ABSTRACT
Joseph Franklin Holson, Jr. TERATOGENIC POTENTIAL OF D-LYSERGIC
ACID DIETHYLAMIDE (DELYSID) IN THE ALBINO RAT. (Under the direction of
Everett C. Simpson) Department of Biology, 1969.
D-lysergic acid diethylamide (LSD-tartrate) in doses of 5-20 ug
per ki]ogram of body weight was given to pregnant rats (Holtzman
strain) at preimplantation (day 4) and organogenic (day 9) periods.
Twenty rats were given 5 ug LSD/kg b.w. on day 4 (10 rats) or day 9 (10
rats) of pregnancy. Another 20 rats were given similar injections of
20 ug LSD/kg b.w. Thirty pregnant rats served as control animals for
intrauterine treatments with LSD. Ten of these animals were not treated.
The remaining 20 animals were given control intrauterine injections. A
total of 40 pregnant rats were given injections as follov;s:
Control unilateral intrauterine on day 4 of pregnancy
Control unilateral intrauterine on day 9 of pregnancy
LSD unilateral intrauterine on day 4 of pregnancy
LSD unilateral intrauterine on day 9 of pregnancy
The control intrauterine injections provided simulative operational
treatment. Control groups (3 groups of 10 animals each) were utilized
for each experim.ental phase to negate any seasonal variation of
reproductive competence. Examination of the resultant 1,233 fetuses
revealed no significant frequency of congenital defects in the centr’d
or experimental animals. Intrauterine LSD injections on day 4 of
pregnancj'' evoked significantly decreased mean fetal weights. There was
a significant increase of resorption sites in those animals given
intraperitoneal injections of LSD (5.0 ug/kg b.w.) on day 9 of
pregnancy (p<0.05). Results of the intrauterine treatments with LSD
did not substantiate a direct target-tissue mechanism involving
reported LSD teratogenicity. The systemic injections of LSD failed to
cause a significant frequency of detectable malformations in offspring
of treated adults.
TERATOGENIC POTENTIAL OF D-LYSERGIC
ACID DIETHYLAMIDE (DELYSID)
IN THE ALBINO RAT
A Thesis
Presented to
the faculty of the Department of Biology
East Carolina University
In Partial Fulfillment
of the Requirements for the Degree
Master of Arts in Biology
by
Joseph Franklin Holson, Jr.
August 1970
C.^
TERATOGENIC POTENTIAL OF
D-LYSERGIC ACID DIETHYLAMIDE (DELYSID)
IN THE ALBINO RAT
by
Joseph Franklin Holson, Jr.
APPROVED BY:
SUPERVISOR OF THESIS
CHAIRMAN OF THE DEPARTMENT OF BIOLOGY
Dr. Graham ¡y'. Davis
DEAN OF THE GRADUATE SCHOOL
f I
/ Dr. John M. Howéll
/
^50598
ACKNOWLEDGMENT
I express gratitude to the citizens of the state of North
Carolina for providing the facilities and faculty of East Carolina
University which made this study possible and provided an environment
conducive to study.
I also wish to acknowledge the invaluable assistance and advice
so effectively but prudently rendered by Dr. Everett C. Simpson.
Because of his patience and skill, this study was enjoyable as well as
heuristic.
LOVINGLY DEDICATED TO MY MOTHER AND
FATHER, MR. AND MRS. JOSEPH F. HOLS ON,
AND TO MY WIFE, JUDITH, AND DAUGHTERS,
PHAEDRA AND SABRINA
TABLE OF CONTENTS
Page
LIST OF TABLES iv
INTRODUCTION 1
REVIEW OF LITERATURE 3
MATERIALS ANT) METHODS 9
Animals 9
Design of Experiment 9
Experimental Procedures 11
RESULTS 14
Effects of Intraperitoneal Injections of 5.0 ug LSD/kg
Body Weight on Day Four or Nine of Pregnancy 14
Results of Intraperitoneal Injections of 20.0 ug LSD/kg
Body Weight on Day Four or Nine of Pregnancy 16
Results of Unilateral Intrauterine Control and LSD
Injections on Day Four or Nine of Pregnancy 18
DISCUSSION 24
SUMMARY 32
BIBLIOGRAPHY 34
iv
LIST OF TABLES
Table Page
1. Mean values for 5.0 ug LSO/kg body weight injections
on day four or nine of pregnancy 15
2. Mean values for 20.0 ug LSD/kg body weight injections
on day four or nine of pregnancy 17
3. Mean values for control intrauterine (lU) and 2.52 ug
LSO/kg body weight intrauterine injected animals (day
four of pregnancy) 19
4. Mean values for control intrauterine (lU) and 2.52 ug
LSD/kg body weight intrauterine injected animals (day
nine of pregnancy) 21
5. Comparison of mean values for control intrauterine (lU)
and 2.52 ug LSD/kg body weight intrauterine injected
animals (day four of pregnancy) 23
INTRODUCTION
The period between 1958 and 1962 represents a very important time
in the development of Tiammalían experimental teratology. When
Widerkind Lenz discovered the relationship between thalidomide and
birth of numerous malformed infants, scientists realized the need for
more thorough drug studies.
During the last decade there has been widespread increase in abuse
of drug use. One aspect of this increased consumption of drugs can be
relegated to the "younger generation." The model psychotomimetic,
lysergic acid diethylamide (LSD), has attracted much scientific and
social consideration.
LSD was first prepared by Stoll and Hofmann in 1938 (Stoll and
Hofmann, 1965). It was not until a few years later that the psycho-
tropic properties of LSD were detected by Hofman while re-investigating
the drug (Stoll and Hofmann, 1965). More recently, LSD has had limited
use in the therapeutic treatment of alcoholics and as an analgesic
(Pfeiffer and Murphree, 1965). Of primary concern in this investigation
is the possible teratogenicity of LSD in the albino rat. It is
generally accepted that inferences can be made to the human from
research involving the rat.
Hov/aver, consideration is maintained with reference to existence
of inter-spacifIQ variables which might pertain to interpretation of
this research. It must be emphasized that the probability of a
pregnant woman consuming LSD at an early stage of pregnancy is high
enough to warrant its investigation. This early stage of pregnancy.
2
from onset of tissue differentiation until eight weeks of age, is an
especially critical insult period for the human (Wilson, 1964). This
period of pregnancy is often the "uncertain period" (Auerbach and
Rugowski, 1967). Therefore, an unknowing mother might endanger fetal
development by ingesting LSD. Likewise, it is necessary to realize the
potential of this drug with respect to its continued use in psycho-
therapy.
Specifically, this research was concerned with any effect of LSD
upon fetal development in the albino rat. Major consideration was
given to the mode of treatment, dosage, and the stage of pregnancy at
which the LSD was administered.
THE REVIEW OF LITERATURE
The initial report of LSD teratogenicity was by George J.
Alexander, 196?. In these experiments, female rats of the
Wistar strain (250 g.) were mated with healthy males (450 g.)* All
experimental animals were given subcutaneous injections of 5 ug LSD/kg
of body weight. This dosage was selected because it corresponds in the
rat to the human hallucinogenic dose (100-400 ug per person--!.7 to
6.7 ug/kg body weight). Control animals received subcutaneous injec-
tions of physiological saline.
The first two experiments consisted of a total of 10 rats which
were treated with the LSD injections on day four of pregnancy. Of the
first five animals, only one was reported to have delivered a normal
litter. The other four rats either contained resorbed fetuses, had
stillborn fetuses, or gave birth to runted offspring. In experiment
two, three of the five rats either aborted or gave birth to varying
numbers of stillborn fetuses. Two litters were normal, except that one
litter was unusually small (4 offspring). The third group of animals
was Injected on days 8, 9, 10, 11, or 12 of pregnancy. There were no
observed effects upon the offspring of animals in this last group. The
treated animals delivered 51 apparently healthy offspring, and the
control animals gave birth to 65 ratlets. Gross observation of the
young from all these experiments revealed no malformations. There was
some stunting of development in the case of the three stillborn off-
spring. Offspring of animals treated with LSD had comparable weights
4
to the control offspring, but some failed to grow as rapidly as the
control animals.
The injection of LSD-tartrate into mice during early stages of
pregnancy caused a 57 percent frequency of abnormal embryos (Auerbach
and Rugowski, 1967). Mice of the strains BALB/Cau, C57BL6/Au,
C3H/HeAu, and Fj (BALB/Cx57BL) were utilized for this investigation.
The appearance of a copulatory plug was designated as day 0 of
pregnancy. Before injection, the LSD-tartrate was diluted in Tyrode's
solution to obtain suitable concentrations. All animals were given I.P.
control or I.P. LSO injections on day 7 of pregnancy. The embryos were
removed and examined by gross inspection 4 days following the injection.
-6 -8
The doses of LSD injected ranged from 1x10 gto5xl0 g. Treated
animals exhibited a 57 percent incidence of deformities, while the
control animals had fewer than 10 percent malformations. In all cases,
malformations were characteristic brain defects and additional
abnormalities of the lower jaw, shifts in eye position, and modified
facial contours. To determine stage specificity of the produced
malformations, mice were given injections of LSD on days 6, 8, or 9 of
pregnancy. Brain abnormalities were observed in the day 6 treated
animals, but no gross observable effects were discerned in offspring of
animals treated later than day 7 of pregnancy.
Congenital malformations induced by Bromolysergic acid, mescaline,
and lysergic acid diethylamide in the hamster were reported by W. F.
Geber (1967). Pregnant hamsters were housed in quiet quarters and
illuminated by natural light (roof window). The alkaloids used as
insult agents were dissolved in sterile physiological saline solution
prior to injection. Injections were given subcutaneously on the 8th
5
day of pregnancy. All control animals were injected with sterile saline
in the same manner. After single injections they were returned to
quarters and Caesarian sectioned on day 12 of pregnancy. The dose
range of LSD given was from 84 ug/kg body weight to 240 ug/kg body
weight. No major abnormalities were detected in the 300 control
fetuses, but there was a limited number of stillborn fetuses, resorbed
fetuses, and runts. In the experimental animals, abnormalities of this
nature were much greater on a percentage basis. An average of 6.2% of
experimental (LSD-treated fetuses) exhibited one or more types of
congenital malformation. Approximately 10% of the malformed fetuses
possessed more than one type congenital defect. The types of gross
defects observed were exencephaly, spina bifida, interparietal
meningocele, omphalocele, hydrocephalus, myelocele, edema along the
spinal axis, generalized edema and hemorrhages of brain areas (parietal
and frontal) and the neck region. This study indicated no correlation
between dose (ug/kg of body weight) of the drug given to the parent and
percentage of malformations contained in litters of treated animals. A
dose-response relationship did occur in reference to two other para-
meters. As litter size decreased, resorption rate and fetal mortality
rate increased. Runting was augmented as drug dosage was increased.
Lysergic acid diethylamide (Delysid) administered to pregnant
Wistar rats during periods of organogenesis did not cause a significant
frequency of congenital defects (Warkany and Takacs, 1968). LSD was
administered orally or i ntraperitonea1ly in single doses to pregnant
rats on day 7f 8, or 9 of gestation or in multiple doses from day 7
through day 12 of pregnancy. The dosage of individual treatments
ranged from 1.5 to 300 ug. Of the 55 pregnant rats treated, 4 had
6
litters completely resorbed; 47 rats were sacrificed on day 21 and the
young were removed; four were allowed to go to term and these offspring
were raised (44 pups were born, some were raised and bred). The mean
weight of the 21-day fetuses was 3.53 + 0,44 g (controls, 4.14 + 0.71 g).
The mean litter size of the 21-day fetuses was 10.2 + 1.8 (controls,
10.0 + 2.8). There was no significant statistical difference between
the control and experimental fetuses. The remaining 504 fetuses were
cleared or dissected (409 dissected and 35 cleared) and considered
normal. There was no discernible pattern common to the four abnormal
fetuses; one was hydrocephalic; one had short extremities and
syndactylism; and the remaining two were small (1.7 and 2.1 g).
Warkany and Takacs (1968) also gave LSD Injections to pregnant
Wistar rats on day 4 or 5 of pregnancy. Thirty-four rats were given
doses ranging from 1 to 100 ug LSD (Delysid). Two of the treated rats
had no litters. Of 355 offspring from the 32 treated rats, 296 v;ere
removed on day 21 of pregnancy by Caesarean section. Except for one
small fetus, all appeared normal following gross inspection, dissection,
or clearing. Thirty-five young were delivered and raised, all
appeared normal and healthy. There was a resorption rate of 5.9% which
was not different from those animals treated with saline solution
injections.
Fabro and Sieber (1968) did not find lysergide tartrate terato-
genic in the New Zealand white rabbit. Lysergide tartrate was
administered orally (20 or 100 ug/kg b.w.) to pregnant does for various
intervals both early and late in pregnancy. Animals given lysergide
tartrate did not significantly differ from the control animals in
respect to average number of implantations, incidence of resorption.
7
and number of malformations. There was a significant decrease in the
average fetal weight of offspring from lysergide tartrate treated
mothers (100 ug/kg b.w. on days 7 to 3 of pregnancy).
High doses (5 ug) of LSD-25 administered to Sv/iss-Webster mice on
days 6, 7, 8, or 9 of pregnancy induced a significantly high incidence
of abnormalities of the anterior subcapsular lens (Hanaw^y, 1969).
Single interperitoneal doses (5 ug) of LSD-25 were given to a total of
18 mice on gestation days 6, 7, 8, or 9. The embryos were removed on
day 18 and the whole heads were fixed for subsequent histological
analysis. Earlier experiments had shown that treatment with LSD-25 on
days 4 or 5 had no observable effect on the developing lens. However,
fetuses treated on days 6, 7. 8, or 9 (prior to closure of the neural
tube and differentiation of the eye) revealed drastic effects. Animals
injected on day 6 had 8l% incidence, day 8 had 55%, and day 9 revealed
79% incidence of abnormal subcapsular lens development. The observed
lens defect was an accumulation of globular, eosinophilic material
outside the anterior lens epithelium and under the capsule. Accumula-
tion of this substance over the total anterior surface area of the lens
occurred in 50% of the abnormal eyes. Hyperplasia was common in the
germinal zones and often the lens bow was posteriorly broadened.
Roux, ^ £j_. (1970), treated pregnant rats, mice, and hamsters with
doses of LSD ranging from 5-500 ug/kg b.w. over varied intervals of
pregnancy. No deleterious effects were noted in offspring treated with
the low or high doses of LSD. Fetal mortality, mean weight, and
incidence of malformations were not significantly altered in either of
these species when treated throughout their respective pre-implantation
or organogenic periods of development. LSD was given to 22 pregnant
8
hamsters, 98 pregnant rats, and 67 pregnant mice. Some I89 hamster
fetuses, 521 mouse fetuses, and 1,003 rat fetuses were examined prior
to partuition, with no significant variation of the considered para-
meters.
There have been several clinical reports (case studies) on human
pregnancies following LSD ingestion. These reports are conflicting and
in general not v/el1 controlled; thus they are not reported in detail
here. Cohen, aj.. (1967), found increased frequencies of chromosomal
aberrations, but no congenital malformations in two offspring whose
mothers consumed LSD during the third and fourth months of pregnancy.
Zellweger, _et ¿j_. (1967), encountered a malformed girl infant whose
mother and father had taken LSD. The mother had ingested LSD on the
25th day after her last menstrual period and three times between the
45th and 98th days. The infant had unilateral fibular aplastic
syndrome. Chromatid breaks were found in peripheral white blood cells
of the father (2 breaks), mother (5 breaks), and child (3 breaks).
Since the mother ingested the second dose of LSD during the period
critical for the production of leg deformities, one might suspect a
causal relationship to exist. Sato and Pergament (1968) studied the
birth of an apparently normal girl child to a mother who had taken LSD
on the 43rd and 57th days of pregnancy. Karyotype studies did not
discern significant frequencies of chromosomal abnormalities.
MATERIALS AND METHODS
Animals
Albino nulliparous female rats (200-305 g) of the Holtzman strain
were used for this research. Males of proven fertility were employed
for mating activity. All females were housed separately from males
until they had two or more successive 4-5 day estrus cycles. These
females were selected as they entered proestrus and housed with one
male in designated cages. Males and females were numbered with ear
markings and charts were kept pertaining to their cage position in the
battery. Vaginal lavages for all females housed with males were
obtained daily between 8:00 and 12:00 A.M.
Animals were housed in a room with 14 hours of light (5:00 A.M.-
7:00 P.M.) and 10 hours of darkness (7:00 P.M.-5:00 A.M.). Care was
exercised to keep extraneous activity and noise to a minimum.
Unnecessary handling or moving of the animals was avoided to maintain a
minimum of environmental stress. All animals had free access to water
and received Purina Laboratory Chow ad lib.
Design of Experiment
Part I of this investigation involved three groups of 10 animals
each. One group of pregnant subjects was given control injections of
sterile physiological saline on days 4 and 9 of pregnancy. These
injections were based on body weight-LSD volume basis as rendered to
the experimental animals. Injections were given intraperitoneal1 y
(I.P.). Another group of 10 pregnant rats (experimental group 1) was
10
given one I.P. injection of 5 ug LSD-tartrate/kg body weight on day 4
of pregnancy. The remaining group (experimental group 2) of animals
was administered 5.0 ug LSD/kg body weight on day 9 of pregnancy.
Day 4 of pregnancy represents the critical preimplantation and
fetal membrane development period, and day 9 is the vital period of
neurogenesis and osteogenesis (Nicholas, 1962). Typically, days 4 and
9 are insult-susceptible periods of development in the rat (Wilson,
1964). The injection doses of LSD-tartrate were extrapolated, on a
body weight basis from the hallucinogenic dose for the human (Pfeiffer
and Murphree, 1965).
Part II also involved three groups of 10 animals each. The
control group rats (control group 2) were given I.P. injections of
sterile physiological saline on day 4 and day 9 of pregnancy. Experi-
mental groups 3 and 4 were treated as were experimental groups 1 and 2
except that the dosage of LSD was increased 4x that used in Part I
(20 ug/kg b.w.). This dosage of LSD is comparable to very potent
hallucinogenic human doses (Cohen, 1967). The dose given experimental
groups 3 and 4 animals was 20.0 ug LSD/kg b.w. The LD^q (LSD) for the
rat is 16 mg/kg b.w. (Cohen, 1967).
Part III of this investigation consisted of one control group and
four experimental groups. Control group number 3 involved no treatment.
These 10 animals were bred, weighed at particular stages of pregnancy
and finally autopsied. Experimental groups 5 and 7 were administered
unilateral intrauterine injections of sterile physiological saline.
Group number 5 received treatment on day 4 of pregnancy, and group
number 7 received the injection on day 9 of pregnancy. Experimental
groups 6 and 8 received unilateral intrauterine injections of LSD on
days 4 and 9 of pregnancy, respectively. Injection doses for groups 6
and 8 were based on 2.52 ug LSD/kg of body weight. Control intra-
uterine injections for groups 5 and 7 were of corresponding volumes to
injections in groups 6 and 8. The intrauterine injections of LSD and
of physiological saline were volume-matched to minimize any adverse
effects due to mechanical stress of the intrauterine injection. One-
half (5 animals) of each group was injected in the right uterine horn,
and the remaining 5 animals received the injection in the left uterine
horn.
Control unilateral intrauterine injections provided simulative
operational treatment for groups 5 and 7. Contention was that stress
afforded by the 4th or 9th day operation might affect development of
fetuses in the LSD treated animals. Therefore, groups 5 and 7 were
utilized as secondary controls and served to effectively demonstrate
any resultant operational effects.
The unilateral treatment of pregnant rats on either day 4 or 9
provided another control facet. With LSD presented to only one uterine
horn, the contralateral horn provided a more discriminating control
situation. Caesarian sectioning quickly revealed the difference in
treated versus untreated uterine positions.
Experimental Procedures
Vaginal Lavages
Vaginal lavages were taken dally from those females housed with
males. Vaginal lavages were always taken between the hours of 8:00 A.M.
and 12:00 P.M. Vaginal cell stages were determined according to
Turner (1966). Approximately ^cc of warm water was introduced into the
12
vagina and asperated by means of a small pipette 3 or 4 times to obtain
an optimal amount of sloughed cells. One drop of the lavage was placed
on a microscope slide. Since the rats were numbered, several lavages
were placed on the same slide. Each lavage was examined microscopi-
cally under lOX and the stage of estrus cycle recorded. Presence of
spermatozoa in the vaginal lavages or of a copulatory plug established
day 0 of pregnancy.
Parenteral Injections
All injections were administered intraperitoneally with a sterile
1 cc capacity tuberculin syringe and 26 gauge needle.
Laparotomy Technique and Intrauterine In j ections
The animals were anesthesized with intraperitoneal injections of
aqueous chloral hydrate (120 mg/cc; dosage equals 30 mg/100 g body
weight). The pelvic region of the abdomen was shaved and swabbed with
70% alcohoKand a mid-ventral longitudinal incision approximately 3 cm
in length was made about 1 cm anterior to the vaginal orifice. The
uterine horns were carefully exteriorized with broad tipped forceps.
In all 9-day pregnant animals both the number and approximate position
of the embryos were recorded. The appropriate uterine horn was then
injected in its anterior one-third. The intrauterine injection was
performed with a sterile tuberculin syringe (27 gauge needle). The
uterine cornua were then replaced into the abdominal cavity, the muscle
wall sutured, and the skin incision closed with sterile surgical clamps
(11 mm). Post-operative care entailed isolation of the animal for
about one hour or until the animal was active enough not to be injured
by cage companions.
13
Autopsy
All animals were autopsied one day prior to term (20th day of
pregnancy). Each animal was weighed and then killed with an overdose
of ether, but promptly removed from the ether with the cessation of
cardiac activity to prevent fetal death. A mid-ventral longitudinal
incision through the abdominal wall was made from the vaginal orifice
to the xiphoid process of the sternum. The uterine cornua were
exteriorized and the number and position of fetuses was recorded.
Fetuses were then checked for a heart beat to determine whether they
were living. Each ovary was examined and the number of corpora lútea
noted. The uterus, containing fetuses, was removed and weighed and all
fetuses were removed from the uterus and the condition of the endo-
metrium observed. The presence and location of resorption sites were
recorded. Metrial gland positions and numbers were compared to fetal
numbers and positions. The uterus was then weighed. Fetuses were
separated from the fetal membranes and the umbilical cord severed near
the abdomen of the fetus. Fetuses were weighed and body lengths
measured with a vernier caliper, after which they were individually and
punctiliously examined for gross malformations. Abnormal appearance of
the placenta, umbilical cord, fetal membranes, and of fetal skin were
noted. Fetuses having abnormalities as well as every fourth fetus
removed were placed in 95% alcohol and kept for subsequent clearing
procedures (Wilson, 1964). The alizarin Red S staining and clearing
procedure was used (Humason, 1967).
RESULTS
Part I
Effects of Intraper i toneal In]ect îons of 5 »0 ug LSD/kg Body Weight on
Day Four or Nine of Pregnancy
A summary of the mean values for all parameters considered in Part
I is presented in Table 1. Significant difference was found between
the weight gains of the parents in the control group and the day 4
injected parents (p<0.01). That the maternal weight gains of experi-
mental group 1, during pregnancy, v;ere less than control group 1 is
perhaps due to the smaller litter size of the experimental group. This
smaller litter size is the result of fetal resorption within the
experimental group. However, fetal resorption was not significantly
greater in the day 4 injected group than in the control group. The
fertilization factor (percent corpora lútea represented at autopsy by
fetuses) is 11.6% lower in the treated group of animals. This reduced
aspect of fecundity is partially due to the greater fetal mortality of
the day 4 treated animals. Since the resorption rate is slight, there
is a possibility that treatment with LSD at the critical pre-imp 1 anta-
tion period may have interfered with necessary or integral processes of
successful implantation. Average fetal weights and number of resorp-
tions and runts did not significantly differ in the control group and
the day 4 treated group.
Experimental group 2 (9th day injections) revealed a significantly
lowered average weight gain as compared to the control group (p<0.01).
Again it would appear that the significant resorption rate of this
group v/as responsible for the weight gain discrepancy. The number of
15
Table 1. Mean values for 5.0 ug LSD/kg body weight
inject ions on day four or nine of pregnancy
Control I.P. LSD i nj . LS D inj .
i nj . on on day 4 on day 9
days 4 & 9 (Exp. Grp. 1) (Exp. Grp. 2)
Weight Gain (g) 142.6 114.7*^'^ 95.9.'nV
No. Fetuses 12.6 11.3 11.4
No. Corpora Lútea 14.0 14.4 15.4
Mean Fetal 4.4 4.4 4.1
Weight (g)
Mean Fetal 3.8 3.8 3.9
Length (cm)
No. Resorptions 0.0 0.3 0.6*
No. Runts 0.0 0.1 0.5
Ferti1ization 90.0 78.4 74.1
Factor (%)
* (p<0.05)
VwV (p<0.01)
(10 animals per group)
runts in the day 9 treated group would lower the mean maternal weight
gain, thereby decreasing the summed mean value for the group. The mean
fetal weight was less in this group, thereby affecting the maternal
weight gain mean. There was no significant change in the mean fetal
length for this series of experiments.
The number of resorption sites was significantly increased in the
day 9 injected group as compared to the control group (p^O.05). The
reduction of the fertilization factor was obviously due to the increase
in fetal mortality. Augmented fetal mortality was represented by the
numerous resorption sites.
Part 1 I
Results of Intraperitonea1 Inject ions of 20.0 ug LSD/kq Body Weight on
Day Four or Nine of Pregnancy
Table 2 consists of the mean values for measurements taken from
the control and two experimental groups testing the effect of an
increased dose (20 ug LSD/kg b.w.) of LSD on fetal development. Appli-
cation of Student t-test analysis for these categories yielded no
significant differences. These results imply that this dose-level of
LSD did not act teratogenical ly. Although the day 4 and 9 injected
groups had relatively large resorption rates, there was a corresponding
increase of resorption in the control animals for this experiment over
those in control group 1. Perhaps this alteration of fecundity can be
related to seasonal influences. The fertilization factors for these
groups show that there was very little variation of reproductive
competence within the groups tested. The day 4 treated group had the
highest resorption rate and thus the lowest fertilization factor.
17
Table 2. Mean va]ues for 20 ug LSD/kg body weight
inject ions on day four or nine of pregnancy
Control 1.P. LS D i nj. LS D i nj .
i nj . on on day 4 on day 9
days 4 & 9 (Exp. Grp. 3) (Exp. Grp. 4)
Weight Gain 121.55 115.6 103.1
No. Fetuses 11.6 11.0 12.1
No. Corpora Lútea 13.4 15.0 14.6
Mean Fetal 4.1 3.9 4.1
Weight (g)
Mean Fetal 3.9 3.9 3.9
Length (cm)
No. Resorptions 0.4 0.9 0.5
No. Runts 0.0 0.6 1.0
Ferti1ization 86.3 73.8 82.9
Factor (%)
(10 animals per group)
18
Part I I I
Resu1ts of Unilateral Intrauterine Control and LSD Injections on Day
Four or Mine of Pregnancy
Control and LSD Uni 1ateral Intrauterine Treatment on Day Four of
Pregnancy
Unilateral intrauterine treatment with LSD was employed to eluci-
date the potential direct or indirect modality of LSD activity.
Subjecting the developing embryo to an environment rich with LSD could
also present data which would provide negative or positive evidence for
its proposed chromosomal effects as reported by Shakkebaek, et al.
(1968).
The intrauterine application of LSD was accompanied by two types
of control groups. Control group 3 was the basic control group. Group
3 animals were bred but were not treated experimentally. This
untampered group of animals served to exhibit any seasonal or unrealized
exogenous influences upon reproductive capacity. Control unilateral
intrauterine injections on day 4 of pregnancy simulated experimental
surgical stress. The simulative treatment was necessary to negate any
effect due to the laparotomy performed during intrauterine treatments
with LSD. Without the intrauterine controls, results of LSD intra-
uterine experiments would have been less defined as to causation and
any physical effect induced by uterine distension. Although intra-
uterine injection volumes were minimized, some degree of uterine
expansion was observed.
Summed data (mean values) from unilateral intrauterine injections
on day 4 of pregnancy is presented in Table 3. The maternal weight
gains are significantly lowered (p<0.01) in the group given an
intrauterine injection of LSD on day 4 of pregnancy (experimental group
19
Table 3. Mean values for control intrauterine (IU) and
2.52 ug LSD/kq body weight intrauterine injected
animals (day four of pregnancy)
Controls 1U control inj. lU LSD inj .
(No inj .) on day 4 on day 4
(Exp. Grp. 5) (Exp. Grp. 6)
Weight Gain (g) 113.3 90.0 65.3**
No. Fetuses 13.4 8.0 7.1
No. Corpora 15.2 12.9 12.9
Lútea
Mean Fetal 4.0 4.2* 3.9
Weight (g)
Mean Fetal 3.9 3.8 3.8
Length (cm)
No. Resorptions 0.4 2.5** 3.6**
No. Runts 0.0 0.0 0.0
Ferti1ization 88.4 61.8 55.8
Factor {%)
Comments: one malformed placenta in lU LSD group
(p «01)
* (p<0.05)
(10 animals per group)
20
6). The decreased maternal weight gain is largely due to the high
resorption rate of this group, which represents a significant increase
over the control animals (control group 3). Neither the number of
fetuses nor the number of corpora lútea of the control or LSD injected
groups differed significantly from those values of the control group.
The mean fetal weight of the control intrauterine treated group
(experimental group 5) does significantly differ from that of the
control group (p<0.05). This increased mean fetal weight for the
control intrauterine treated animals may be the result of decreased
litter size, since reduced litter size often results in larger off-
spring. No runts were observed in control group 1 or experimental
groups 5 and 6. One fetus, in group number 6, which appeared normal in
all respects, had an elongated and discolored placenta. Experimental
groups 5 and 6 did exhibit large decreases in their fertilization
factors. Obviously, these decreases were due to the greater fetal
mortality represented by the increased resorption rates.
Control and LSD Uni lateral Intrauterine Inj ections on Day Nine of
Pregnancy
Results of control and LSD intrauterine injections on day 9 of
pregnancy are given in Table 4. None of the categories in either case
gave significant statistical differences when compared with control
group 3. From the data in Table 4, it is apparent that laparotomy did
not affect fetal development at day 9 as drastically as at day 4 of
pregnancy. Litter size, average fetal weight, and average fetal length
were closely correlated throughout the three groups. The resorption
rate increase in LSD injected animals, though not statistically
significant, is a noticeable variation from the control values. The
21
Table 4. Mean va 1ues for control intrauterine (IU) and
2.52 uq LSD/kq body weight intrauterine injected
animals (day nine of pregnancy)
Controls 1U control inj. 1U LS D inj.
(No inj.) on day 9 on day 9
(Exp. Grp. 7) (Exp . Grp. 8)
Weight Gain (g) 113.3 93.9 107.0
No. Fetuses 13.4 12.4 11.3
No. Corpora 15.2 13.7 13.8
Lútea
Mean Fetal 4.0 4.1 4.1
Weight (g)
Mean Fetal 3.9 3.8 3.9
Length (cm)
No. Resorptions 0.4 0.3 0.6
No. Runts 0.0 0.0 0.5
Ferti1Î zation 88.4 90.4 82.0
Factor (%)
Comments: one spina bifida in ID LSD group
(10 animals per group)
22
LSD treated group also displayed a sizeable Increment in runting. The
fertilization factor was relatively uniform in all three groups. One
LSD treated rat contained a malformed fetus which was dead at autopsy
(experimental group 8). Spina bifida was the primary congenital
defect, as well as dorso-ventral compression and underdeveloped limbs.
There was a total of 643 fetuses from LSD treated animals (only one
anomalous condition was observed). A total of 580 offspring were
taken from parents comprising the various control groups; no spontaneous
congenital malformations were detected.
Table 5 represents a statistical comparison (Student t-test)
between the day 4 control and LSD intrauterine injected groups of
animals (experimental groups 5 and 6). There were no significant
differences between data of day 9 control and LSD intrauterine treated
animals. Therefore, these two groups (experimental groups 7 and 8)
were not statistically compared. Comparison of experimental groups 5
and 6 showed a significant difference between the mean fetal weight
categories only. The LSD treated animals produced offspring which were
significantly lighter (0.3 g mean). Though not statistically different,
there is a noticeable increase of the resorption rate for LSD treated
animals over those given control injections.
23
Table 5. Comparison of mean values for control intrauterine
(IU) and 2.52 uq LSD/kq of body weight intrauterine
injected animals (day four of pregnancy)
1U control inj. 1U LSD inj .
on day 4 on day 4
(Exp. Grp. 5) (Exp. Grp. 6)
Weight Gain (g) 90.0 65.3
No. Fetuses 00 • o 7.1
No. Corpora 12.9 12.9
Lútea
Mean Fetal 4.2 3.9,v*
Weight (g)
Mean Fetal 3.8 3.8
Length (cm)
No. Resorptions 2.5 3.6
No. Runts 0.0 0.0
Ferti1ization 61.8 55.8
Factor (%)
Comments: one malformed placenta in lU LSD group
(p<0.01)
(10 animals per group)
DISCUSSION
Part 1 of this study was conducted to reproduce the previously
reported teratogenic action of LSD (Alexander, e_t , 1967, and
Auerbach and Rugowski, 1967). The original intention was to reproduce
this teratogenic effect and to subsequently research its causal
mechanisms, though initial findings indicated a need for modification
of this approach. Part II was conducted (4x dose of LSD) to investi-
gate the possibility of a dose-response relationship in our laboratory
animals which might have explained the apparent lack of LSD teratogen!-
city in the described experiments. Experiments involving the increased
dosages of LSD (Part II) did not yield data which supported the
proposed teratogenicity of LSD. Part III (unilateral intrauterine
injections) v;as designed and performed to further investigate the
potential teratogenicity of LSD. Unilateral injections of LSD were
used in order to obtain greater restriction of control, the contra-
lateral uterine horn serving as an added control feature. As suggested
by Saxen and Rapóla (1969), the best possible control might be to use
embryos located in the same uterus (opposite uterine horn). The
technique of Brent (1965, uterine clamping) was not employed for these
intrauterine tests to avoid additional variables. Intrauterine appli-
cation of LSD was utilized because this allowed the presentation of LSD
directly to the developing conceptus without prior biotransformation of
large portions of the injected LSD. According to Pfeiffer and Murphree
(1965), LSD is rapidly transformed to 2-hydroxy-LSD (no significant
psychotomimetic activity) by the liver microsomes. According to the
25
autoradiographic studies of Idanpaan-Heikkila and Schoolar (1969)»
l4
C-LSD readily penetrates cell membranes and therefore should gain
ready access through the extra-embryonic membranes. Idanpaan-
Heikkila and Schoolar (1969) also demonstrated that 2.5% (early in
14
pregnancy) of I.V. injected C-LSD passed the placenta into the fetus
and 5.0% entered the fetus later in pregnancy. These amounts passed
the placental-fetal interface within five minutes following injection.
Another important aspect of the intrauterine LSD experiments is
that the technique allowed delimitation of the direct versus the
indirect modality of proposed LSD effects. Obviously, a teratogenic
agent may exert its influence in a "target tissue" fashion or it might
affect development indirectly via "generalized physiological stress."
Any deleterious influence LSD might have upon embryogenesis should be
dramatically evident in the late fetus following intrauterine applica-
tion of the drug.
The parenteral (IP) injections of 5.0 ug LSD/kg b.w. on day 4 or 9
of pregnancy (Part l) were not teratogenic under these experimental
conditions. Strictly speaking, teratogenic implies the causation of
anomalous (monster) development. Significant numbers of developmental
abnormalities were not detected in the offspring of the treated rats.
However, there were significant statistical differences between
control and experimental animals with reference to two other parameters
listed in Table 1. The maternal weight gains of both the day 4 and 9
groups were significantly lower than the control animals. This appears
to be a reflection of the increased resorption frequencies demonstrated
by these two experimental groups. The experimental groups (1 and 2)
displayed reduced fertilization factors. This appeared to be
26
correlated to the augmented resorption frequencies. Thus, injections
of 5.0 ug LSO/kg b.w. on day 9 of pregnancy increased fetal mortality
in these experiments. It should be realized that this rate of resorp-
tion (as In experimental group 2) is not actually very high (6.0
resorptions for ten animals) when compared to the resorption frequencies
of subsequent control groups (control 2=4 resorptions, -and control 3 =
3 resorptions). Control group 1 appears atypical in that there were no
resorptions in the 10 litters, whereas rats in this laboratory minimally
approximate 2-3% spontaneous resorption rates.
The fertilization factor for experimental group 1 (day 4 injection
of LSD) revealed an 11.6% drop from the control value. This group of
animals did not have a significant rate of resorption; therefore, it
would appear that LSD treatment at this period of development (pre-
implantation) reduced fecundity—possibly by upsetting mechanisms
critical to successful implantation. This is speculation, since these
experiments do not provide unquestionable evidence for this hypothesis.
Because this experiment (experimental group 2) involved admini-
stration of LSD during the osteogenic and neurogenic phases of rat
development, one would expect teratogenic insults to appear in the form
of gross malformations (spina bifida, exencephaly, etc.). No anomalies
were observed, thus any insult afforded by LSD treatment was radical
enough to terminate development and not induce non-lethal developmental
alterations.
LSD did not act teratogenically when given rats on days 4 and 9 of
pregnancy at a dose level of 20 ug LSD/kg b.w. Examination of the data
from these experiments (Table 2) indicates that LSD was apparently
ineffectual with respect to reproduction in this group of animals. Not
27
one of the measured parameters revealed any apparent alteration which
might have resulted from LSD activity. These results are quite
similar to those published by Warkany and Takacs (1968) and Roux,
et (1970). It is difficult to explain a decrease in effect of LSD
at higher doses as compared with results of experiments in Part I.
There are dose-effect thresholds but no known instances where too great
a dose of the substance renders it non-teratogenic. Part II results
when compared to Part I results are somewhat conflicting. However, the
results in Part It are quite clear—LSD did not act teratogenically.
The data obtained from experiments in Part I are somewhat more
ambiguous.
Part I I I of this investigation consisted of a series of experi-
ments involving the intrauterine application of LSD on days 4 and 9 of
pregnancy. Part III was comprised of one control group of animals
(control group 3), which was not experimentally manipulated and four
experimental groups (groups 5. 6, 7, and 8). Experimental groups 5 and
7 were mock or control treated groups. These two groups functioned to
negate any effects due to surgical trauma brought about by the
laparotomies performed. Groups 5 and 7 received volume-matched intra-
uterine injections of sterile physiological saline on days 4 and 9 of
pregnancy, respectively. Groups 5 and 7 were then matched to the
intrauterine LSD injected groups (6 and 8). To date, these are the
first published experiments utilizing unilateral intrauterine injections
to investigate the teratogenic potential of LSD. Analysis of raw data
collected revealed no detectable significant differences between
treated and untreated uterine horns in any of the experimental groups.
28
Table 3 presents data comparing control group 3 to day 4 control
and LSD intrauterine injected groups (5 and 6). The mean maternal
weight was significantly lowered (p<0.01) in the LSD injected animals
as compared to the untampered control animals. The lowered maternal
mean weight gain is obviously a result of the increased fetal mortality
as illustrated by resorption frequency. Control and LSD injected
animals displayed significantly augmented levels of fetal mortality
when compared with controls. Thus, it appears that the laparotomy or
at least some facet of the technique disturbed normal reproductive
processes. Reduced fecundity is dramatica1ly i11 ustrated by the
radical drop in the fertilization factor for experimental groups 5 and
6. These decrements were 26.6% and 32.6%, respectively, the greater
reduction being in the LSD treated group. The fertilized ova were not
implanted at the time of operation (day 4), so that the mechanical
process of aggitation at the time of injection may have contributed to
reduced fecundity in groups 5 and 6.
There were significant differences between resorption rates for
control and LSD intrauterine treated groups when compared to the
untampered control group (p<0.01). Some proportion of this increased
resorption may have been evoked by surgical manipulations. Table 5,
which represents the comparison of data from control and LSD intra-
uterine (day 4) experiments (5 and 6), illustrates significant
difference (p<0.01) when comparing the mean fetal weight of the LSD
and control treated groups. Intrauterine LSD injection on day 4 of
pregnancy appeared to effect a decrease in the mean fetal weight. The
differential mean fetal weight value was the only parameter displaying
29
any significant statistical variation between the control and LSD
intrauterine injected groups.
Intrauterine injections of LSD on day 9 of pregnancy did not
exhibit deleterious effects, as shown in Table 4. The laparotomies
performed for experiments on day 9 of pregnancy did not interfere with
development as was the case in the day 4 intrauterine experiments.
Results of intrauterine LSD injections on day 9 of pregnancy did not
indicate that LSD acted teratogenica11 y. The single incidence of
malformation (spina bifida) which occurred did not constitute sufficient
evidence to declare LSD a teratogen. Conversely, this single incidence
of malformation would tend to dispute the alleged teratogenicity of LSD
in the rat. Injections of LSD were given during the organogenic period
of development in the rat, a period which is typically sensitive to
drug insults (Saxen and Rapóla, 1969). In these experiments LSD did
not elicit deranged development, as shown by the lack of malformations.
Furthermore, LSD did not appear to interfere with development to the
extent of halting its progression, as evidenced by the absence of
significantly increased fetal mortality in experimental animals.
None of the previously described experiments indicated that
injection of LSD into pregnant rats would induce malformations in their
offspring. However, LSD injections did produce statistically signif-
leant differences between control and experimental animals with respect
to two other parameters. LSD injection (5.0 ug/kg b.w.) on day 4 of
pregnancy did increase fetal mortality significantly (p<0.05).
Whether this is to be accepted as valid proof of an abortifacient
effect by LSD depends on one's statistical viewpoint. However, 20 ug
LSD/kg b.w. injection did not evoke significantly increased fetal
30
mortality in experiments 3 and 4. Prenatal mortality was increased
over the control conditions but not at statistically significant
levels. In all groups which were treated with LSD, fetal mortality was
usually increased slightly.
The second parameter which significantly varied from control
values in animals injected with LSD was mean fetal wéight. The mean
fetal weight of offspring from day 4 intrauterine LSD injected adults
was significantly decreased. This parameter was significantly different
at the p<0.01 level, agreeing with results published by i-jbro and
Sieber (1968) for LSD experiments with the New Zealand white rabbit.
Fabro and Sieber's results indicated reduced fetal weights for offspring
of LSD treated does, which might be considered as involving a general
growth inhibiting factor. Experiments by Yielding, ^ (1969), have
indicated that LSD binds to deoxyribonucleic acid (DNA) in vitro. The
LSD absorption spectrum was not depressed by the addition of RNA to an
LSD solution. These experiments, with imaginative interpretation,
might lend some consideration for an LSD role in growth inhibition.
One Important reservation, the extent of fetal examination, might
be mentioned, since this project did not entail histological analysis
of offspring. Serial sectioning is the ideal technique for searching
out subtle malformations, but it is not a practical procedure (Wilson,
1965). The techniques of examination used in this study are those
currently accepted and utilized by the majority of teratologists.
Therefore, it is with some reservation that these results have been
Interpreted as not supporting the proposed teratogenicity of LSD in the
rat. These experiments have indicated the inability of LSD to induce
31
gross malformations in offspring of treated rats under the described
conditions.
SUMMARY
The effect of lysergic acid diethylamide (LSD) upon the developing
rat embryo (Holtzman strain) was studied. All injections were given on
days 4 or 9 of pregnancy. Both systemic (5.0 ug and 20.0 ug LSD/kg
b.w.) and intrauterine modes (2.52 ug/kg b.w.) of application were
tested.
Developmental malformations or congenital anomalies represent one
of the most immediate manifestations of reproductive failure. This was
the basis upon which adverse effects of LSD upon development were
monitored.
Based on data accumulated from autopsies and fetal examinations
during the described experiments, the following inferences were made:
1. In the described experiments LSD did not elicit
significant incidence of detectable anomalies.
2. Injection of 5.0 ug LSD/kg b.w. on day 9 of pregnancy
significantly increased prenatal mortality (p^0.05)
under the described experimental conditions. This
statement is made with reservations because of the
level of significance obtained and because the control
animals (control group 1) of this experiment were not
typical of subsequent control animals with respect to
their resorption rates.
3. Unilateral lU injections of 2.52 ug LSD/kg b.w. on day
4 of pregnancy reduced mean fetal weights.
33
4. These results are not complete proof that LSD is
non-teratogenic. Care must be exercised when extra-
polation from these data to human concern is desired.
This is especially important since this strain of
rat appears relatively resistant to exogenous
influences as exhibited by their slight rates of
spontaneous malformation and fetal mortality.
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JSÙSS2