EPA-600/1-80-032
September 1980
PESTICIDE EFFECTS ON PRENATAL CARDIOVASCULAR PHYSIOLOGY
I. An Electrocardiographic Study of
Mi rex-Exposed Rat Fetuses and Newborns
II. An Analysis of the Causes of Perinatal Deaths
Induced by Prenatal Exposure to Mi rex
by
Casimer T. Grabowski
Department of Biology
University of Miami
Coral Gables, Florida 33124
EPA Grant No. R-8053010
Project Officer
Dr. Neil Chernoff
Experimental Biology Division
Health Effects Research Laboratory
Research Triangle Park, NC 27711
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S.ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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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 the use.
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FORWARD
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 establishment of sound regulatory policy. These regulations
serve to enhance the quality of our environment in 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 in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation, environ-
mental carcinogenesis and the toxicology of pesticides as well as other
chemical pollutants. The Laboratory participates in 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 in
use, conducts research on hazardous and toxic materials, and is primarily
responsible for providing the health basis for non-ionizing radiation
standards. Direct support to the regulatory function of the Agency is
provided in 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.
The report details research into the effects of Mirex, an insecticide,
on the development of the cardiovascular system of rodents. It details
changes in electrocardiogram patterns of fetuses whose mothers were exposed
to this insecticide during different periods of gestation.
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PREFACE
One of the great problems of contemporary Teratology is the develop-
ment of both criteria and tests for adequately evaluating developmental
toxicity. This document describes (1) the development of a new testing
procedure, electrocardiography applied to fetuses and newborns of small
laboratory animals; and (2) the application of this method to a study of
the developmental toxicity of the pesticide Mirex.
In Part I the results of testing 18-1/2 day rat fetuses are described.
It is shown that a high incidence of cardiovascular problems are present
even in healthy-appearing, Mirex exposed fetuses. In Part II this
technique is applied to the perinatal period. It is shown that the cardio-
vascular problems which originate during the last third of gestation
persist to parturition and help to explain the high incidence of perinatal
death induced by Mirex. The usefulness of the ECG test for both diagnostic
and analytical purposes is demonstrated bv these studies.
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TABLE OF CONTENTS
Page
Forward ---------------------------- 3
Preface ---------------------------- 4
Part I - An electrocardiographic study of cardiovascular
problems in Mirex-exposed rat fetuses -------- 6
Abstract ------------------------ 6
Introduction ---------------------- 7
Materials and Methods ------------------ 8
Results 12
Discussion ----------------------- 18
Literature Cited -------------------- 24
Part II - An analysis of the causes of perinatal deaths
Induced by Prenatal exposure to Mirex -------- 27
Abstract ------------------------ 27
Introduction ---------------------- 28
Materials and Methods ------------------ 28
Results ------------------------- 29
Discussion ----------------------- 42
Literature Cited -------------------- 45
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PART I
AN ELECTROCARDIOGRAPHIC STUDY OF CARDIOVASCULAR PROBLEMS
IN MIREX-EXPOSED RAT FETUSES
ABSTRACT
Sperm-positive rats were intubated with Mirex in oil (5 to 10 mg/kg)
on days 8-1/2 to 15-1/2. Controls were untreated or oil-fed. Testing was
done on dav 18-1/2. Fetuses were sequentially exposed and ECGs obtained
with the fetus attached to the placenta and uterus. Counterparts of
standard leads, I, II and II were used. Fetuses were weighed and examined
afterwards. Swollen fetuses were rated on a scale of 1 (slight edema under
chin) to 5 (3 mm edema across back). ECGs from 81 controls and 205 Mirex
fetuses were obtained. They were evaluated for rate of heart beat, regu-
larity of beat, PR intervals and other features. One control exhibited an
abnormality, a transitory period of premature atrial contractions. Mirex-
fed fetuses exhibited tachycardia, closely correlated with degree of edema.
The heart rate increased from 150/min in controls to 180 in slightly
swollen to 224 in swollen fetuses. Mean PR intervals increased with degree
of swelling and with dose. The frequency of first degree heart block was
also dose-related, ranging from 20% to 77%. Second degree heart blocks
were found in 8%, 3% showed arrhythmias, and one had atrial flutter/
fibrillation. These cardiovascular problems seem primarily related to the
Mirex-induced edema and demonstrate that fetal edema is not innocuous.
These data demonstrate the usefulness of fetal electrocardiography to
detect functional teratology.
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INTRODUCTION
The developmental toxicity of the pesticide Mirex (dodecachloro-
octahydro - 1,3,4,-metheno-2H-cyclobuta (c,d) pentalene)talso sold as a com-
ponent of Ferriamicide, has been the subject of several studies. Gaines and
Kimbrough ('70) reported a reduction in litter size in rats fed Mirex during
gestation, and cataracts and a lowered viability in surviving neonates.
Khera et al. ('76) found that feeding pregnant rats 1.5 to 12.5 mg/kg of
Mirex on days 6 to 15 of gestation produced fetal death, skeletal and visual
abnormalities, and fetal edema. Chernoff et al ('79) fed pregnant rats 5 to
38 mg/kg of Mirex on days 7 to 16 and found increased fetal mortality,
decreased weight, and a significant incidence of enlarged cerebral ventricles,
undescended testicles and skeletal abnormalities. They also found a dose-
related incidence of edematous fetuses, ranging from 6 to 75%.
Khera et al ('76) did not attribute any significance to the edema.
Chernoff et al ('79) commented that some of the severely edematous fetuses
were dead, but others had beating hearts and responded to tactile stimulation.
Embryonic and fetal edema is induced by many agents which are also terato-
genic in amphibia, birds, and several species of mammals including humans
(Giroud et al. *55; Grabowski, '70 and '77). Edema in the early stages of
development has been traced to several types of abnormal development in a
sequence of events termed the edema syndrome (Grabowski, '64, '70; Turbow,
'66; Jaffee, *74). However, the consequences of edema during fetal stages
are poorly understood. Mirex induces conspicuous edema in the later stages
of development, therefore it seemed a good agent to study the effects of
prolonged swelling on the fetus.
The initial phase of the Mirex study concentrated on the fetal heart
since hypoxia-induced edema in the 3 to 5 day chick embryo has been shown to
induce hypervolemia, hypertension, and changes in the rate of heart beat
(Grabowski and Schroeder '68; Grabowski et al '69). The primary techniques
used to study the heart was the fetal electrocardiogram. We have previously
shown that anatomical and functional cardiac disorders could be prenatally
detected with this method in rat fetuses exposed to Trypan blue (Grabowski
and Tunstall '77). Mirex-induced edema, in the present study, was found to
be closely associated with a variety of cardiovascular problems, especially a
high incidence of first and second degree heart blocks. The usefulness of
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the ECG method to supplement standard teratological techniques is demon-
strated .
MATERIALS AND METHODS
Long-Evans rats obtained from the Blue Spruce Farms, Altamont, N. Y.
were used. Animals were maintained at 24°C + 1°; 40 to 60% relative humidity;
and on a 12 hour daylight period. They were housed in polypropylene cages on
Sanicel bedding and fed Purina Lab Chow and water aid libitum. Virgin females
in estrus, as determined by vaginal smear, were housed overnight with males
and examined the following morning for the presence of sperm in the vagina.
Rats were considered 1/2 day pregnant on the morning they were found to be
sperm-positive.
The Mirex used was of commercial purity and was obtained from the Mis-
sissippi State Laboratory, Mississippi State University. It was dissolved in
peanut oil and fed to the rats by gastric intubation. Treated rats were
intubated daily with 5,6,7 or 10 mg/kg Mirex in 1/2 cc peanut oil. Initially
animals were fed on days 6-1/2 to 15-1/2 of pregnancy. Later this was re-
duced to days 8-1/2 to 15-1/2 because we found this was just as effective as
the longer treatment period. The doses for each rat were based on their
weights on the day before the feeding regimen was started. Controls were
given either no treatment or 1/2 cc plain peanut oil on days 8-1/2 to 15-1/2.
Daily records of maternal weight and food and water intake were kept. All
testing was done on day 18-1/2 of pregnancy.
Some preliminary results of these ECG studies have been published
(Grabowski and Tunstall, '77; Grabowski, '78, '79), but since this is the
first full report, the equipment and procedures are described in detail. The
basic unit is a Tektronix 122 preamplifier powered with a Tektronix 125 Power
Supply. The voltage gain on the preamplifier was set at lOOOx; the high fre-
quency response set at 50 cycles and low frequency response at 80 cycles.
The electrodes consisted of about 15mm of .007 inch pure silver wire soldered
to the two leads of a miniature shielded cable (fig. 1). The terminal por-
tion of the shielding was twisted into a ground, to which another piece of
silver wire was also soldered. The output of the preamplifier was monitored
on an oscilloscope for convenience, but all measurements were made on
permanent records obtained on a Brush 220 recorder. One mm of deviation on
this instrument is equivalent to 1 mv at full sensitivity, but it was usually
used at half of this value. The recorder delivers the chart at speeds of 1,
5, 25 and 125 mm/sec with an accuracy of + 0.25%. The preamplifier, rat and
fetuses were kept in a galvanic cage which was kept warm (36°C) with an
infra-red lamp placed overhead.
Various combinations of leads were tried, but the only ones routinely
used in this particular study were comparable to leads I, II, III of conven-
tional human electrocardiography. These are, respectively, shoulder to
shoulder, right shoulder to left thigh, and left shoulder to left thigh (fig.
2). Electrodes were inserted into muscle to a depth of about 1 mm. The
ground wire was inserted into the perineal area.
On day 18-1/2 of pregnancy the rats were anesthetized with an IP injec-
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4^ J
Fig. 1. An 18-1/2 day rat fetus
In position for ECG record-
ing. The electrodes are in
position I. Note the umbili-
cal cord (arrow) extending
to the placenta which is
still attached to the uterus,
W
5 mm
Fig. 2. Scale drawing of rat
fetus of 18-1/2 days
shown with drawing of the
heart superimposed on the
chest wall. The place-
ment of the electrodes for
positions I, II and II
are indicated.
Abbreviations
G, ground electrode
placement
U, umbilicus
Squares, positive electrode
placement
Circles, negative electrode
placement
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jection of sodium pentobarbital, 30 rag/kg. Laparotomies were started about 15
minutes later. All further procedures were timed beginning with the first
incision. The fetuses were removed one at a time and placed into a shaped
holder attached to a micromanipulator (fig. 1). Placental attachment to the
uterus was maintained and the umbilical cord arranged with minimal possible
tension. This arrangement eliminates electrical noise due to maternal muscle
movements. The cable, attached to another micromanipulator, was positioned
over the fetus and the electrodes inserted. The chart was first run at a
speed of 5 mm/sec to check the rate of heart beat. Recordings from leads I,
II and III were successively made at 25 and 125 mm/sec, and then the rate of
heart beat was rechecked at 5 mm/sec. The time required to prepare each fetus
was 1 to 2-1/2 minutes. Tracings were taken for a period of about 1-1/2
minutes, sometimes longer if a problem was detected. It took about 20 to 30
minutes to check a full litter. Occasionally fetuses were rechecked in
between litter mates or at the end of the session. Data from these were not
used for the quantitative summaries because the rate of heart beat slows down
considerably after the fetus is detached from the uterus. All quantitative
measurements were made from lead II tracings. This study is primarily based
on an analysis of ECGs from 81 control fetuses and 205 Mirex-fed fetuses.
All fetuses were weighed at the conclusion of the recording session.
They were then placed in a dish of saline and examined under a stereoscopic
microscope for the presence of visible abnormalities and edema. Several ways
to quantitate the edema were tried. Wet weights did not correlate well with
edema because treated fetuses sometimes exhibited a moderate degree of growth
retardation (see e.g. the wet weight data in Table 2). A good visual estimate
of subcutaneous swelling could be made by examining immersed fetuses because
the skin is very transparent at this stage. A rating system was adopted which
ranked fetuses on a scale of 1 to 5, defined as follows:
0 - no edema apparent;
1° - slight swelling, mostly under the chin;
2° - visible edema under chin, along belly and back;
3° - edema extends to head and upper arm, 1/2 to 1 mm of
edema visible across back;
4° - 1-1/2 to 2mm of edema across back;
5° - 2-1/2 to 3mm of edema across back.
Two operators independently rating the fetuses would agree in their estimates
to within 1/2 degree. Later in the study, this scale was correlated with
percent water determinations by taking a series of fetuses treated with from
5 to 7 mg of Mirex/kg body wt., rating them on the edema scale, obtaining wet
weights and then drying at 70°C to constant weight.
Thoracic dissections were performed under a stereomicroscope on about
half of the fetuses in both control and treated groups. Three fetuses, a
control, a normal-appearing treated and a swollen treated, were preserved in
Bodian's solution, sectioned at 15 u and stained with Ehrlich's hematoxylin
and Eosin. Significance was determined by the t-test.
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Fig. 3. A swollen 4° rat fetus, 18-1/2 days,
along with a normal appearing litter
mate on the left. Both came from a
mother who was fed 6 mg/kg Mirex per
day. The subcutaneous edema in this
photo is particularly evident across
the forehead, chin and neck and
abdominal wall.
Fig. 4. Cross section through the anterior
thoracic wall of an edematous fetus.
Note the loose, space-filled, subcu-
taneous connective tissue, even
though considerable shrinkage of this
region has taken place from the
histological treatment. Note also
that the edematous tissue extends
between some of the superficial
muscles.
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RESULTS
Lethal and Teratogenic Effects of Mirex
The death rate was only slightly above normal in the 5 and 6 rag/kg
groups but quite high in the 7 and 10 mg/kg group ( Table 1). Some treated
fetuses appeared undersized and retarded. Seven fetuses (out of 205) were
microphthalmia. No other externally visible abnormalities were noted.
Mirex-induced edema
The degree of edema observed in 18-1/2 day fetuses never reached the
extremes observed in near term fetuses exposed to Mirex, when some individuals
appear swollen to twice normal size (Khera et al. '76; Chernoff et al. '79).
For the most part the edema observed would be considered slight and could be
readily missed without careful examination under saline (fig. 3). About half
of the controls exhibited 1° edema, never more, but 85% of the treated fetuses
were appreciably edematous. Half of the treated and control fetuses were
dissected after ECG testing. There were no grossly discernible differences
in the size of the thorax, abdomen or viscera between normal and edematous
fetuses. The edema seemed largely confined to subcutaneous layers. This was
confirmed by examination of the serial sections. The edematous areas were
extensive areas of very loose connective tissue, sometimes extending into and
between the superficial musculature (fig. 4).
As the investigation progressed, it became apparent thfet even a slight
degree of edema could have a detectable physiological effect on the fetus.
It became desirable to correlate the edema scale with total water determina-
tions. The mean water content of 18-1/2 day controls was 89.09% (Table 2).
Edematous fetuses from Mirex-treated mothers showed regular increases to
89.77% water at level 2 edema, up to 91.17% at level 4. The increments are
not great, but this is not surprising since the controls are already 89%
water. The difference between controls and level 1 treated fetuses was not
significent, but that between controls and level 2, 3 and 4 fetuses was
(P <.001). Similarly the difference between treated fetuses of levels 2 and
3 was also significant (P <.001) as was the difference between treated fetuses
of levels 3 and 4. In effect, the water content data of Table 2 represent a
calibration of the edema scale.
Characteristics of the ECG of normal rat fetuses
The ECG of the 18-1/2 day rat fetus is mature and clearly shows P, QRS,
and T components. The tracings look somewhat different than those of the
human adult, presumably because of the small size of the heart and the fact
that the electrodes are placed very close to it (fig. 2). The QRS interval
in adult humans is about 0.08 seconds versus 0.02 sec. in the rat fetus.
Consequently the individual components cannot always be easily visualized at
a chart speed of 25 mm/sec, which is the standard clinical rate. The record
for every fetus contains segments run at 3 different speeds, 5, 25 and 125mm/
sec. Records obtained at the 5 mm/sec rate are convenient for measuring rate
of heart beat and visualizing arrhythmias (fig. 7a). Those at the 25 mm/sec
rate are good for visualizing the T wave and for detecting some abnormalities
(fig. 7b). Records obtained at the 125 mm/sec rate are the only ones in which
the P and QRS complexes are sufficiently spread out to be examined with
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Table 1
Lethal and Teratogenic Effects of Mirex Treatment
Number Maternal Number Living
Treatment attempted deaths litters fetuses Resorptions Microphthalmos
None
Oil only
MIREX-5mg/kg
on days 6-1/2 to 15-1/2
on days 8-1/2 to 15-1/2
MIREX-6mg/kg
on days 8-1/2 to 15-1/2
MIREX-7mg/kg
on days 6-1/2 to 15-1/2
on days 8-1/2 to 15-1/2
MIREX-lOmg/kg
on days 6-1/2 to 15-1/2
3
5
10
11
10
0
0
0
0
2
8
34
47
23
70
60
0
49
13
80
33
48
0
3
0
0
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Table 2
Wet Weights and Water Content of 18-1/2 Day Fetuses
Wet Weight
% h2o
No.
Mean +
SEM
Mean + SEM
Controls
23
1.320g
+ .015
89.09 + .096
Treated*,
1°
Edema
3
1 • 373g
+ .065
89.53 + .353
Treated,
2°
Edema
39
1.242
+ .034
89.77**+ .087
Treated,
3°
Edema
31
1.278
+ .033
90.52**+ .108
Treated,
4°
Edema
30
1 .357
+ .030
91.17**+ .088
* Treated with Mirex, 5 to 7 mg/kg/day on days 8-1/2 to 15-1/2 of
gestat ion.
** Significantly higher than controls (P<.001). The increments between
2° - 3° and 3° - 4° were also significant (P<.001).
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clarity (fig. 7c).
The rate of heart beat was obtained from the ECG records taken as soon
as possible after the fetus was exposed and at the conclusion of the record-
ing session. The heart rate usually starts to slow down gradually after fetal
exposure, but sometimes remains constant or even speeds up slightly. The
before and after measurement of heart rate almost always agreed within + 10%
The mean rate of heart beat of 18-1/2 day fetuses (untreated controls) was
149 + 4.95 (SEM) beats per minute.
The ECGs obtained from 81 control fetuses of this study were normal
with but a single exception. One untreated control fetus developed a tran-
sient episode of premature atrial contractions, most of which were blocked.
The abnormality started 30 seconds after recording was started. Two minutes
later the beat returned to normal. This fetus was rechecked 10, 20 and 40
minutes after being removed from the mother. The rate of beat was reduced at
these times, but the beat was regular and ECG normal. This particular
abnormality was not found in any treated fetus of this series. It was also
the only abnormality found in over 300 ECGs of control fetuses taken in this
and other experimental series.
ECGs of Mirex-treated fetuses
Edema and the rate of heart beat. The heart rate in treated fetuses
was more rapid than normal and this increase was related to the degree of
visible edema (fig. 5). The mean rate in both treated and untreated controls
is 150 beats/min. It jumps to 181 beats/min. in the slightly swollen (2°)
and 224 beats/min. in the grossly swollen (4°) treated fetuses. There was a
drop in mean rate of heart beat in severely swollen fetuses (5°) due to a
very low rate (down to 144 beats/min.) in a few individuals. All the
differences in the rates of heart beat between fetuses with 1° edema or
higher and treated controls were significant (P <.001), but that between
treated fetuses without visible edema and controls was not.
First degree heart block in treated fetuses. The PR interval in normal
humans varies somewhat with age and rate of heart beat. The range for
newborns is 0.07 to 0.14 seconds with an average of 0.11 sec (Schaffer and
Avery, '77). The range for adults is 0.14 to 0.18 seconds (Litman, '72). A
prolongation of the PR intervals beyond 0.20 seconds in adults indicates a
delay in impulse conduction from the sinus nodes to the ventricles and is
called a 1° heart block.
The mean PR interval in untreated control rat fetuses was .069 + .008
(SD)seconds and in oil-fed controls was .065 + .006 seconds. The difference
between the two is probably due to subtle improvements in technique as the
study progressed. The mean PR interval of all controls was .067 + .007 sec.,
about 8 mm on the records taken at a chart speed of 125 mm/sec (fig. 7c). A
1° heart block in 18-1/2 day rat fetuses was defined as any PR interval
greater than the mean interval in controls (.067 sec) plus 3 standard
deviation intervals (.021 sec), i.e. 0.088 seconds. This is a convenient
interval to measure because it is 11 mm on the 125 mm/sec records.
The PR interval in Mirex-treated fetuses was considerably more variable
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11«
.10
.09
z .08
07
.06.
IUTC
Itc
T
0
I
1
T
2
I
5
DEGREE OF EDEMA
Fig. 6. Mean PR intervals (+ SEM) in the ECG's of Mirex-treated fetuses
with different degrees of edema.
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225.
200.
Z
i
\
CO
h-
<•
LU
CD
uj 175i
K
<
a:
cc
<
150.
125
o
i
T
2
DEGREE OF
3 4
EDEMA
Fig. 5. The rate of heart beat (mean + SEM) in Mirex-treated fetuses with
different degrees of edema.
Abbreviations
TC, Treated controls
UTC, Untreated controls
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than that of controls. Though in many of them the PR interval fell within
the normal range, in others it was prolonged (figs. 8 and 9), sometimes up to
3-1/2 times normal. First degree heart blocks, using the criterion defined
above, were common and related to both dose and degree of edema. The
frequency of 1° heart block ranged from 20% in the 5 mg/kg group to 77% in the
10 mg/kg group (Table 3). An increase in mean PR interval was also dose-
related (Table 3). There was a proportional relationship between PR
intervals and the degree of edema present in the fetuses (fig. 6). The
differences between the mean PR intervals of the ECGs of all edematous fetuses
(1° through 5°) were significantly greater (P <.001) than those of oil fed
controls.
Second degree heart blocks and other abnormalities. Some of the 1°
heart blocks apparently led to a 2° block, in which a P wave is not followed
by a QRS complex. This indicates that an atrial contraction has not been
followed by a ventricular contraction. Such cardiac pathology occurred in
from 4 to 10% of the groups of Mirex-treated fetuses. All of the cases of 2°
heart blocks were Type I (Wenckebach), in which the missing beat is preceded
by progressively longer PR intervals (fig. 9). Fetuses with 2° heart blocks
were often rechecked for periods of up to 20 minutes after being removed from
the uterus. The irregularities inevitably persisted.
A single case of atrial flutter or fibrillation was found in one of the
8 severely swollen (5°) fetuses (fig. 10). Several kinds of arrhythmias,
presumably pacemaker problems, were found in the ECGs of 5 individuals
scattered through the dosage groups (figs. 11a and b).
Morphological Observations. No gross differences were apparent in the
size and proportion of the thoracic cavity in the dissected fetuses of
treated and control groups. There were no grossly visible differences in the
appearance of the heart. The serial sections through the control and two
treated fetuses did not show any obvious differences between their hearts.
DISCUSSION
Mlrex toxicity
The developmental toxicity of Mirex fed to pregnant rats has been well
documented (Gaines and Kimbrough, '70; Khera et al. '76; and Chernoff et al.
*79). Lowered pre- and postnatal viability, prenatal edema as well as a few
anatomical abnormalities have been reported. Mirex-induced microphthalmos
(4% in this study) had not been observed before. The major new observations
of this study have been (1) a description of the Mirex-induced cardiovascular
changes; and (2) a quantitation of the occurrence of edema. The scale of
edema ratings, which fortuitously was adopted early in the study, eventually
made feasible a number of correlations between edema and other problems. The
maximum amount of edema which we observed in 18-1/2 day fetuses was never as
extreme as that shown by Chernoff et al. ('79). This is due to the fact that
the latter made observations on the 20th day of gestation and affected
fetuses apparently continue to swell throughout gestation. We have noted a
much higher incidence of edematous fetuses than either Khera et al. ('76) or
Chernoff et al. ('79) even though our dosage was in the moderate range. This
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Table 3
PR
intervals
and heart block in Mirex-fed rat fetuses
Number
Mean PR interval
in seconds + SEM 1 heart block
2° heart block
Untreated
Controls
34
.069 + .0014
Treated Controls
47
.065 + .0009
5 mg Mirex
93
.079 + .0019 20%
4%
6 mg Mirex
60
.091 + .0023 50%
10%
7 mg Mirex
49
.086 + .0036 27%
4%
10 mg Mirex 13 .110 + .0069 77% 8%
-------
rr'rr
' r
V--1
r\.-
PR
r x-
J
QRS T
A
Jf
¦4~
— PR-
_V—
1(1
PR-
r-v.
j.
-PR"—
I....
v~
r
Fig. 7. Electrocardiogram of a normal 18-1/2 day rat fetus, taken at chart
speeds of (a) 5 mm/sec; (b) 25 mm/sec; and (c) 125 mm/sec. The rate
of heart beat was 172/min. The P, QRS, and T components are labeled In
7c, along with the PR interval (.064 sec). Actual size.
Fig. 8. Electrocardiogram of a Mirex-treated fetus (5mg/kg per day) showing
a pronounced 1° heart block. Note the extremely long PR interval
(0.16 sec). Segments of the 5, 25 and 125mm/sec record are shown.
The waviness in the base line is due to drift in the amplifier. Edema
2°. Heart rate 168/min.
Fig. 9. Electrocardiogram of a Mirex-treated fetus (6 mg/kg per day) show-
ing a 2° heart block. The missing QRS complexes are indicated by
arrows. Note that the two PR intervals, PR' and PR", are of different
duration. Edema 4°. Heart rate 210/min. Segments of the 5, 25, 125
mm/sec record are shown.
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Wto*
11a
~|r J, v~|r -—|r ¦-—I" Y
>v--—|f* V —
Fig. 10. Electrocardiogram of a Mirex-treated fetus (6 mg/kg) with either
atrial flutter or fibrillation. The P waves are occurring at a rate
of 36/second. Edema 5°. Heart rate 204/min. Segments of the 25 and
125mm/sec record are shown.
Fig. 11. Electrocardiograms of two treated fetuses with arrhythmia. (a) A
mild irregularity in a fetus exposed to 6 mg/kg/day. Note the varied
intervals between successive QRS waves. Edema 2°. Heart rate 228.
Chart speed 25 mm/sec. (b) A very irregular beat due to an
arrhythmia along with a 2° heart block in a fetus exposed to 10 mg/kg/
day. Edema 5°. Heart rate 120. Chart speed 5 mm/sec. These ECGs
illustrate the variety of conditions detectable by this method.
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probably reflects our emphasis on edema as well as method of observation.
In addition to the edema, several kinds of cardiovascular problems were
detected in Mirex-fed fetuses. Some of these, such as the arrhythmias,are
probably benign. Others, such as 2° heart block and atrial flutter/
fibrillation, are more serious and apparently are responsible for at least
some of the high incidence of perinatal mortality induced by Mirex treatment
(Grabowski, '79). These cardiovascular disturbances appear to be a signifi-
cant facet of Mirex toxicity in the prenatal period.
The physiological basis for the Mirex-induced cardiovascular changes.
The most prominent cardiovascular symptoms observed were tachycardia and
1° and 2° heart blocks. It is difficult to ascribe a possible cause to these
symptoms on the basis of human pathology. Among the many causes of tachy-
cardia which could possibly be operative in this situation are anoxia, hypo-
tension, and cardiac failure (Marriott and Myerburg, '70). There is no reason
to suspect the first two, but the latter is a possibility. Chernoff et al.
('79) observed enlarged cerebral ventricles in Mirex-fed fetuses, and this
could possibly result in increased intracranial pressure. However, increased
intracranial pressure leads to bradycardia in adults rather than tachycardia.
The list of agents which can induce heart blocks is also long and includes
vagal stimulation, anoxia, ischemic heart disease, and hyperkalemia (Marriott
and Myerburg, '70). Though rat fetuses are very susceptible to hyperkalemia,
especially after exposure to hypoxia, (Chernoff & Grabowski, '71, Grabowski,
'73), we have found only a slight elevation in serum potassium in Mirex-
treated fetuses (5.1 mEq/1 + 1.4 in 18-1/2 day controls; 6.2 + 1.6 in trea- v
ted). This would seem to eliminate either hyperkalemia or anoxia as possible
explanations of the observed data. Measurements of fetal blood pressure,
and blood volume will be necessary to decide between the remaining alter-
natives. Such studies are underway. The only clue available at the moment
is that the correlation between the degree of visible edema observed and
degree of tachycardia and prolonged PR intervals is too close to be totally
spurious.
Prenatal edema and Its consequences
Mirex was the primary subject of this investigation, but also, this was
a study of fetal edema and its physiological effects. Embryonic and fetal
edema can be induced by numerous agents in several groups of vertebrates as
well as by genetic factors. Edema in the embryonic stage can have lethal and
teratogenic effects due either to the distention of hollow organs during a
critical period of development or to the mechanical effects of persistent
hematomas near or within developing structures (Jost, '53; Giroud et al, '55;
Grabowski, '70 and '77, and Jaffee, *74).
The significance of edema produced in the fetal stages is, in contrast,
poorly understood and usually dismissed without comment. The present study
shows that fetal edema is associated with significant cardiovascular effects.
The enlarged cerebral ventricles in Mirex-fed fetuses (Chernoff et al. '79)
are probably another manifestation of fetal edema. Such enlargements could
affect the function of the central nervous system. Other hollow organs could
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be similarly affected. Posner and Darr ('70) have suggested that the edema
induced by the antihistamine, chlorcyclizine, is responsible for the high
incidence of cleft palate produced by this agent because the swelling of
facial tissues interferes with their morphogenetic movements.
Fetal edema in mammals is not uncommon. Some of the agents which induce
conspicuous edema include chlorcyclizine (King et al. *65); thalidomide
(Khera, *75); clamping of uterine blood vessels (Leist and Grauwiler, '74);
carbon monoxide and vasopressin (Grabowski, unpublished). It is occasionally
observed in human babies (Potter '61, Schaffer and Avery *77). Studies on the
physical and chemical properties of fetal blood are under way which may help
understand why mammalian fetuses are susceptible to this type of fluid distur-
bance .
The potential of fetal electrocardiography
in developmental toxicity studies
The domain of teratology has been enlarged in recent years to include
functional as well as anatomical disorders induced by various environmental
factors (Wilson '73). Suitable procedures for the systematic detection and
evaluation of functional disorders in experimental animals are not, as yet,
common. Fetal electrocardiography could become a relatively simple, standar-
dized procedure for detecting functional disorders of the cardiovascular
system. Numerous specific disorders can be detected and measurements of the
rate of heart beat, whether feeble or excessive, could give some indication
of the general state of health of the fetus. It can also be applied to new-
borns (Grabowski, *79). The ECG equipment required is neither expensive nor
esoteric. Ihe skill required is not beyond the reach of a good technician.
A litter can be tested in about half an hour, and the individuals could still
be subsequently used for necropsy or skeletal analysis. This method could be
a valuable adjunct to traditional procedures.
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LITERATURE CITED
Chernoff, N., and Grabowski, C. T., 1971 Physiological responses of the
rat fetus to maternal injections of epinephrine and vasopressin.
British J. Pharmacol., 4_3^ 270-278.
Chernoff, N., Linder, R. E., Scotti, T. M., Rogers, E. H. , Carver, B. D.,
and Kavlock, R. J. 1979 Fetotoxicity and cataractogenicity of Mirex
in rats and mice with notes on Kepone. Environmental Research, 18:
257-269.
Gaines, T. B., and Kimbrough, R. D. 1970 Oral toxicity of Mirex in adult
and suckling rats. Arch. Environ. Health, 2JL_: 7-14.
Giroud, A., Lefebvres, J., Prost, H. and Dupuis, R. 1955 Malformations
des membres due a des lesions vasculaires chez le foetus de rat
deficient en acide pantothenique. J. Embryol. Exp. Morphol., 3^:
1-12.
Grabowski, C. T. 1964 The etiology of hypoxia-induced malformations in
the chick embryo. J. Exp. Zool., 157; 307-326.
Grabowski, C. T. 1970 Embryonic oxygen deficiency — a physiological ap-
proach to analysis of teratological mechanisms. Adv. Teratol., 4_:
125-169.
1973 "etal cardiac physiology and hypoxia-induced
hyperkalemia. Teratol. 7_: A16,
1977 Altered electrolyte and fluid balance. Handbook
Teratol., 2: 153-170. eds. Wilson and Fraser.
1978 3CG analysis of effedts of Mirex on cardiovascular
physiology of rat fetuses. Teratol., 17: 34A.
1979 An analysis of the causes of perinatal deaths
induced by prenatal exposure to Mirex. Teratol., 27A.
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Grabowski, C. T. , and Schroeder, R. E. 1968 A time-lapse photographic
study of chick embryos exposed to teratogenic doses of hypoxia. J.
Embryol. exp. Morph., JL9_: 347-362.
Grabowski, C. T. , Tsai, E. N. C., and Toben, H. R. 1969 The effects of
teratogenic doses of hypoxia on the blood pressure of chick embryos.
Teratol., 2: 67-76.
Grabowski, C. T., and Tuistall, A. C. 1977 An electrocardiographic study
of rat fetuses treated with Trypan blue. Teratol., _L5_: 32A.
Jaffee, 0. C. 1974 The effects of moderate hypoxia and moderate hypoxia
plus hypercapnea oi cardiac development in chick embryos. Teratol.,
Kh 275-282.
Jost, A. 1953 La degenerescense des extremites du foetus de rat sous des
actions hormonales (acroblapsie experimentale) et la theorie des
bulles myelencephaliques de Bonnevie. Arch franc. Pediat., 10:
865-870.
Khera, K. S. 1975 Fetal cardiovascular and other defects induced by
Thalidomide in cats. Teratol., _11_: 65-72 .
Khera, K. S., Villeneuve, D. C., Terry, G., Panopio, L. , Nash, L., and
Trivett, G. 1976 Mirex: a teratogenicity, dominant lethal and
tissue distribution study in rats. Fd. Cosmet. Toxicol., _1^_: 25-29.
King, C. T. G., Weaver, S. A., and Narrod, S. A. 1965 Antihistamines and
teratogenicity in the rat. J. Pharmacol. Exp. Ther., 147: 391-398.
Leist, K. H., and Grauwiler, J., 1974 Fetal pathology in rats following
uterine-vessel clamping on day 14 of gestation. Teratol. _10: 55-67.
Litman, B. 1972 Electrocardiography, sixth edition, Yearbook Medical
Publishers, Inc., Chicago.
Marriott, H. J. L., and Myerburg, R. J. 1970 Recognition and Treatment
of Cardiac Arrhythmias and Conduction Disturbances. Ch. 32C in The
Heart, J. W. Hurst and R. B. Logue, eds. McGraw-Hill Book Co.,
New York, pp 489-5 36.
Potter, E. L. 1961 Pathology of the Fetus and Infant, second edition.
Year Book Medical Publishers, Inc., Chicago.
Posner, H. S., and Darr, A. 1970 Fetal edema from benzhydrylpiperazines
as a possible cause of oral-facial malformations in rats. Toxicol.
Appl. Pharmacol. 17: 67-75.
Schaffer, A. J., and Avery, M. E. 1977 Diseases of the Newborn, fourth
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edition. W. B. Saunders Company, Philadelphia.
Turbow, M. W. 1966 Trypan blue-induced teratogenesis of rat embryos
cultivated in vitro. J. Embryol. Exp. Morph. JJk 387-396.
Wilson, J. G. 1973 Environment and Birth Defects, Academic Press, New
York.
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PART II
AN ANALYSIS OF THE CAUSES OF PERINATAL DEATHS
INDUCED BY PRENATAL EXPOSURE TO MIREX
ABSTRACT
Electrocardiograms of normal and Mlrex exposed rats (6 mg/kg/day of
Insecticide on days 8 to 15 of gestation) were obtained on day 21-1/2 of
gestation, at birth and for several days afterwards. Data on 252 control
fetuses and newborns were compared with that on 219 treated fetuses. On
day 21-1/2, a high incidence of cardiovascular problems was found in
treated fetuses, e.g. 21% first degree heart blocks 2% second degree heart
blocks, and 6% third degree blocks, as well as a fair number (24%) of dead
and dying individuals. Df those that were allowed to be born, every attempt
was made to obtain ECGs within 5 min. of birth. Pups were then weighed and
tattooed so that individuals could be followed to day 5. Of the treated
pups, 27% were stillborn, 17% died within 6 hours of birth, another 15%
within 48 hours. At birth 20% had first degree heart blocks, and 4% had
second degree blocks. All those with second degree blocks died shortly
after birth. Some of the other deaths were also correlated with cardio-
vascular problems. Respiratory problems were also common and 4% died from
failure to breath. Cardiovascular and respiratory problems are responsible
for the majority of the high incidence of perinatal deaths induced by
prenatal exposure to a moderate dose of Mirex.
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INTRODUCTION
Mirex is not a potent teratogen in the classic sense, since even
high doses induce only a low incidence of visible malformations. However,
this pesticide does induce a very high rate of perinatal mortality (Gaines
and Kimbrough, 1970; Khera et al. 1976; Chernoff et al. 1979). Grabowski
and Payne (1980) found a high incidence of cardiovascular problems,
diagnosed by electrocardiogram, in 18-1/2 day fetuses exposed to Mirex. In
the present study, an attempt was made to determine if these problems
persisted into the perinatal period and to see if they help to explain the
deaths induced by exposure to a moderate dose of Mirex (6mg/kg). A
positive correlation was found.
MATERIAL AND METHODS
Long-Evans rats obtained from Blue Spruce Farms, Altamont, New York
were used. Animals were maintained at 24°C + 1°, 40-60% relative humidity
and on a 12 hour daylight period. They were housed in polypropylene cages
on Sanicel bedding and fed Purina Lab Chow and water ad libitum. Virgin
females in estrus, as determined by vaginal smear, were housed overnight
with males and examined the following morning for the presence of sperm in
the vagina. Rats were considered a half-day pregnant on the morning they
were found to be sperm-positive.
The Mirex used was of commercial purity and was obtained through the
Mississippi State Laboratory, Mississippi State University. It was
dissolved in peanut oil and fed to the rats by gastric intubation. Treated
rats were intubated daily with 6mg per kg Mirex on 1/2 cc peanut oil. This
treatment was given on days 8-1/2 and 15-1/2. The dosage for each rat was
based on their weight the day before the feeding regimen was started. Con-
trols were given either no treatment or 1/2 cc plain peanut oil on days
8-1/2 - 15-1/2. Daily records of weight and food intake were kept.
On day 21-1/2 of pregnancy, that is the morning before the pups were
supposed to be born, some control and treated rats were subjected to
laparotomy and pups tested as previously described (Part I ; Grabowski
and Payne, 1980).
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Every attempt was made to get an ECG record on pups as soon as pos-
sible after birth and also to get an accurate record of stillborns before
maternal cannibalism could possibly occur. This was accomplished by fre-
quently observing the mothers on day 21-1/2 until birth began. Occasion-
ally we would take a full term rat home at night and bring her back to the
laboratory after parturition had started. One or two hours might elapse
before the first pups were checked in those cases, but most of the time
the pups were checked within 5 min. of delivery.
Initially, we felt the only feasible way to get the electrocardio-
grams from active newborns was to anesthetize them with a small amount of
sodium pentathol injected subcutaneously. This automatically became a
terminal experiment because the mother would refuse to re-accept the
babies and would cannibalize them. The anesthetized pups, however, made it
possible to get quality records with all three leads, I, II, III. Midway
through the study we found that ECGs could be obtained from unanesthetized
pups by placing the electrodes across their backs while they were in a
prone position on their abdomens, making skin contact with the aid of
electrode gel, and working rapidly during brief periods of inactivity. The
records obtained in this manner are not as neat and clean as those from
anesthetized rats because the movement of the animal interferes, but they
are adequate for analysis. Only recordings from Lead II were attempted.
Electrocardiograms of the newborn were obtained shortly after the mother
had separated the placenta, which usually occurred with 2-5 minutes after
birth. The pups were weighed after the recordings were made, tattooed in
a standardized pattern on the hips, and then returned to the female after
the whole litter was checked. Electrocardiograms and weights were also
checked on days 1, 2 and 5. Weights were checked on days 15 and 30. The
tattoo marks lasted several days and before they were covered by fur, the
saddle markings became apparent. Sketches were made of these so that
throughout the observation period each specific individual could be identi-
fied.
This study is based on observations of 161 live newborns obtained
from control litters, 116 treated newborns (Table 1), and 128 fetuses at
day 21-1/2 (78 controls and 50 treated) (Table 6). Differences between
untreated and oil fed controls were negligible and hence all data from
controls were treated as a single group. Statistical comparisons were made
using the Student "t" test.
RESULTS
Newborns. The mean rate of heart beat in the 161 controls (both
groups) was 2 56 beats per minutes and the mean PR interval as obtained from
the ECG tracings was 0.061 seconds (Fig. 2; Table 2). Two types of
arrhythmia were found. In one type, the one or two beats associated with
the peak of a breathing affort were displaced, usually occurring somewhat
prematurely (Fig. la). These anomalous beats could be readily correlated
with breathing activity because respiratory activity shows up clearly on
the ECG record. In the other type of arrhythmia, the rate of heart beat would
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Table 1
Summary of Newborn Data
Untreated Oil-fed Mirex
Controls Controls Treated
Mothers
No. of litters attempted 10 6 24
Not pregnant 2 0 5
Died during treatment 0 0 4
No. of litters delivered 8 6 15
Newborns
No. Alive 86 75 116
No. Stillborn 3 0 32
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Table 2
Weighing
grams
Heart rate
beats/min
PR interval
seconds
Comparison of control and Mirex-treated newborns
during the first 5 postnatal days
Newborn
(Control) 5.71+.53
(Treated) 5.6+.63
Day 1
6.44+1.15
6.1+.69
(Control) 256.42+37 330+30
(Treated) 221+38 302+27
Day 2
7.34+.75
6.5+.78
359+25
341+26
(Control) .061+.0078 .053+.0048 .050+.0048
(Treated) .078+.0192 .054+.0032 .049+.0046
Day 5
10.57+1.19
10.7+1.88
345+49
342+19
.051+.0080
.047+.0014
Means + S.D. The number of newborns was 161 in the control groups (both
untreated and oil fed) and 116 in the treated. There were between 40 and
60 individuals in each of the day 1 to 5 groups.
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b
Figure 1. Arrhythmias found in newborn control rats. (a) Misplaced beats
with breathing efforts. Note also that breathing movements (arrows) are
clearly evident in the ECG tracing. (b) Changes in rate of heart
beat at 5 to 15 second intervals. The rates vary in this segment from
144 to 216 beats per min. Both segments taken at 5mm/sec chart rate.
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speed up or slow down somewhat for periods of 15 to 45 seconds (Fig. lb).
These arrhythmias occurred in about 15% of the control animals. Whenever
the neonates were rechecked, it was found that a spontaneous remission
would occur 15 to 40 minutes after birth.
Two untreated controls were obviously in distress when born. The
heartbeat was 40 beats per minute in one, and 27 and irregular in the other.
PR intervals of these individuals were, respectively, 0.16 sec and 0.27 sec,
easily classified as severe first degree heartblocks. The first died
within 30 minutes of birth and the other within 34 minutes of birth. Data
on these two individuals were not included with the other controls in the
calculations of mean heart rate and PR intervals, since one of the func-
tions of those particular calculations was to determine the norm of
healthy individuals. These two were obviously very unhealthy.
Changes in heart rate and ECGs during the first 5 days. The heart
rate and the ECG characteristics of controls become more stable within the
first 12 to 24 hours after birth. The mean heart rate in the controls in-
creased to 330 beats per min on the day after birth. The PR interval is
much shorter than that of newborns and the standard deviation is narrower.
All these features indicate a stabilization from the trauma of being born.
No deaths occurred and no abnormalities of ECG whatever were found in mem-
bers of this group. Neither heart rate nor PR intervals changed in any
appreciable fashion between days 1 to 5 (Table 2).
Electrocardiograms of Mirex-Treated Individuals
There were 24 females treated with Mirex but 5 turned out to be not
pregnant and 4 died during the treatment. A total of 148 individuals were
born to the remaining 15 mothers (Table 4). Thirty-two of these were
stillborn. All of them apparently died within less than a day before birth.
In some of these the heart could still be stimulated to contract by pinch-
ing it with tweezers, indicating a very recent death. The mean weight at
birth of the 116 born alive was just slightly lower than that of controls
(Table 2). The mean rate of heartbeat was 220 beats per min., significantly
lower than that of the controls. The mean of the PR intervals of the
treated was .078 sec., appreciably greater than that of controls. The
range of variation was also much greater. The criteria for first degree
heart block were calculated by the same method as that used for 18-1/2 day
fetuses (Grabowski and Payne 1980; Part 1 of this report). I.e. any indi-
vidual with a PR interval greater than 0.084 sec, which is the mean of the
healthy controls, 0.061 sec, plus 3 standard deviations of 0.0078 sec., was
considered to have a first degree heart block. This interval, 0.084 sec.,
was a convenient one to measure because it is 10.5 mm on the ECG tracing
taken at 125 mm/sec. None of the PR intervals of the controls (except for
the two dying newborns) exceeded this figure but 23 of the Mirex-treated
newborns did (Figs. 3 and 5; Table 3).
Seven newborns had second degree heart blocks (Fig. 4). None of
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Table 3
Cardiac Abnormalitiles in Mirex-treated Newborns at Birth
Conditions No. %
First degree heart block* 23** 20.0
Second degree heart block 4** 3.5
Premature atrial contractions 1** 0.9
* PR interval of .084 sec or more, i.e. mean of controls plus 3 standard
deviations.
** Out of total of 116 live, newborns.
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Figure 2. Electrocardiogram of a control newborn, 5 min. old. Rate of
heart beat, 228. Segments of the ECG were taken at (a) 5, (b) 25,
and (c) 125mm/sec.
4
w-,. .1 'I
Figure 3. ECG of a Mirex-treated newborn 4 min. old, showing a firse degree
heart block. HR, 162 beats/min. Segments taken at different speeds
as in Fig. 2.
Figure 4. ECG of a Mirex-treated newborn, 5 min. old, showing severe second
degree heart block. HR, 84-114 beats/min. Note the greatly prolonged
PR interval. The missing QRS complexes are indicated by arrows. The
block persisted until the pups died, 35 min. after birth. Segments
taken at different speeds as in Fig. 2.
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these seven recovered and all died within 60 minutes after birth. One
individual was born with premature atrial contractions which disappeared
within 30 minutes after birth. Four individuals had inverted QRS com-
plexes (Fig. 5). Two of these had a normal ECG by 15 minutes after birth,
but the other two died without recovery.
Observations on treated neonates for several days after birth. Just
as with the controls, the majority of newborns stabilized by day one and
the heart rates and PR intervals remained more or less constant through
day five. The ECG characteristics were very close to those of the control
group. The mean weights of the treated group were not significantly dif-
ferent than those of controls over the five-day period (Table 2).
Two individuals, both from the same litter, developed classic hydro-
cephaly after birth. This was first noted when they were two weeks old.
The condition progressively became more prominent and both were sacrificed
and autopsied on day 45. The cranium was greatly enlarged in both and the
cerebral cortex was reduced to a very thin layer, only a millimeter thick.
In keeping over 1,000 individuals to approximately 2 months of age, no
control individual ever displayed this condition. One rat, prenatally ex-
posed to carbon monoxide, developed a comparable case of hydrocephaly.
Causes of Postnatal Death
A major goal of this study was to determine the causes of the high
perinatal death rate so characteristic of Mirex treatment. A number of
correlations were possible in the postnatal period. In this section, only
the 65 treated newborns which were not anesthetized could be compared,
because the anesthetized pups were killed by their mothers.
Eleven individuals died within a few hours of birth (Table 4). Four
individuals never respired. This was evident from their ECG records as
well as by visual observations. These four were obviously doomed to die.
Two of these also had second degree heart blocks at birth, one had a first
degree heart block and the fourth had a normal ECG at birth, although all
developed progressively more severe heart blocks as the hypoxic stress
increased (Fig. 6). Three other newborns who did respire had second degree
heart blocks at birth (Fig. 4). None of these three gave any indication
of recovery from the blocks and all died within sixty minutes after birth.
One individual had a moderate first degree heart block and died later. One
individual had an umbilical hernia. It's ECG was normal and the heart beat
was regular and strong, but it was cannibalized by its mother a few hours
after birth. It is impossible to say whether it died of natural (i.e.
internal) causes or from maternal action. Two individuals died within
three hours after birth without apparent cause, at least their electro-
cardiograms and respiratory rates were perfectly normal at the time they
were checked (Table 5).
It is obvious that some of these premature deaths were associated
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Table 4
Perinatal Mortality
Before birth
Within 6 hours after birth
Between 6 to 24 hours
Between 1 to 2 days
Between 2 to 5 days
Controls*
3/164 (1.8%)
2/161 (1.2%)
0
0
0
Mirex
Treated
32/148 (22%)**
11/65 (17%)***
6/54 (11%)
2/48 (4%)
0
* Both Untreated and oil fed controls
** The total pool of Mirex-treated newborns
*** Only the 65 unanesthetized pups were counted in this and
subsequent groups
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Table 5
Symptoms Prior to Death in 65 Treated Newborns
Day 0 Failure to begin respiration 4
Second degree heart block 3
First degree heart block 1
Cannibalization (umbilical 1
hernia)
No symptoms apparent 2
Day 1 First degree block at birth 1
No symptoms apparent 5
Day 2 Abnormally long QT interval 1
on day 1
No apparent cause 1
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Figure 5. ECG of an Mirex-treated newborn, 30 min. old. Showing first
degree heart block and inverted QRS complexes. Heart rate 96/min.
Segment at 125 mm/sec. This pup died 65 minutes after birth.
Figure 6. ECG of a Mirex-treated newborn, 4 min. old. No respiration
started and the heart beat is feeble and erratic. Segments at
(a) 5 mm/sec and (c) 125 mm/sec.
I i | i I H"fr
Figure 7. ECG of a Mirex-treated fetus at 21-1/2 days of gestation. Probably
a third degree heart block with only feeble P waves present.
-------
with the observed cardiovascular symptoms. The abnormal ECGs may indicate
that a serious cardiovascular disorder existed in these newborns and was a
primary cause of death, or simply by a secondary reflection of some other
illness.
Severe edema was not associated with these postnatal deaths since
grossly swollen fetuses were never born alive. Some of those that died
later did have some visible edema, but other sick animals did not appear
to have any.
Respiratory problems were a major cause of postnatal deaths. Four
pups totally failed to begin respiration. Numerous others appeared to
have difficulty initiating respiration, something rarely observed in the
controls. Two individuals were observed to have a period of respiratory
distress about 4 to 6 hours after birth, ie. they were visibly cyanotic
and gasping for breath, but they did recover.
Lungs were preserved and sectioned from three pups that died between
4 to 24 hours after birth without any previous history of cardiovascular
symptoms or respiratory distress that was observed. There was considerable
hemorrhage in the tissues and the majority of the alveoli (about 75%) were
collapsed. There was just a suggestion of hyaline membranes. However, the
tissues displayed considerable edema and a pediatric pathologist suggested
that alveolar collapse occurred due to "edemic congestion". It is possible
that other pups also died from respiratory distress that did not happen to
be observed.
Six individuals died between 6 to 24 hours after birth. Only one of
these had a prior history of any kind. It had a 1° heart block at birth.
An additional two individuals died on day two. One of these had an abnor-
mally long OT interval, about 4 times normal, the day previous to death.
This may be a significant observation because an abnormal QT interval is
thought to be one of the causes of premature infant death in humans. In
our experience testing the ECGs of several hundred fetuses, only one other
individual with such a long QT interval was found. This was in an 18-1/2
day fetus that had been prenatally treated with Trypan Blue.
Prenatal Observations
Many of the newborns looked as if they had died during or just prior
to delivery. To confirm this, a number of observations were made of indi-
viduals examined on the morning of day 21-1/2 of gestation, ie. about 6 to
18 hours before birth would have occurred. The fetuses from 4 litters of
untreated controls and 5 litters of oil-fed controls were compared to
those from 11 Mirex-fed mothers (Table 6). Again the differences between
the two control groups were inconsequential e.g the mean heart rate in
untreated controls was 185 +33.5 SD beats per min. and 179 +34.3 in oil-
fed controls. Therefore the data from the two control groups are con-
sidered together.
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Table 6
Mortality and ECG Data in Fetuses
Examined on Day 21-1/2 of Gestation
Number of fetuses
Number dead
Number alive
Controls
No.
78
0
78
Mirex-treated
No. %
103
18 18%
First degree heart block
Second degree heart block
Third degree heart block
0 18 21%*
0 2 2 %*
0 5 6%*
* out of the 85 living
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There were no dead or dying within the group of the 78 control
fetuses. Their mean PR interval was .067 sec + .0079 (SD). Of the 93 fe-
tuses recovered from treated mothers, 18 were dead. All nine members of
one litter were dead, the other nine were from two other litters. None of
the 18 appeared morphologically retarded compared to their litter mates,
hence could not have been dead for more than 1-2 days. Though early re-
sorptions were counted, they are not being cited here because the aim of
this experiment is to determine perinatal death rate.
The incidence of cardiovascular problems detected in the ECGs was
high. Eighteen of the 85 living fetuses had a first degree heart block,
i.e. the PR interval was greater than .090 seconds (the mean of the
controls, 0.067 sec + 3 S.D.s of .0079 sec). Two fetuses had a second
degree heart block. An additional 5 fetuses seemed to be on the verge of
death. Their heart rates were very slow (10 to 80 beats per min.), feeble,
irregular, and typically consisted of only one component, presumably P
waves (Fig. 7). This could be either a third degree heart block or an ad-
vanced stage of heart failure. It is doubtful that this latter group in
particular would have survived the rigors of birth.
DISCUSSION
Cardiovascular Problems and Perinatal Death
This study was an attempt to analyze the factors which are directly
responsible for the high rate of perinatal mortality induced by the pesti-
cide Mirex. It is also a study in functional teratology.
Gaines and Kimbrough (1970) found high levels of pre- and post-natal
deaths in rats after extended exposure to low levels of Mirex. Litter size
was reduced by about 25% in females fed 25 ppm Mirex mixed with their food
(about 1.8 to 2.8 mg/kg/day) throughout gestation and lactation. Of those
born alive, only 53 to 61% survived to weaning. Khera et al. (1976) found
prenatal death rates of 8.4% after rats were fed 6.0 mg/kg/day Mirex on
days 6 to 15 of gestation (compared to 3.7% in controls) and 40% after 12.5
mg/kg/day. Chernoff et al. (1979) found prenatal death rates ranging from
16.8% after exposure of rats to 7 mg/kg/day to 100% after exposure to 38
mg/kg/day. In the present study of rats prenatally exposed to 6 mg/kg/day
of Mirex, 19% were dead just before birth and another 6% appeared on the
verge of death. This is in agreement with a stillborn rate of 22% of those
allowed to go to birth.
Of those born alive, 17% died within a few hours after birth and an
additional 15% by 2 days after birth. A scattering of morphological ab-
normalities have been described in all of these studies, but, apart from
edema, these are not sufficiently frequent or severe to account for these
death rates,
A clue to a possible cause of these late prenatal and early postnatal
deaths was found in the studies described in Part I of this report (see
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also Grabowski and Payne, 1980). In 18-1/2 day fetuses exposed to moderate
doses of Mirex (5 to 7 mg/kg/day) the incidence of first degree heart block
ranged from 20 to 50% and the incidence of second degree heart block was 4
to 10%. This high incidence of cardiovascular problems, which was very
closely correlated with degree of visible edema, could verv well affect
perinatal health and vitality. The ECG study of fetuses at day 21-1/2
showed that these cardiovascular symptoms persist up to birth at about the
same frequency. In addition, by day 21, there were significant numbers of
dead and dying fetuses.
Most of the newborns (20%) who were born with first degree heart block
recovered during the first day after birth. Some of these did die, indi-
cating that this disorder, which is not necessarily serious in itself, was
at least indicatory of a health problem. A second degree block is more
serious, and all pups who were born with this problem died within an hour
of birth. Therefore, it is apparent that the cardiovascular problems which
were evident at 18-1/2 days do persist into the perinatal period, and are
correlated with some perinatal death.
The cause of the first and second degree heart blocks induced by
Mirex is problematical. None of the usual explanations for these problems
in adults, such as hyperkalemia and hypoxia, seem to apply (see Discussion,
part 1). Though the mean PR interval at 18-1/2 days was very closely
correlated with degree of edema, this is not quite as apparent during the
perinatal period. This is partly due to the fact that the grossly swollen
individuals die before or during birth. Many of the newborns which showed
moderate and severe cardiovascular symptoms had little subcutaneous
swelling evident.
Respiratory Distress
A surprising discovery was that treated newborns exhibited a fairly
high incidence of respiratory distress. Four pups (4%) failed to begin
respiration. It is perhaps significant that two of these four also had
second degree heart blocks immediately after birth, evident before the
respiratory failure could have induced sufficient hypoxia to account for
this symptom. Numerous other treated newborns had difficulty in initiating
respiration. Two others recovered from classic symptoms of Respiratory
Distress Syndrome (RDS) several hours after birth. Three others that died
between 4 to 24 hours after birth without any symptoms that were observed
showed lung collapse due to edemic congestion. Some of the others which
died during the first day after birth may have done so from RDS which was
detected.
It would appear that one of the consequences of fetal edema is
pulmonary congestion which can lead to respiratory distress at birth or
shortly thereafter.
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The ECG Technique and Perinatal Studies
This technique was very useful in this Mirex study. Objective
evaluations of cardiac activity, ie. rate of heart beat, PR intervals,
heart blocks etc., were readily obtained in permanent record form which
could be examined at leisure, and re-examined if desired. A record of
breathing activity was also obtained in the newborns with no extra effort.
Both sets of observations yielded valuable data in evaluating the develop-
mental toxicity of Mirex. The only difficult phase of this procedure was
waiting for the dams to give birth, which usually occurred at night.
Keeping the animals in a room in which the duirnal cycle was reversed, a
relatively simple procedure, would help to ensure that most of them would
go into parturition during a normal work day.
The ECG technique also provided clear documentation of functional
problems induced by Mirex which do not have any readily apparent morpho-
logical basis, i.e. examples of functional teratology. Whether the
physiological problems described have permanently affected the survivors
has not yet been ascertained. However, it is clear that many affected
individuals were killed by these functional problems and others nearly so.
The Developmental Toxicity of Mirex
This study extends further the observations of others that Mirex
induces a high rate of perinatal death. The moderate dose of Mirex used,
6 mg/kg/day on days 8 to 15, induced a prenatal death rate of 22-25% and a
postnatal rate of 32%. It is noteworthy that many of these deaths would
normally be missed in standard studies. Most necropsy studies on rats are
performed on day 20-1/2 of gestation. These would miss the prenatal
mortality that occurs just before and during birth, as well as the postnatal
deaths. Even routine postnatal studies would miss some dead pups because
of maternal cannibalism (see e.g. Khera and Ruddick 1973.
This study also provides evidence that most of these perinatal deaths
are related to either (1) cardiovascular problems, or (2) respiratory
failure. The evidence that the cardiovascular problems are associated with
the observed edema is circumstanial, but strong. The respiratory failure
seems also to be associated with pulmonary edema, it would appear, then
that knowing the basis for the development of fetal edema will be neces-
sary to more fully understand the developmental toxicity of Mirex and the
numerous other agents which induce this problem ( Grabowski 1977).
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LITERATURE CITED
Chernoff, N., Linder, R. E. , Scotti, T. M., Rogers, E. H. , Carver, B. D.,
and Kavlock, R. J. 1979 Fetotoxicity and cataractogenicity of Mirex
in rats and mice with notes on Kepone. Environmental Research, 18;
257-269.
Gaines, T. R., and Kimbrough, R. D. 1970. Oral toxicity of Mirex in adult
and suckling rats. Arch. Environ, 2jjj 7-14.
Grabowski, C. T. 1977 Altered electrolyte and fluid balance. Handbook
Teratol. 2_: 153-170, eds: Wilson & Fraser.
Grabowski, C. T., and Payne, D. B. 1980 An Electrocardiographic study of
cardiovascular problems in Mirex-fed rat fetuses. Teratol. (in press)
Khera, K. S., and Ruddick, J. A. 1973 Polychlorodibenzo-p-dioxins:
Perinatal Effects and the Dominant Lethal Test in Wistar Rats. In:
Chlorodioxins-Origin and Fate, E. A. Blair, ed. Adv. in Chemistry
Series, No. 120 pp. 70-84.
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO. 2.
EPA-600/1-80-032
3. RECIPIENT'S ACCESSION NO.
PB81 1047 3 9
4. TITLE ANO SUBTITLE
Pesticide Effects on Prenatal Cardiovascular
Physioloay.
5. REPORT DATE
SEPT.1980 Issuing Date.
6. PERFORMING ORGANIZATION COOE
7. AUTHOR(S)
Casimer T. Grabowski, Ph.D., Professor of Biology,
Dept. of Biology, Univ. of Miami, Coral Gables, Fla.
8. PERFORMING ORGANIZATION REPORT NO.
. - . .. _ -i
Department of Biology
University of Miami
Coral Gables, Florida 33124
1LA629
11. CONTRACT,'GRANT NO.
R-805301010
12. SPONSORING AGENCY NAME AND ADORESS
Environmental Protection Agency (Neil Chernoff)
Health Effects Research Laboratory
Environmental Research Center
Research Triangle Park, NC 27711
13. TYPE OF REPORT ANO PERIOD COVEREO
14. SPONSORING AGENCY COOE
600/11
15. supplementary notes
Project Officer: Neil Chernoff
16. ABSTRACT
Sperm-positive rats were intubated with Mirex in oil (5 to 10 mg/kg) on days 8-1/2 to
15-1/2. Controls were untreated or oil-fed. Testing was done on day 18-1/2. Fetuses
were sequentially exposed and ECG's obtained with the fetus attached to the placenta and
uterus. Counterparts of standard leads, I, II and II were used. Fetuses were weighed
and examined afterwards. Swollen fetuses were rated on a scale of 1 (slight edema under
chin) to 5 (3 mm edema across back). ECG's from 81 controls and 205 Mirex fetuses were
obtained. They were evaluated for rate of heart beat, regularity of beat, PR intervals
and other features. One control exhibited an abnormality, a transitory period of prema-
ture atrial contractions. Mi rex-fed fetuses exhibited tachycardia, closely correlated
with degree of edema. The heart rate increased from 150/min in controls to 180 in slightly
swollen to 224 in swollen fetuses. Mean PR intervals increased with degree of swelling
and with dose. The frequency of first degree heart block was also dose-related, ranging
from 20% to 77%. Second degree heart blocks were found in 8%, 3% showed arrhythmias, and
one had atrial flutter/fibrillation. These cardiovascular problems seem primarily related
to the Mirex-induced edema and demonstrate that fetal edema is not innocuous. These data
demonstrate the usefulness of fetal electrocardiography to detect functional teratology.
17.
KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
b.lOGNTIFIERS/OPEN ENDED TERMS
c. COSATl Field/Group
Rat fetuses
Mi rex
Electrocardi ography
06T
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