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POLYMBROMINATED BIPHENYL TOXICOSIS IN SWINE: EFFECTS
ON SOME ASPECTS OF THE IMMUNE SYSTEM IN

LACTATING SOWS AND THEIR OFFSPRING

BY

Shirley Kay Howard

A THESIS

.Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Pathology

1979

ABSTRACT
POLYBROMINATED BIPHENYL TOXICOSIS IN SWINE: EFFECTS

ON SOME ASPECTS OF THE IMMUNE SYSTEM IN
LACTATING SOWS AND THEIR OFFSPRING

BY

Shirley Kay Howard

Sows were fed diets containing 0, 100, or 200 ppm of Firemaster
BP-6, a mixture of polybrominated biphenyls (PBB), during the last
half of gestation and the following 4 weeks of lactation. Mitogen
stimulation (phytohemagglutinin, lipopolysaccharide, pokeweed
mitogen) of peripheral blood lymphocytes was studied. Lymphocytes
from sows fed PBB for 12 weeks showed significantly decreased
mitogen responses. Mitogen responses of lymphocytes from piglets
of sows fed PBB were normal at birth, but responses were signifi-
cantly decreased when the piglets were 4 weeks of age. Hematological
parameters were normal. Bactericidal activity was assayed in whole
blood from the sows once during gestation and twice during lacta-
tion, and from the piglets at 2 and 4 weeks of age. No significant
changes in bactericidal activity occurred in sows fed PBB, or in

their nursing piglets.

Dedicated with love to my husband, Don,

and my daughter, Terri

ii

ACKNOWLEDGEMENTS

I wish to express my appreciation to Dr. C. K. Whitehair, my
major professor, for his support and counsel during my course of
study.

Special thanks go to Dr. Stuart Sleight for his guidance,
patience, and encouragement during the completion of my research.
I also wish to thank Martha Thomas and Dr. G. R. Carter,
members of my guidance committee, for their helpful comments and

suggestions.

My deepest appreciation to my friend and fellow student,
Pedro Werner, who was always willing to unselfishly give of himself
and his time. His encouragement and thoughtful cooperation were
invaluable.

To Dr. Toby Jones of the Department of Surgery, I wish to
express my appreciation for the technical assistance and words of
encouragement she provided. My thanks also to Dr. Charles Cress
of the Department of Soil Science for his expert help with sta—
tistical evaluation of the research data, and to Dr. Bruce Baker
of the Department of Anatomy for allowing me to use his laboratory
facilities.

Appreciation is also due to fellow students Araquen Telles
and Soesanto Mangkoewidjojo, and to my co-worker, Linda Stegherr,

for their assistance and support.

iii

I am grateful to the Michigan Department of Agriculture for
their financial support of my research.

My special thanks and deep appreciation to my husband, Don,
and my daughter, Terri, for their support, love, and understanding

throughout my studies.

iv

INTRODUCTION

TABLE OF CONTENTS

LITERATURE REVIEW. . . . . . . . . . . . . . . . . .

The Polybrominated Biphenyls (PBB). . . . . .

Assays

Chemistry. . . . . . . . . . . . . . .
Kinetics . . . . . . . . . . . . . . .
Toxicosis. . . . . . . . . . . . . .

Human Exposure . . . . . . . . . . . .
Effects on the Immune System . . . . .
of Immune Function . . . . . . . . . .
Assessment of Phagocytic Cell Function

Assessment of Lymphocyte Response to Mitogens.

MATERIALS AND METHODS. . . . . . . . . . . . . . . .

Experimental Design . . . . . . . . . . . . .
Mitogen Stimulation of Lymphocytes. . . . . .
Bactericidal Assay. . . . . . . . . . . . . .
Cholesterol . . . . . . . . . . . . . . . . .
Statistical Evaluation. . . . . . . . . . . .

RESULTS. . .

Mitogen Stimulation of Lymphocytes. . . . . .
Bactericidal Assay. . . . . . . . . . . . . .
Cholesterol . . . . . . . . . . . . . . . . .

DISCUSSION .

SUMMARY. . .

APPENDIX . .

REFERENCES .

VITA . . . .

Page

15
15
15
l7
l9
19
20
20
20
24
27
32
33
37

42

LIST OF TABLES
Table Page

1 Lymphocyte responses to mitogens in sows after 12
weeks of feeding experimental diets . . . . . . . . . . . 21

2 White blood cell counts in sows fed PBB and in
their piglets . . . . . . . . . . . . . . . . . . . . . . 22

3 Lymphocyte responses to mitogens at birth and 4 weeks
of age in piglets of sows fed PBB . . . . . . . . . . . . 23

4 Serum cholesterol in sows fed PBB and in their piglets. . 26
1A Bactericidal assay in sows fed PBB. . . . . . . . . . . . 33
2A Bactericidal assay in piglets of sows fed PBB . . . . . . 34

3A Ratio of adrenal weight (gm) to body weight (kg) of
sows fed PBB and of their piglets . . . . . . . . . . . . 35

4A Total and differential white cell counts in sows fed
PBB and in their piglets. . . . . . . . . . . . . . . . . 36

vi

Figure

1

LIST OF FIGURES

Gas chromatogram of Firemaster BP-6 on an OV-lOl
column, using an electron capture detector. . . . . . . . 4

Bactericidal activity in whole blood of sows after
12 weeks of feeding . . . . . . . . . . . . . . . . . . . 25

vii

INTRODUCTION

The lack of knowledge of the metabolism, toxicity, and inter—
actions of common industrial compounds decreases our ability to
cope with the unexpected chemical contamination of the environment.
The accidental mixing of polybrominated biphenyls (PBB) into live-
stock feed in Michigan in 1973, and the resulting contamination of
the food chain, brought about a difficult crisis and was made worse
by the lack of information regarding these chemicals. More than
34,000 cattle and 1.5 million chickens were destroyed, as well as
many tons of milk, eggs, cheese, and other farm products. There is
continuing anxiety and concern about the unknown effects on the
health of man and livestock exposed to PBB.

During 1973 and 1974, an estimated 10,000 Michigan residents,
mostly farm families and their neighbors, were exposed to high
levels of PBB, and another unknown number of persons were exposed
to lower concentrations. Numerous reports of ill health among
persons and farm animals exposed to PBB stimulated research into
the toxicity of these chemicals. Investigations into the kinetics,
metabolism, and environmental distribution of these chemicals
resulted in valuable information for dealing with this accidental
contamination of the environment. However, many questions regarding
the effects of exposure to PBB on health still remain unanswered,

and research continues. The objectives of this thesis were to

investigate the effects of the ingestion of PBB upon some aspects
of the immune system of swine during gestation and lactation, and
to determine the toxicity of PBB to the developing immune system in
piglets exposed to PBB by placental transfer and consumption of

breast milk.

LITERATURE REVIEW

The Polybrominated Biphenyls (PBB)

 

The following is a general review of relevant information about
PBB, with emphasis on the literature available regarding the transfer
of PBB to young animals via the placenta or breast milk, and the

influence of PBB on immune functions.

Chemistry

The flame retardant that was substituted for magnesium oxide
in a livestock feed supplement was produced by Michigan Chemical
Company under the trade name "Firemaster BP—6." It is a mixture of
polybrominated biphenyls (Figure l), the major fraction (60—70%)
being a 2,2',4,4',5,5'-hexabromobiphenyl. This mixture of brominated
biphenyls is insoluble in water but highly soluble in organic sol—
vents and fats. Calcium polysilicate was added to the mixture as
an anticaking agent, but there are few other impurities. Very
small amounts of hexa-, penta-, and tetrabromonaphthalenes have
been identified, but there has been no evidence of the presence of

any highly toxic substances such as the dibenzofurans (19,22,27).

Kinetics
Polybrominated biphenyls are apparently poorly absorbed from
the intestine and much of that ingested is excreted in the feces

(59). Some components of the mixture may be more effectively

FIRE MASTER BP’6

4

 

 

8
23 67

 

 

 

 

 

«r-
-r-

)-
db
Ji-

TIME (MIN)

Figure 1. Gas chromatogram of Firemaster BP-6 on an
OV-l column, using an electron capture detector (Varion 3700).
The major component (peak 4) is a 2,2',4,4',5,5'- hexabromobiphenyl.

5

absorbed than others, as residues in tissues have a higher concen-
tration of hexabromo fractions and a markedly lower concentration
of the heptabromo fraction relative to the standard Firemaster
mixture (18). The rate of metabolism of PBB in the body, like that
of the polychlorinated biphenyls (PCB), is very low. Also similar
to PCB, the distribution of residues in the tissues is closely
correlated to the fat content of the tissues. Exceptions are the
liver, in which concentrations are higher than would be expected,
and the brain, in which residue concentrations are low despite the
high lipid content of the tissue. It has been suggested that the
low concentration of residues in the brain is due either to the
effectiveness of the "blood-brain barrier" or to the presence of
different forms of lipids in the brain (17,30,43).

Storage of PBB residues in adipose tissue results in a body
fat concentration to blood concentration ratio of approximately
1000:l (17). In lactating animals and laying chickens, milk and
eggs are the major source of elimination of PBB from the body. The
ratio of milk fat concentration to body fat concentration is approxi—
mately 0.42:1 in cattle (17,18). Fries et a1. (18) did extensive
studies on the kinetics of PBB and found that milk fat concentration
in cattle reached a steady state at about 4 times the diet concen—
tration after 20 to 30 days of feeding. Milk fat residues have
almost 25 times more hexabromo— than hepta— and octabromo-isomers,
suggesting that the less halogenated components of Firemaster BP-6
are more easily transferred across biological membranes (17,18).

In cattle and rats, PBB crosses the placental barrier in the
blood, accumulating in the fetal tissues and blood at a concentration

of about l/3 that of the dam (17,43). Exposure of the suckling

6

young to breast milk of a dam fed PBB raises the concentration of
the residues in tissues and blood to equal or greater than those
of the dam. Because milk fat concentrations are so much higher
than blood concentrations, PBB transferred through the milk is
considered a more important source of exposure than placental
transfer for offspring of animals consuming PBB (43).

Fries et al. (13,17) have reported that when exposure to PBB
is stopped, the residue concentration in milk fat declines in a
two-phase, first order pattern like that of PCB. The rapid initial
drop is followed by a more gradual but continued decline, resulting
in a half-life of approximately 60 days in cattle. The rate of
elimination is influenced by several factors: level of milk pro-
duction, total amount of body fat, and changes in body fat concen-
tration. Some limited studies have been made of the influence of
different compounds on the elimination of PBB from the body. Cook
et a1. (9) reported that administration of activated carbon, sodium
phenobarbital or vitamins A, D and E did not affect the rate of

excretion in milk or feces, or the half-life of residues in body fat.

Toxicosis

Before the accidental contamination of the environment with
polybrominated biphenyls in Michigan in 1973, little work had been
done on the toxicity of these chemicals. In investigating the
suitability of some of the PBB isomers as flame retardants, octa-
bromobiphenyls were tested for chloracne-type activity and there
was no response other than a slight reddening of the skin (40).
Aftosmis et a1. (1) also found low acute skin toxicity with octa-

bromobiphenyl and hexabromobiphenyl, but reported enlarged livers

7

in animals exposed to hexabromobiphenyl. After exposure of farm
animals to very high levels of PBB in contaminated feeds, there
were many reports of adverse health effects in these animals.
These included decreased milk production, loss of weight, increased
urination and lacrimation, hematomas, abscesses, poor wound healing,
and problems associated with gestation (19,23,27,35). It has been
estimated that these animals were exposed to concentrations of PBB
as high as 13,000 ppm. Experimental exposure of animals to lower
concentrations (1 to 500 ppm) has generally resulted in a much lower
level of toxicity and the failure to demonstrate many of the severe
clinical symptoms reported in the heavily contaminated farm animals.
Sleight and Sanger (48) reported that rats fed concentrations of
up to 100 ppm PBB for 60 days had no overt clinical signs of
disease. Similar results for other species have been reported
(19,27,35), and surveys of the health status of cattle exposed to
low concentrations of PBB failed to uncover any consistent clinical
signs (35). However, toxicity may vary among species, as guinea
pigs apparently have a greater sensitivity to low levels of PBB (19).

Less overt signs of the toxicity of PBB have been reported in
various species, even at low concentrations. Pathologic effects on
the liver have been consistently observed (19,48). Changes appear
to be dose dependent and include an increased liver to body weight
ratio and swelling, hyperplasia, and vacuolation of the hepatocytes.
Some changes in serum alkaline phosphatase, blood urea nitrogen,
and serum glutamic oxalocetic transaminase have been reported in
animals exposed to dietary concentrations of 100 to 500 ppm PBB
(14,19,48). Several investigators have reported that PBB, like

PCB, is a potent stimulator of the hepatic microsomal enzyme system

8

(ll,37,45,47). An important result of this induction of the mixed-
function oxidase system is its effect on the hepatic metabolism
of drugs and other xenobiotics (7).

Polybrominated biphenyl is known to be transported across the
placenta to the fetus (17,43). These chemicals do not appear to
be potent teratogens in rats or mice, but reports of the severity
of embryotoxicity and teratogenicity are somewhat contradictory.
Cattle exposed to contaminated feed were reported to suffer
increased calf losses, abortions, and malformed fetuses (23,27,35).
With dietary concentrations of 1000 ppm, some laboratory animals
have decreased fetal weight and decreased numbers of live offspring,
but no severe teratogenic effects. When using lower concentrations
of PBB (100 ug/kg body weight), other investigators found no

embryotoxic or teratogenic effects (19).

Human Exposure

 

An unknown quantity of PBB-contaminated dairy products and meat
entered the food chain in Michigan before the contaminated farms
were quarantined. An estimated 10,000 Michigan farm residents were
exposed to high concentrations of PBB during this period. Fries
et al. (17), having worked extensively with the kinetics of halo-
genated hydrocarbons, estimated that the most highly exposed people
consumed from 5 to 15 grams of PBB over a 230—day period through
consumption of contaminated milk alone. At the time the quarantine
was imposed in 1974, some of the farm residents had blood concen-
trations of PBB as high as 2.3 ppm (27). An unknown number of
persons was exposed to lower concentrations of PBB in contaminated

food products. A Michigan Department of Public Health study (5)

9
found detectable levels of PBB in 96% of human breast milk samples
tested in the Lower Peninsula and in 43% of those from the Upper
Peninsula. From these data, it is estimated that 8 million of
Michigan's 9.1 million residents have detectable body burdens of
PBB .

Many of the persons exposed to high levels of PBB complained
of health problems. These complaints, including easy fatigability,
muscle and joint pain, nervousness, irritability, skin rashes,
headache, and decreased resistance to infections, are similar to
the symptoms observed in persons accidentally exposed to high
concentrations of PCB (29,34). Several studies of the PBB-exposed
population were conducted by government agencies. The Michigan
Department of Public Health did a short-term health study of
persons on quarantined farms in 1975 and a more extensive study
in 1976. Intensive medical examinations of exposed persons were
made by research teams from the Center for Disease Control and
Michigan's medical schools. In these studies there were no identi-
fiable increases in the frequency or severity of health problems
in persons exposed to PBB. It was concluded that there was no set
of symptoms or physical or biochemical abnormalities which could
be associated with exposure to PBB (6,36). In 1978, the Michigan
Department of Public Health began a long-term study of the human

health effects of PBB under a Health, Education, and Welfare grant.

Effects on the Immune System

 

In areas of environmental contamination with PBB, the poten-
tial toxic immunosuppression by these chemicals is of great

importance to the assessment of human health status and to the

10
animal industries. The reports of immunosuppressive effects of
the closely related compounds, polychlorinated biphenyls (PCB),
give reason to suspect that PBB may also have toxic effects on the
immune system. Some mixtures of PCB cause a reduction in thymic
and splenic weights, depletion of lymphocytes in lymphoid tissues,
a severe reduction in humoral immunity and delayed-type hypersensi-
tivity, and a decreased resistance to infection and endotoxins
(29,31,55,56).

Data on the effects of PBB on the immune system are contra-
dictory. This may be due to the different species involved,
variability of age and immune development, or difference in length
and amount of exposure to the chemicals. Luster et al. (32),
working at the National Institutes of Environmental Health Sciences,
reported decreased mitogen responses, immunoglobulin levels and
antibody production in rats and mice given oral doses of PBB.

These animals were exposed to 3 and 30 mg/kg body weight/day for

30 days. In a study of PBB-exposed Michigan dairy farm residents
conducted by a research team from the Mount Sinai School of
Medicine, Bekesi et a1. (4) reported decreased mitogen and mixed-
lymphocyte responses and alterations of lymphocyte membrane markers
in 60% of those tested. Since the tests were conducted four years
after the exposure, the results suggest an ongoing immunosuppression
or a failure to correct acute toxic injury to the immune system.
Fraker (16) found that exposure of mice to dietary levels as low

as 10 ppm for 30 days reduces humoral immune response to about

1/3 that of normal mice. There was no apparent effect on delayed
hypersensitivity. In contrast, Kately and Bazzell (24) found no

long-term effects on the immune system of cattle with body burdens

ll
of up to 30 ppm PBB in the body fat. They reported no significant
changes in mitogen responses, immunoglobulin levels, surface markers
or in Vivo antibody production. In a collaborative study conducted
by the Michigan Department of Public Health and the University of
Michigan in 1977 (26), lymphocyte function was assessed in persons
with high and low blood serum concentrations of PBB compared to
persons with no detectable blood serum concentrations. As a group,
those with measurable blood serum levels of PBB did not have any
alterations of absolute T and B lymphocyte counts and lymphocyte
responses to mitogens were not depressed. However, 15% of these

individuals did have some depression of in vitro lymphocyte responses.

Assays of Immune Function

 

Following is a brief overview of the methods used to assess
the function of phagocytic cells, and the use of mitogen stimulation
of lymphocytes to monitor their ability to respond to antigenic

stimulation.

Assessment of Phagocytic Cell Function

 

Due to early reports of increased numbers of infections and
slow-healing skin lesions in farm animals consuming PBB-contaminated
feeds, there was an interest in determining the effect of exposure
to PBB upon phagocytic cell function. Many in vitro systems have
been developed to assess phagocytic cell activities, such as
motility, adherence, ingestion, and the destruction of ingested
material. Abnormalities may occur in any of these processes. The
Boyden chamber has been widely used to assess in vitro response of
neutrophils to chemotactic factors. It is known that complement and

antibody play important roles in phagocyte adherence and, thus, the

12
presence of complement and antibody Fc receptors on neutrophils
has been related to normal phagocytic adherence. Measurements
used to assess ingestion include direct counting of particles,
estimation of cell-bound radioactivity after ingestion of radio-
labeled particles, and the spectrophotometric measurement of
ingested lipid particles (50,52).

The final stage of phagocytosis, the destruction of the
engulfed material, is dependent upon normal motility, recognition
and ingestion, as well as several intracellular metabolic systems.
The nitroblue tetrazolium dye reduction test and chemiluminescence
monitor metabolic processes presumably involved in this final
stage of phagocytosis, whereas the bactericidal assay directly
measures intracellular killing of engulfed organisms. By varying
the test organisms, the bactericidal assay can be used to assess
a variety of bactericidal activities. The assay may be done with
isolated neutrophils, but this approach ignores several physiologic
factors that may contribute to normal in vivo phagocyte function
(8,39). The use of whole blood allows the investigation of phago—
cytic function in the presence of natural opsonins and takes into
account the importance of the interaction of neutrophils with other
leukocytes and blood components. By maintaining the phagocyte/
bacteria ratio in the range of those of naturally occurring
bacteremias, a closer approximatly to an in vivo system is achieved.
The smaller volume of blood required allows the use of the assay
for small animals and for repeated measures.

Keusch et a1. (28) developed a simplified whole blood bacteri-
cidal assay using serum resistant strains of Escherichia coli and

Staphylococcus aureus. Designed to help in the diagnosis of chronic

l3
granulomatous disease, the assay is also sensitive enough to
detect the non-symptomatic heterozygote carrier. Thus, this assay
seems appropriate to investigate any PBB-induced toxic changes in
phagocytes severe enough to contribute to the reported increase

in incidence of infections.

Assessment of Lymphocyte Response to Mitogens

 

Numerous environmental contaminants, from pesticides to heavy
metals, have been reported to have deleterious effects on the
immune system (56). The mechanism of immunosuppression of these
toxic chemicals is unknown, but in most cases it appears to be a
functional alteration of the cells involved in the immune process.
The toxic effects may be principally in the humoral or in the cell
mediated immune functions, or in both systems. The mode of action
may be direct alteration of the lymphocytes or an indirect effect
through adrenal glucocorticoids or changes in metabolic patterns.

Stimulation of isolated lymphocytes with antigens or non-
specific activators is an in vitro technique used to assess cellular
immune function. It measures the functional capability of the
lymphocytes to respond to an antigenic stimulus by undergoing
blastogenesis and proliferation. A number of plant lectins and
other substances have been used as non-specific stimulants to
lymphocytes, requiring no previous sensitization of the cells to
the antigen. There is evidence that B and T lymphocytes of some
species are somewhat selectively stimulated by these mitogens (51).
Phytohemagglutinin (PHA), a plant mitogen made from the kidney bean
Phaseolus vulgaris, appears to be predominantly a T cell mitogen,

only secondarily stimulating B cells. Pokeweed mitogen (PWM), made

14
from Phytolacca americana, may activate principally the B cells
of some species, while apparently stimulating both E and T cells
in other species. Lipopolysaccharides (LPS) have been reported to
be B cell mitogens in some species. Due to lymphocyte subpopula-
tion interactions, this apparent selectivity of activation by
mitogens is by no means complete, and there is considerable species
variation.

Although responses and conditions for culture vary, plant
mitogens have been widely used to assess cellular immune response
in many species. Symons et al. (54) reported successful stimula-
tion of porcine peripheral blood lymphocytes with PHA, PWM, LPS,
and anti-immunoglobulin serum. However, they found considerable
individual variability in magnitude of response to these mitogens
even with optimal assay conditions. They did not attempt to
identify subpopulations responding to the different mitogens.

Blastogenesis and proliferation of lymphocytes following
mitogen stimulation is most often assessed by monitoring DNA
synthesis. DNA synthesis begins within 24 hours after PHA exposure
in human lymphocytes and reaches a peak between 48 and 72 hours
(41). The incorporation of tritiated thymidine into the culture
media allows the quantitation of DNA synthesis in the cells by
liquid scintillation. Although this assay must be rigidly stan-
dardized for cell density, mitogen concentration, and incubation
conditions, it is more reliable and less laborious than the morpho-
logical determination of blastogenesis. Mitogen stimulation of
peripheral blood lymphocytes provides a sensitive and practical
technique to monitor functional capability of the lymphocytes to

respond to antigenic stimulation.

MATERIALS AND METHODS

Experimental Design

 

Sows were fed 2.5 kg/day of a standard ration containing 0,
100, or 200 ppm Firemaster BP-6 (Michigan Chemical Company) during
the last half of gestation and the following 4 weeks of lactation
(a total of 12 weeks). These dietary concentrations of PBB are
equivalent to approximately 1.25 and 2.5 mg/kg body weight per day.
Firemaster BP-6 was pulverized and mixed with a ground standard
Michigan State University swine ration. Two sows were fed a ration
containing no PBB, 3 were fed a ration containing 100 ppm PBB,
and 2 were fed a ration containing 200 ppm PBB.

At parturition, one-third of each litter was randomly selected
and necropsied immediately (colostrum deprived). The remainder
of the litter and the dam were necropsied at 4 weeks postpartum.
Blood and tissue samples were taken at that time for evaluation
of various parameters. A total of 21 piglets from the control
sows, 24 piglets from the sows fed 100 ppm, and 15 piglets from

the sows fed 200 ppm were evaluated.

Mitogen Stimulation of Lymphocytes

 

At necropsy of piglets and sows, blood was collected into
preservative-free heparin (10 units/ml, Sherwood Medical Industries)
by cardiac puncture of piglets and from the jugular vein of sows.

Fifteen milliliters of blood was mixed with an equal volume of

15

l6
phosphate buffered saline (PBS) and layered onto 10 ml ficoll/
hypaque (24 parts of 9% ficoll, Sigma Chemical Co., to 10 parts
of 33.9% hypaque, Winthrop Laboratories) in a 50 ml plastic centri-
fuge tube (Corning) and centrifuged for 40 minutes at 400 x g
at 18 to 20 C. The mononuclear cell layer was removed to a sterile
plastic tube and washed 3 times with 10 m1 sterile PBS, centri-
fuging for 10 minutes at 150 x g at 18 to 20 C. Washed cells were
suspended in 2 ml of cell culture media (RPMI 1640, Flow Labora-
tories) containing 10% fetal calf sera, 10,000 Units penicillin,
10,000 pg streptomycin, and 200 mM glutamine (Grand Island
Biological Company). A hemacytometer was used to count the cells
and the cell suspensions were diluted as necessary in RPMI to
obtain a final concentration of l to 2 x 106 cells/ml. Viability
was assessed by trypan blue exclusion and ranged from 90 to 99%.
Cell suspensions were greater than 98% mononuclear cells.

One milliliter of the standardized cell suspension was
pipetted into each of 12 sterile plastic tubes with Morton
closures (Falcon Plastics). The first 3 tubes were used as
unstimulated control cultures. To each 3 of the remaining tubes,
0.1 m1 of the appropriate mitogen was added: phytohemagglutinin,
25 ug/ml (Burroughs Wellcome), pokeweed mitogen, final dilution
of 1:40 (Grand Island Biological Company), and Escherichia coli
lipopolysaccharide, 10 ug/ml (Difco Laboratories). Tubes were
loosely capped and incubated at 35 to 37 C in 5% CO2 for 60 hours.
One uCi of [3H]thymidine in aqueous solution (Sp Act 6.7 Ci/mol,
New England Nuclear) was added to each culture tube, and the tubes

were incubated for an additional 12 hours.

l7

Cultures were terminated by suspension in cold PBS and resedi-
mentation by centrifugation at 400 x g for 10 minutes. After 2
washes with PBS, 1 drop of 2% bovine serum albumin and 1 m1 of
6% trichloroacetic acid (TCA) were added to each tube. After
centrifugation at 1000 x g for 2 minutes, and removal of the
supernatant, the precipitate was resolubilized in 1N NaOH. Pre-
cipitation and solubilization were repeated twice more, and the
final precipitate dissolved in 0.2 ml tissue solubilizer (Unisol,
IsoLab Inc.). One milliliter of glass distilled absolute methanol
was added to each tube and the contents mixed and transferred to
a scintillation vial containing 10 ml scintillation fluid (Complement,
IsoLab Inc.). Liquid scintillation counting was done with a
Beckman LS-230 counter. Data were expressed as mean counts per
minute of the 3 replicate cultures i the standard error of the
mean. Counts were normalized by subtracting the counts per minute

of control cultures from those of stimulated cultures.

Bactericidal Assay

 

Blood samples were collected into heparinized syringes from
the ear vein of the sows and the superior vena cava of the piglets.
Samples were obtained from the sows after 4, 10, and 12 weeks of
feeding the experimental diets, and from the piglets at 2 and 4
weeks of age. Total and differential white blood cell counts were
performed by standard methods.

Whole blood was assayed for bactericidal activity according
to the method of Keusch et a1. (28). Escherichia coli 286 and
Staphylococcus aureus 986663 were determined to be resistant to

porcine serum bactericidal activity by standard viability studies.

18
For use in the bactericidal assay, bacteria were grown overnight
in trypticase soy broth, sedimented by centrifugation at 100 x g,
washed 3 times in PBS, and resuspended in PBS to an optical density
of 0.6 at 620 nm (PE Coleman 44). This standardized suspension
was diluted 1:5000 in PBS, resulting in a concentration of
approximately 105 organisms/ml.

For the assay, 0.1 m1 of the diluted bacterial suspension
was added to 0.9 ml heparinized whole blood in a capped sterile
glass tube. After thorough mixing, a 0.1 ml (zero-time) sample
was removed and added to 9.9 ml sterile distilled water. After
agitation on a vortex mixer, aliquots were added to molten trypti—
case soy agar, mixed, and poured into petri dishes, yielding
dilutions of 10.2 and 10-3. Plates were incubated 18 to 24 hours
at 37 C, and then colonies were counted to determine the number of
viable bacteria.

After removal of the zero-time sample, the blood/bacteria
mixture was immediately incubated on a tilting aliquot mixer
(Lab—Tek) at 37 C. After 1 and 2 hours of incubation, samples
were removed and pour plates prepared as described above. At the
2-hour sampling, a 10-1 dilution was also prepared.

As a bacterial viability control, a second tube with whole
blood and bacteria was prepared and polyanethol sulfonate added at
a final concentration of 0.25% to inhibit phagocytosis and intra-
cellular killing. One control for Staphylococcus and one for

E. coli was prepared each time the assay was performed.

19

Cholesterol

 

Blood samples were taken for cholesterol assay at necropsy of
sows and piglets. Serum was separated from the cells as soon as
possible. Kinetic assays were performed on the Gemani Mini Centrifugal
Analyzer (Electron Nucleonics Inc.) at the laboratory of the Michigan

State University Veterinary Clinical Center.

Statistical Evaluation
Data are presented as means i standard error of the mean (SEM).
Statistical evaluation of data by analysis of variance, followed
by comparison of means by Duncan multiple range tests, was performed
by computer using the Statistical Package for Social Science (SPSS,
Northwestern University). Differences were considered significant

when at the level p<0.05.

RESULTS

Mitogen Stimulation of Lymphocytes

 

Results of the stimulation of peripheral blood lymphocytes
with mitogens are recorded in Table l. The responses to pokeweed
mitogen (PWM) and phytohemagglutinin (PHA) of the lymphocytes of
sows fed PBB for 12 weeks were significantly decreased (p<0.05).
Lymphocytes from sows fed 200 ppm had the greatest decrease in
mitogen response. Lymphocyte stimulation by E. coli lipopolysac-
charide was minimal, and there was no significant difference in
lymphocyte response between sows fed PBB and those fed no PBB.
Total leukocyte counts and absolute numbers of lymphocytes (Table
2) were similar in all sows and all were within normal ranges.

Peripheral blood lymphocyte responses from piglets of all
sows to PWM and PHA were similar at birth. However, after 4 weeks
of lactation, lymphocyte responses to PWM in piglets of sows fed
PBB were significantly decreased (p<0.002). No significant dif-
ferences in response to PHA were detected. Lymphocyte responses
to PWM were the most depressed in piglets of sows fed 200 ppm PBB.
No lymphocyte stimulation by E. coli lipopolysaccharide was observed

with any of the piglets at either birth or at 4 weeks of age.

Bactericidal Assay

 

There was no statistically significant difference between the

bactericidal activity of blood from sows fed PBB and those not fed

20

21

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mo~.umo
ounw
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Uoom.o H ooo.mom ooom H oom.n ooo.m H ooo.NmH arm A mmom m oom
muooo.mm H ooo.mmm Doom.H H oov.m ooo.m A ooo.mHm mmm H mmmh m 00H
ooo.o~ H ooo.mmm 00m.a H 00m.HH ooo.wm H ooo.mmm Hon H moms N o
:mmouflz pmozmx0m mUHMMLUOMm>HOQOQHQ cacflunammmesou>nm Mmucsoo m30m AEQQV
x poumHsEHumGD mo umnfisz cowumu cw mmm

mmucsoo omumasswum

i,

mumac Hmucmefluwmxm mcflpwom mo mxoms NH kumm w30m CA mewoOUHE ou noncommou mphoosmESA .H manme

. I cum
2mm + 2w

 

22

 

«mm H HmH.o Nmm H NHH.m mxomz v m OON

omH.H H ovN.o HHH.H H mmN.m mxmms 4 OH ooH

one H Nmm.m mom H mon.n mxmm3 v NH 0

mmv H Hoo.N mom H mmo.s cuonzm: m ooN

mmH H ONv.H mNm H ovo.m cuonsm: oH OOH

msH H mmv.H HHS H mom.o cuonzm: m o mumHmHm

mHH H HAN.6 oom H mNm.NH mxmmz NH N ooN

ohm H NAN.N on.H H mmm.mH mxmm3 NH m 00H

Ame H HON.A cem.N H oov.NH mxwm3 NH N o mzom
mAmEEV mmu>oonmfihq mHmEEV mouxooxswa Hmuoa mom Ho mHMEHcm Hemmv mBOm pom

comm :0 wEHB mo Hmnfisz COHMMH CM mmm

 

muwamwa Hflonu CH paw mmm pow mBOm CH mucsoo HHmo GOOHQ wuHSS .N mHnme

23

N00.umo

cum
Hmm Q

.muCDOU
pmumHCEHumCC mo CoHuomansm >2 prHHmEHOC muCCoo UmumHCEHum .zmm H Emu mm pmpnouwu muCCoo HHflm

 

 

 

O00H..mH H 000.HNN noom.HH H 000.>NN 00N H mHHH mxwms v 0 00N
Doom.MH H ooo.mmm noo¢.OH H ooocmvm Hum H mmmo mxom3 v m OOH
ooo.MH H ooo.Hom oom.m H ooo.mvm 0mm H Hmoo mme3 V NH 0

000.0 H 00N.mmm 00v.0 H 00N.HmN mmN H msoN cHonzmc 0 00N
con.h H oov.mvm ooo.m H mno.mvm omH H mmmH CHOQ30C v OOH
OOH.o H oon.va coo.m H oom.mmm mmH H ownH Cuonsz v o

ComouHS pmmbxom CHCHHDHoomeCouxzm mmuCDoo pmHMHCEHumCD 004 mumHmHm AEQQV

Mmucsoo pmumHSEHum mo Hmnfisz m3om OH pom

COHHMH CH mmm

mmm pom m30m mo mumeHm CH mom mo mxmoB v pCm CHHHQ um mamouHE ou mmmCommmH muxoonmE>A .m OHQMB

24
PBB at any of the sampling periods during feeding. In some cases
(Figure 2), data suggest a decreased bactericidal activity after
1 hour of incubation, especially in sows fed 100 ppm, but these
differences are not significant at the 5% level. Results of the
bactericidal assay at 3 periods during the feeding are given in
Table 1A in the Appendix.

Whole blood bactericidal activity of all piglets at 2 and 4
weeks of age was generally lower than that of the sows. There
were no significant differences between the bactericidal activity
of blood from piglets from sows fed PBB and that of piglets from

sows fed no PBB (Table 2A, Appendix).

Cholesterol

 

Serum cholesterol values for sows after 12 weeks of feeding
and for piglets at birth and at 4 weeks of age are given in Table
4. Serum cholesterol values for piglets were quite variable, even
within the same litter. Although values were slightly lower in
the sows fed 100 ppm and slightly higher in the newborns of sows

fed 200 ppm, there was no significant overall treatment effect.

 

 

25

 

 

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3.1.2:: 8:385 1er as: 8:885
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26

 

 

 

 

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mm Ivn H H mm mxmwz NH N 00N
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HHc\Emv HOHonmHOCU comm Co mEHB mo HmQECZ COHHMH CH mmm
mHonHm HHmCu CH UCm mmm pom m3om CH HOkummHOCo ECHmm .¢ mHnme

DISCUSSION

The effects of exposure to PBB on the immune system are
important in assessing the impact on health of environmental con-
tamination with these industrial chemicals. The immunosuppressive
activity of similar environmental chemicals and the frequent
health complaints among persons exposed to PBB have emphasized the
need to further investigate the possible immunotoxicity of PBB.
Immune suppression following ingestion of PBB has been reported
in rats and mice (16,32) and in man (4). The ingestion of PBB
apparently also alters some in vitro lymphocyte responses in swine.
Lymphocytes of sows fed PBB for 12 weeks had significantly decreased
responses to plant mitogens. This depression of in vitro cell-
mediated response is similar to that reported in rats and mice
exposed to PBB for a shorter period of time, and to that of some
individuals accidentally exposed to PBB in Michigan. However, normal
lymphocyte mitogen responses were reported in studies of environ-
mentally contaminated cattle and in studies of PBB exposed human
populations by other investigators (24,26).

The possibility of secondary exposure of animals to PBB by
passage of the chemicals through the placenta or breast milk is an
important concern. Several researchers have reported that PBB is
passed through the placenta and is accumulated in tissues of the
fetus (17,43). Because pigs are immunocompetent at birth (42),
toxicity to the thymus affecting the ontogeny of the immune system

27

28
is most important during fetal development. However, passage of
these lipid-soluble chemicals through the breast milk to the
suckling young is probably of even greater importance in secondary
exposure because the concentration of PBB in breast milk is much
higher than that of the blood. Although only the sows were fed
PBB, the alteration of lymphocyte responses to mitogens that was
observed in sows was also demonstrable in their suckling offspring.
Lymphocytes from newborn piglets of sows fed PBB had normal
responses to mitogens. However, lymphocytes from piglets that had
nursed these sows for 4 weeks had significantly decreased mitogen
responses, suggesting the importance of exposure to PBB through
consumption of breast milk in pigs.

Alteration of lipid metabolism has also been suggested as a
mechanism of immunosuppression. The correlation of altered lipid
metabolism and immunodepression in conditions such as obesity,
diabetes mellitus, aging, cancer and chemical toxicity suggests
a causal relationship (13). Increased cholesterol and low density
lipoproteins have been reported as effects of various toxic
chemicals and carcinogens (13,25), and it has been shown that these
lipids inhibit both lymphocyte responses to mitogens (10,15) and
the phagocytic activity of macrophages (12). Exposure to PCB
increases cholesterol, phospholipids, and neutral lipids in the
liver, and cholesterol in the serum (19,27). In contrast,
decreased blood cholesterol levels have been reported in PBB con-
taminated cattle and in monkeys experimentally exposed to PBB
(2,27). Sleight et al. (49) reported an elevation of serum
cholesterol and an alteration of serum lipoprotein electrophero-

grams in rats fed 100 ppm PBB for 60 days. In the present study,

29
no significant changes in serum cholesterol levels were observed
in the sows fed PBB or in their piglets.

Reported increased incidences of infections and skin lesions
in environmentally contaminated animals warranted an investigation
of the effects of PBB on phagocytic cell function. Few studies
have reported on the effects of toxic chemicals on phagocytic cells.
In animals exposed to organophosphates or carbamate, Street et a1.
(53) found decreased phagocytic activity in blood neutrophils and
in phagocytic cells of the reticuloendothelial system. No data
have been reported on the effects of PBB on phagocytic cell func-
tion. Results of the present investigation indicate that ingestion
of 100 to 200 ppm PBB for 12 weeks has little, if any, effect on
the bactericidal function of porcine neutrophils. As the assay
used is sensitive enough to detect the symptom-free carriers of
genetic defects of phagocytes (28), it seems unlikely that toxic
effects of PBB on phagocytes could contribute significantly to any
increase in infections after PBB exposure.

The mechanism of the functional alteration of lymphocytes is
unknown. Some investigators (32) have proposed an indirect effect
of PBB upon immune function through the stimulation of adrenal
glucocorticoid production. In many animals, enlargement of the
adrenal glands and increased levels of serum glucocorticoids are
responses to stress. Glucocorticoids slow release of cells from
lymphoid organs to the blood, inhibit lymphoid cell mitosis, and
can cause degenerative changes in lymphocytes resulting in lysis
(l3). Depletion of lymphocytes in lymphoid tissues has been
reported in chickens exposed to PBB (44), and Moorhead et a1. (38)

found thymic atrophy in cattle fed PBB. In rats fed up to 100 ppm

30
PBB for 60 days, Sleight et a1. (49) found no changes in adrenal
weight or morphology, and Fraker (16) reported that serum gluco-
corticoid levels in mice exposed to PBB were not elevated although
the mice were markedly immunosuppressed. In sows fed PBB and in
their piglets, there was no increase in adrenal/body weight ratio
(Table 3A, Appendix), and total lymphocyte counts were not depressed
(Table 2). Histopathological examination revealed no premature
thymic involution or cellular depletion of lymphoid tissues in
sows or piglets (57).

Further studies are needed to investigate the mechanism and
permanence of the alteration of lymphocyte responses by ingestion
of PBB. Changes in lipid metabolism including alterations in serum
lipoprotein fractions and lipid content of lymphocytes should be
studied. The effect of serum or serum lipid fractions from animals
ingesting PBB upon in vitro lymphocyte responses should also be
investigated. An assessment of the role of some nutritional
factors such as vitamin A, vitamin E, or iron might also be
warranted.

To more accurately assess the impact of the immunosuppressive
effect of PBB upon the health status of the human population
exposed to these chemicals, it will be necessary to determine if
this effect of acute toxicity is also present with ingestion of
lower concentrations of PBB over a long period of time. However,
the use of in vitro methods to evaluate low level immunotoxicity
is difficult because of the relative insensitivity of most of these
assays. Any long-term alteration of in vivo lymphocyte function
would have importance in two aspects of human health: 1) the

ability to resist infection and eliminate foreign materials from

31
the body, and 2) the efficacy of the immune surveillance system in
eliminating inappropriate or malignant clones of cells. Alterations
of the ability to resist infectious agents could be investigated

by using a known pathogen with an established LD , and specific

50
pathogen-free laboratory animals chronically exposed to low concen-
trations of PBB. The immune surveillance system is important in
controlling the development of malignant clones, and thus influences
the rate of cancer. It is also presumed to be important in
eliminating undesirable clones of lymphoid cells which could give
rise to autoimmune reactions. The response of PBB exposed animals
to certain tumor lines, or the use of allograft rejection, would

be helpful in evaluating the effect of PBB on this aspect of
immunocompetence. The use of these in vivo techniques would yield

valuable information needed to assess the long-term effects of

ingestion of PBB on human health status.

SUMMARY

The ingestion of polybrominated biphenyls apparently alters
in vitro lymphocyte responses in swine. This immunotoxic effect
can also be demonstrated in the young nourished by milk of dams
exposed to PBB. It is not known how permanent these alterations
are or if this effect of acute toxicity would also be present in
chronic, low-level exposure to these chemicals. Any long-term
in vivo alteration of the immune response to foreign substances
or a suppression of the immune surveillance system would have
significant consequences for human health and for the animal
industries.

Further studies are needed to investigate the mechanism and
permanence of the alteration of lymphocyte responses by PBB, and
to determine the effect of these industrial chemicals on the
immune surveillance system and the immune response to infectious

agents.

32

APPENDI X

33

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34

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0.N H 0.NH 0.0 H H.NH 0.0 H 0.0H 0 H 00 NH H HN m H 00 0x003 N .000
msooooonCmmum

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35

Table 3A. Ratio of adrenal weight (gm) to body weight (kg) of

sows fed PBB and of their piglets

 

 

PBB in ration
fed sows (ppm) Ratioa
Sows 0 0.09 i .01
100 0.09 i .00
200 0.05 i .00
Piglets
Newborn 0 0.24 i .03
100 0.20 i .02
200 0.18 i .03
4 weeks 0 0.12 i .01
100 0.12 i .01
200 0.11 i .01

 

a
Mean i SEM

36

2mm H C0020

 

 

 

 

 

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0 H N 00 0N 000 H 000.0 0x003 0 NH 0
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0 0 H 0N 00 0N0 H 000.0 :Honzmc 0H 00H
0 0 H 0N H0 HHO H 000.0 :Hon300 0 0 mumeHm
0 0 0 H0 00 000 H 0N0.NH 0x003 NH N 00N
H v 0 mm mm OHm.H H mmm.mH 0x003 NH m OOH
0 0 v 00 N0 000.N H 000.NH 0x003 NH N 0 0300
Ommm om ozoz 02>H 220 0490 0.0520 000 H0 mHmchm Heady
Aw Cmmzv HmHHCmuwmeo 0wu>ooxan H0009 mem C0 weHB m0 Hmnfisz 0300 00m
COHHMH
0H 000
0HOHOHQ HHmzu CH 0C0 mmm 00w 0300 CH 0HCCOU HHwo muHCB HmHHCwummeU 0C0 Hmuoe .dv 0Hnme

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VITA

VITA

The author was born in Higgins, Texas, on September 23, 1947.
She received her primary and secondary education at Shattuck,
Oklahoma. She continued her education at the University of Oklahoma
in Norman, Oklahoma. From 1969 to 1971 she worked in Peace Corps
programs in Bolivia and Nicaragua. Upon returning to the United
States, she completed her professional training at the University
of Oklahoma Health Sciences Center in Oklahoma City, Oklahoma.

She graduated from the University of Oklahoma in 1972 and was
certified with the Registry of Medical Technologists of the
American Society of Clinical Pathologists. Subsequently, she was
employed as a medical technologist at University Hospital in
Oklahoma City, Oklahoma, for two years.

In 1975, the author enrolled in the Clinical Laboratory
Science program at Michigan State University and accepted a posi-
tion as research technologist in the Department of Pathology at
the university.

The author was married to Donald S. Howard in 1968 and has

one daughter, Terri Lynn.

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