(SIGHA-S10)130,130,129
129 OK-TEMPK/0
SIGMA-SIG
AVAl-A
150 A-A-SI2F
GO TH 123
130 IFISIZE-.01)132,132,131
131 SIZE-.01
A-A+.09
155 GO TO 123
C SOLVE FOR FBMAX,
132 FBMAX'0.0
160 OENOM-0.0
DO 133 J-lfN
NB-NPUl
00 133 I«1,NB
FBMAX»FBMAX+FBII»J)+(1.*1./(OK*FF(I,J))**AVAL)
165 133 OENOM«OENOM+1,0
FBMAX-F9MAX/J)ENr)H
A-AVAL
WRITE (3,811) A.OK,FBMAX
811 FORMAT(21H~FIRST APPROXIMATIONS ,/»12H A • ,F10.6,/>
170 112H KO • ,E10.4,/,12H FBMAX • .E10.4)
-------
C BEGIN FITTING CONSTANTS FOR BEST CALCULATED C VALUES IN THE LEAST SQUARES
C SEMSE.
1?5 P1H)«FBMAX
P1I2)«08
Pim-A
Pm»-OK
PU5J-PB
160 C
C PVARU)-*/- MAXIMUM ALLOWABLE EXCURSION FOR THE J'TH FIRST GUESS.
C
602 PVAR(2)».1*P1
605 PVAR(5)«.l*P
185 607 PVAR(l)«a*P
PVAR(3)-.075
(2>
(5)
m
PU3)
WRITE (3,88)
GOTO 60S
190 BOO CONTINUE
C THIS IS THE BEGINNING OF OPTION TWO, READ IN THE
C APPROXIMATIONS FOR FBMAX, OF/OB, A, KO* AND PB
C FOLLOWED BY THE AMOUNT YOU WANT THE CHI-SQUARE FIT
C ROUTINE TO USE TO VARY THE VALUE.
C NOTE - ENTERING AT ZERO FOR THIS VARIANCE WILL FORCE THE PROGRAM
C TO USE THE ESTIMATE THE USER HAS PROVIDED, AND NOT CHANGE IT.
200 WRITE 13,8b)
WRITE (3,59) TITL,INDIC
READ (1,100) N,OP,PF,RANGE
WRITE (3,3) N,QF,PF
205 00 805 KK«1,N
READ (1,101) NP(KK),AB(KK)
NB-NP(KK)
605 READ (1*807) (AMI I,KK),P(I>KK),t«1,NBI
807 FORMAT (2F10.5)
210 READ (1*810) (PHI),1-1,5)
810 FORMAT (5E10.3)
READ (1*810) (PVARU),(*1>5>
Pl(2) » OF/PH2)
Pl(6)«PF
PVA1(6)«0.
C BEGINNING OF THE ITERATIVE CHI-SQUARE IMPROVEMENT ROUTINE
608 CHI SO • 1000.
220 606 00 6?0 1-1*5
FIXm-PVAft(I>/Pl(I)
pm,ii«pim-PVAfim
PTJI.2) • PKM * PVAR(I)
620 CONTINUE
C COMPUTE ALL POSSIBLE COMBINATIONS AND CHI-SQUARE VALUES)
IF (PTI3.2) .GT. 1.) PT(3.2)«1.
-------
00 299 Jl • 1»Z
230 FBHAX » Pm*j|)
00 299 Kl • 1.2
OB • PTC2.K1)
DO 299 LI • 1.2
A « PTOfLl)
235 00 299 HI • 1*2
OK • PTI4»H1]
DO 299 Nl • 1*2
P3 • PT(5.Nir
sunso • o.o
240 R • 0.0
NFLAGS • 0
00 290 NN • l.N
FBHAX*ABINN»
NQ • NP(NN)
DO 2f
245 DO 290 MM • l.NO
AMOL « AH(HH.NN)
B-l.E-12
FTHV • ZEROIN (8.C.F. l.E-12 »
IF (FTHV .60. l.E-12) GOTO 290
260 PCJMM,NN)-(CPF»OF-PB*QB»*FTHV+PO*Qa»AHOL>/t(OF-QB)»FTHV+OB*AMQL)
ft * R 4- 1
SUNSO -'SUMSO * ( P(M«»NN> - PC(MM.NNM**2
290 CONTINUE
b~v u wiv i i rjws.
cusa • 100. * SORT isunso)
291
299
IF (CHSO .EO. O.I GOTO 299
IFICHISO-CHSO) 299*299*291
CHISO - CHSO
KUTBAK • 1
PFITI
PFIT
PFIT
PFIT
PFIT
PFIT
CONT1
I
2
3
4
5
- FBHAX
. OB
• A
- OK
1 • PB
6 -PF
NUE
260
265
299
NZ • 5
409 IF(NZ-O) 410*420*410
410 IFIFIX(NZ)-RANGE) 411*420*420
270 411 NZ » NZ - 1
IFINZ - 0) 409*500*409
420 CONTINUE
DO 424 M • 1*5
424 PKMI • PFlTIHt
27S IFUUTBAK - 1) 421,423*423
421 KUTBAK • 0
DO 422 M • 1,5
422 PVARIH) » .5*PVAR(M)
423 KUTflAK • 0
280 GO TO 606
C PRINT FINAL OUTPUTS.
$00 CONTINUE
2B5 PFITm-OF/OB
-------
PVAR(2»-OF/lQft-PVAR(Z»-OF/QB
SRIT.E_!?»?99>
799 FORMATION
Co
290
295
300
305
310
315
320
325
330
305
WRIT? .
FORMAT(1
112H F
212H
312H
412H
512H
612H
305) CHISQ.(PFIT(I),PVAR(I).I
HO XI-SOUAREO • .EH.8,///,
" - ,E10.
- .ElO.
- .ElO.
LEAST
• 1.6J
SOUARES FIT
j£10.<.
•F10.4.7H
.F10.6.7H
•E10.4.7H
.F10.6.7H
.F10.6.7H
*/-
.ElO.
,E10.
,£10.
.F, 1 .E-12 )
GOTO
: COMPUTE ANO PHINT THE BEST POLARIZATION VALUES,
OB«OF/PFITm
A • PFITI3)
OK * PFIU4)
PB > PFITI5)
PF-PFIT16I
DO 790 NN • l,N
F3M - PFIT(1)»A«
NO « NP(NN)
00 790 MM • l.NO
AMOL • AH(MM.NN)
B • l.E-12
C • l.E-7
FTMV • ZEROIN (B.C. __
IF (FTMV .NE. l.E-12) GOTO 760
PC (MM.NN) • 0.
GOTO 790
760 PC(HM,NN)-(CPF»OF-PB*OB)*FTHV*PB»OB*AMOL1
790 CONTINUE
WRITE (3,306)
306 FQRHAT(IHO)
WRITE (3.3071
307 FOR1AT(7
-------
(XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
.(X»Y»N»A»BfSTOERR)
C COMPUTE LEAST SQUARES FIT TO LINEAR EQUATION
C V « A X * B
jj USING NORMAL EQUATIONS (ONLY BECAUSE SIMPLE LINEAR MODEL USED)
10 C H SUMX R SUMY
C SUMX SUMXX A * SUMXY
DIMENSION X(30)>Vf30)*CALCri30)»RESIOf30t
C COMPUTE SUMS
SUrtX-0.0
SUMt-0.0
20 SUMXX-0.0
SUMXY-0.0
§0 22 I»l»N
UMX«SUMX*X(I
25 SUMXX*SUMXX*X
22 SUHXY-SUMXY+X
ijj C COMPUTE PARAMETERS A AND B
40 ORO-N
DENOH-ORD+SUMXX-SUMX**2
IFIDENOMI
24 A-(ORO*SUMXY-SUMX*SUMYI/OENOM
B.CSUMV*SUMXX-SUIX*SUHXYI/DENOM
C COMPUTE BESIOUES
SUIRES'0.0
00 23 1-I.N
40 CALCY(h«A*xm*B
RESIDlri-Yill-CALCYUI
23 SUMRES-SUMRES»RESIOUi*»2
C COMPUTE THE ERRORS
STOERR*$ORTfSUMRES/CORO>2.))
CO TO 26
?5 fl-SUMY/ORO
26 IFJB) 27,?7,28
90 27 8-1.
28 RETURN
END
-------
1
CXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
REAL FUNCTION ZEROIN tAX.BX,F,TOL)
00
C
C ROOT UNDER FROM COMPUTER METHODS FOR MATHEMATICAL COMPUTATIONS
5 C BY FOPSYTHE. MALCOLM AND MOLER
C PRENTICE-HALL. 1977
C AX AND BX SHOULD BRACKET THE REGION IN WHICH THE ROOT IS TO BE
C FOUND, f IS THE NAME OF AN EXTERNAL FUNCTION PROVIDED BY USER
10 C WHICH SPECIFIES THE FUNCTION WHOSE ROOT IS SOUGHT. TOL IS A
C USER DEFINED ACCURACY REQUEST.
C NOTE: MACHINE DEPENDENT ROUNDOFF ERROP EPS
15 EPS - 7.E--IV
C INITIALIZATION
A»AX
B-BX
FA-F(A)
20 FB-F(B)
C BEGIN STEP
20 C-A
FC-FA
D-B-A
25 E»D
30 IF (ABS(FC) .GE. AQS(FBI) GOT3
C ADJUST SIGNS
55 60 IF (P .GT. 0.) 0--Q
P-ABS(P)
C IS INTERPOLATION ACCEPTABLE?
-------
$ IP^E! A^?.5?E:2?rQG5TSB?omi*oni 60TO T°
60 E-D
D-P/0
GOTO 80
C BISECTION
70 D-XH
65 F-D
C COMPLETE STEP
80 A-B
FA-FB
IF (ABS(D) .GT. TOLII B«B+0
70 IF (ABS(O) .LE. TOLli B-fl*SIGN ** A » (FBM - AMOL » X) - AMOL
_ _ K c I URN
10 END
-------
P|P|R2?NrL BUJOXJDE
3 1.
l.E-09.2179
3.33E-09.1509
9.9E-09.07927
3 3.
l.E-09. 35917
3.33E-09.3248
9.9E-09.2341
3 10.
l.E-09. 4232
3.33E-09.62 ,..._^__....
.8150
.8521
1.094
.6624
.6491
.7031
36 OAYS 147- 94 -1
.02
1.236
1.533
2.762
.B1SO
.6521
1.094
.6624
.6491
.703*1
.84 €+00.7673 £+090.45 E+0
.84. . .I-Q1.7673 f +060.0 E+00
»l
-------
PIPE»ONYL BUTOXIDE 36 DAYS 1*7- 94
WITH 3 ANTIBODIES PF • .0372
3.5350
THE FIRST TERN IS P* FOR DATA SET 1
.253335
.217900
.150900
.079270
FIRST TERM IS Q* FOR DATA SET 1
1.10236
1.23600
1.53300
2.26200
THE FIRST TERM IS P» FOR DATA SET 2
.37*275
.359170
.3?4800
,;»i4lOO
FIRST TERN IS 0« FOR DATA SET 2
.60690
.81500
.85210
1.09400
THE FIRST TERM IS P* FOR DATA SET 3
.421898
.423200
.423100
.399200
FIRST TERH IS 0" FOR DATA SET 3
.67332
.66240
.64910
.70310
PB
.46635
SLOPE" -.91675
STANDARD ERROR « .01722
OB • .6204
SLOPE* .19962
STANDARD ERROR « .00711
FIRST
KO
FUMAX
.7673E+09
.2034E-07
-------
XI-3QUAR60 * ,1<.12U76E*01
LEAST SQUARES FIT
FBHAX
QF/QB
KO
PB
PF
.7247E-OB
8.8262
.7651B7
.60916+09
.451781
.027200
ANTISOOf
.iooooaooE+oi
1 i I i I 1
.1271E-09
.P653F-01
.3938E-02
I2915E-02
0.
MOLAR 1TY
.ioooooooE-oa
.33300000E-08
.99000000E-08
.217900
.190900
.079270
F CALCULATED
.217155
.U5685
.071936
.TOOOOOOOE»01
.10000000E-OB
.33300000E-08
.99000000E-08
,35>9170
.32^800
.23
-------
PIPERONYL BUTQXIDE 36 OAVS 147- 94
PF •
rflTH 3 ANTIBODIES
XI-SOUAPEO • .11947542E+01
3.5250 OF-
LEAST SQUARES FIT
.0272
CO
FflMAX
QF/QB
KO
PR
PF
.6738E-08
6.6000
.774250
.51316+09
.450000
.027200
ANTIBODY
. lOOOOOOOEtOl
+ /-
+ /-
»/-
*/-
+ /-
*/-
.1271E-09
0.
.52t>0t-02
.4796E*07
o.
0.
HOLAR1TY
.100000006-08
.33300000E-08
.990000006-08
P OBSERVED
.217900
.150900
.079270
P CALCULATED
.216044
.147699
.076514
,30000000E»01
.100000006-08
.333000006-08
.990000006-08
.359170
.324800
.234100
.357875
.317228
.237310
.10000000E+02
.100000006-03
.333000006-08
.990000006-08
.473200
.423100
.399200
.426675
.416910
.400521
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-79-039
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
.Effects of Pesticides on Immune Response
5. REPORT DATE
September 1979
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
WalterB. Dandliker, Arthur N. Hicks, Stuart A. Levison
Kris Stewart and R. James Brown
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Biochemistry
Scripps Clinic and Research Foundation
La Jolla, CA 92037
10. PROGRAM ELEMENT NO.
1EAfil5
. CONTRACT/GRANT NO.
11
Grant No. R803885
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
US Environmental Protection Agency
Research Triangle Park, NC 27711
RTP, NC
13. TYPE OF REPORT AND PERIOD COVERED
Final 2/16/77 - 4/30/79
14. SPONSORING AGENCY CODE
EPA/600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The influence of various pesticides on the humoral and cellular immune response to
fluorescein labeled ovalbumin has been analyzed. Pesticides (Aroclor 1260, Dinoseb,
Parathion, pentachloronitrobenzene, piperonyl butoxide, mixed pyrethrins and
Resmethrin) were administered intragastrically in corn oil in one dose (one half of
LD50) before primary immunization. Control groups included those .treated with corn
oil alone or immunosuppressed with Methotrexate. Booster immunizations and test
bleedings were scheduled at weekly intervals thereafter. The cellular immune response
was quantified by redness and swelling, histological examination and by differential
temperature measurements of the foot pads after antigen challenge. The concentration,
binding affinity and heterogeneity of the serum antibody were determined by fluore-
scence polarization measurements. Dinoseb and Parathion depress both the humoral and
cellular response. Methotrexate and pentachloronitrobenzene give a late stimulation,
while Resmethrin an early, sometimes very marked stimulation of the cellular immune
response. Other pesticides showed little or no effect under the conditions tested.
Effects on the humoral response were limited to changes in antibody concentration,
the binding affinity being nearly constant in all instances.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Immune Responses
Cellular immune responses
Humoral immune responses
Dinoseb, parathion, PCB, PCNB, pyrethrin's
methotrexate
Pesticides
Immunosuppression
Immunoglobin
06C,F,T
21. NO. OF PAGES
49
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (This Report)
Unclassified
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
44
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