464 RICHARD L. W I L L I A M S A N D GEORGE M. S T E W A R T

L Coates, .1. Β., Jr., and Beyer, J. C.: Wound ballistics. Office of the Surgeon General Department of the Army, 1962.

2. Medical Statistics Division Report, OTSG, 11 December 1953. 3. Stewart, G. M . : Eye protection against small high-speed missiles. Am. J. Ophth., 51:80 (Jan.) 1961. 4. Rose, H. W., and Stewart, G. M . : Eye protection against small high-speed missiles, Tr. Am. Acad.

Ophth. May-June, 1957. 5. Berens, C.: The Eye and Its Diseases. Saunders, Philadelphia and London, Saunders 1949, ed. 2. 6. Medical Dept., U. S. Army, Surgery in World War II, Ophthalmology and Otolaryngology, Office

of the Surgeon General, Washington, D.C., 1957. 7. Tillett, C. W., Rose, H. W., and Herget, C.: High speed photographic study of perforating ocular

injury by the BB. Am. J. Ophth., 54:675 (Oct.) 1962.

F U R T H E R S T U D I E S O N T H E E F F E C T S O F I N T R A U T E R I N E

V A S O C O N S T R I C T O R S O N T H E F E T A L R A T L E N S *

M A R T H A PITEL, P H . D . , AND SIDNEY L E R M A N , M . D . Rochester, New York

INTRODUCTION

In a previous study by Pitel and Lerman,' it was demonstrated that adrenalin and nor-adrenalin, potent vasoconstrictors, produced partial or complete opacification of the fetal rat lens when injected directly into the fetus during the latter part of gestation. In an attempt to determine whether the mechanism of action was primarily a vasoconstrictive one, various known vasoconstrictors, spe­cifically caffeine, ergot and nicotine, were employed in the present investigation.

Nishimura and Nakai^ have shown that nicotine has a teratogenic effect upon the developing fetal mouse when administered intraperitoneally to the pregnant animal. In­troduction of the teratogen during the critical period from nine to 11 days of pregnancy evoked mainly skeletal defects predominantly of the limbs, such as adactyly, syndactyly, brachydactyly and so forth. Similar anom­alous lesions plus a high percentage of hematomas were obtained by the same in­vestigators'''* with large doses of caffeine.

Landauer^ has indicated that nicotine also plays a teratogenic role in provoking abnor­malities of the cervical vertebrae in chicken

* From the Departments of Anatomy and Sur­gery (Ophthalmology) of the University of Roch­ester School of Medicine and Dentistry. This study was supported by U.S.P.H.S. grant B-3067.

embryos when injected directly. It is inter­esting to note that no ocular defects were reported by these investigators.

Opacities of the lens were, however, pro­duced in newborn rabbits by the Bulgarian investigators Vassilev, Dabor and Rankov" upon intravenous injection of ergotamine tartrate into the pregnant animal on the 10th day of gestation.

Thus known vasoconstrictors have pro­duced anomalies in the offspring when intro­duced into the mother at a critical period of gestation early in pregnancy.

Although all the substances employed in the studies just described and in the present study produce vasoconstriction, they do so by different modes of action. Caffeine is a potent stitmilator of the vasomotor center in the medulla; whereas, ergot acts directly on the peripheral blood vessels.^ Nicotine, on the other hand, induces peripheral vaso­constriction by central vasomotor and sympa­thetic ganglionic stimulation with a release of adrenalin from the adrenal medulla, as well as a direct constrictor effect on the blood vessels.'

MATERIALS AND METHODS

A. HISTOPATHOLOGIC STUDIES

Dated pregnant females of the Holtzman and Carworth strains of white albino rats

E F F E C T S O F I N T R A U T E R I N E V A S O C O N S T R I C T O R S 465

T A B L E 1

HISTOPATHOLOGIC STUDIES

Vasoconstrictors Fetuses Injected

(No.)

Day of Gestation Injected

Dose Range

(gammas)

Strains Injected (No.) Fetuses Alive Fetuses Dead Vasoconstrictors

Fetuses Injected

(No.)

Day of Gestation Injected

Dose Range

(gammas) Carworth Holtzman No. % No. % Caffeine sodium

benzoate 64 16th 17th 18th

25-50 50-150

105-300

8 8

19

3 26 35 55 29 45

Ergotamine bitartrate 51

17th 18th 19th

5-50 5-125

25-5

6 19

19

7 17 33 34 67

Nicotine sulfate

32 16th 17th

10 10 8

14 10

13 41 19 59

TOTAL 147 65 44 82 56

were employed in this experiment. A total of 147 fetuses were injected with one of the following vasoconstrictors: caffeine sodium benzoate, ergotamine tartrate and nicotine sulfate. Saline was injected into the con­trols. All fetuses were injected via the in­trauterine method as previously described.* The dosage range differed for each vasocon­strictor injected on the 16th, 17th, 18th or 19th day of gestation as indicated in Table 1.

All o f the pregnant animals were killed on the 21st day of gestation via ether anesthesia. The fetuses were examined macroscopically for gross abnormalities such as ocular defects and limb abnormalities. They were then de­capitated and the heads were placed in Bouin's fixative for at least five days, im­bedded in paraffin, sectioned at 7.0 μ, and stained with hematoxylin and eosin. Micro­scopic sections were then examined for anomalies.

B . BIOCHEMICAL STUDIES

A total of 111 fetuses (table 3 ) (Hol tz­man strain) were injected with 50 gamma caffeine on the 17th day of gestation and were divided into three groups to be killed on the 18th, 19th and 20th day of gestation, respectively. The control groups, consisting of a total of 122 fetuses, were injected with saline and treated in a similar manner. A third category of fetuses (normal groups)

received no injections and were killed at the same time periods.

All the lenses were carefully removed and treated according to the methods described in previous communications.*'^ The number of lenses employed in the incubation experi­ments varied between three and six per in­cubation vessel.

RESULTS

A . HISTOPATHOLOGIC STUDIES

O f the vasoconstrictors selected for this study, only caffeine exerted a teratogenic effect upon the developing fetal lens. The findings are described later under histo­pathologic findings. The fetal deaths were considerable, with slightly more than half of the animals dead on the 21st day of gesta­tion. The highest percentage of deaths (67 percent) were found in those fetuses in­jected with the ergot preparation (table 1 ) . Killing some of the animals 24 and 48 hours after injection decreased the fetal loss to one third (33 percent, as shown in Table 3 ) .

Since the greatest number of fetuses ( 7 7 ) were injected on the 17th day, the greatest number of cataractous changes occurred in this particular group (table 2 ) . The varia­tion in dosage did not seem to have any appreciable influence upon the teratogenic action of the vasoconstrictor administered. But there was a marked difference in the

466 M A R T H A PITEL A N D SIDNEY L E R M A N

TABLE 2

HISTOPATHOLOGIC STUDIES

Caffeine Sodium

Benzoate (Dosage in gammas)

Day of Gestation Injection

Fetuses Fetuses Alive (No.)

Fetuses Dead (No.)

Eye Opacities

Strain of Animal

Caffeine Sodium

Benzoate (Dosage in gammas)

Day of Gestation Injection

Day of Death

In­jected (No.)

Fetuses Alive (No.)

Fetuses Dead (No.) Complete

No. % Partial

No. % Slight

No. % Normal No. %

Carworth Carworth Carworth

25-50 80-150

123-300

16th 17th 18th

21st 21st 21st

8 8

19

0 2

12

8 6 7 1 8 Ξ Ξ 11 92

Holtz man Holtzman Holtz man

50 50

80-125

16th 17th 17th

21st 21st 21st

3 20

6

1 17 3

2 3 3

1 6 6 35 1 100 4 24 6 35

3 100

strain response (table 2 ) . The Carworth animals are more resistant than the Holtz­man strain.

One finding which is worthy of mention here and will be discussed in detail in a fu­ture publication is that of the effect upon the developing limbs of the fetus. Hemor­

rhagic lesions and complete necrosis were noted in several fetuses injected with each of the selected vasoconstrictors.

Histopathologic findings. The lenses from the groups of fetuses injected with nicotine and ergot were normal except for one which showed severe degenerative changes. The histologic structure of the lenses as a whole revealed slight vacuolization of the lens fibers, which was also found in the control lenses. Thus the majority of the lenses were considered essentially normal (figs. 1 to 3 ) .

In one animal complete opacification o f the lens was produced by nicotine sulfate ( 1 0 gamma) injected on the 17th day of ges­tation. Necrotic debris, gross vacuolization of the lens fibers and pyknotic nuclei were found throughout the lens (fig. 4 ) .

Cataractous changes were most prevalent in the group injected with caffeine (particu­larly in the Holtzman strain). Table 2 indi-

Fig. 1 (Pitel and Lerman). Minimal changes in lens fibers. Fetus injected with SO gammas of ergot preparation on the 17th day of gestation and killed on the 19th day.

Fig. 2 (Pitel and Lerman). Lens normal. Fetus injected with 10 gamma of nicotine preparation on the 17th day of gestation and killed on the 21st day.

E F F E C T S O F I N T R A U T E R I N E V A S O C O N S T R I C T O R S 467

T A B L E 3*

BIOCHEMICAL STUDIES

Experimentais Controls

Day of Gestation

Day of Gestation

Fetuses Injected

Fetuses Alive

Fetuses Dead

Fetuses Injected

Fetuses Alive

Fetuses Dead

Injected Killed No. No. % No. % No. No. % No. % 17th 17th 17th

18th 19th 21st

60 37 68

34 29 48

57 78 71

26 8

20

43 22 29

49 29 44

32 20 32

65 67 73

17 9

12

35 23 27

TOTAL 165 111 67 54 33 122 84 69 38 31

* All fetuses were of the Holtzman strain. The experimental animals were injected with SO gammas of caffeine and the controls with saline.

cates that 11 out of 17 fetuses injected with 50 γ of caffeine had some degree of cata­ractous change (fig. 5). Partial opacification of the lens was observed in only one fetus of the Carworth strain which had received 300 γ of caffeine on the 18th day of gesta­tion.

In Figure 6 the lens fibers in the equatorial region extending from the anterior to pos­terior poles show extensive degeneration with proliferation of the anterior epithelial cells in the area of fiber destruction. A hem­orrhage into the anterior chamber has also occurred.

B. BIOCHEMICAL STUDIES

A comparison of the total protein levels in the three groups of lenses under study is shown in Figure 7. It is interesting to note that by the 20th day of gestation the concen­tration of total protein in the normal, saline and caffeine lenses had reached similar levels. However, if this data is further analyzed with respect to the concentration of soluble and insoluble protein in these lenses (fig. 8), it becomes apparent that there is an actual decrease in the soluble protein level of the saline and caffeine lenses as compared with the normal group, while the concentration of insoluble protein is significantly higher in the two experimental groups.

The concentration of ATP per lens re­mains relatively imchanged in all three groups during the 18th and 19th day of ges­

tation (table 4) but, after this period, the level of ATP in the saline and caffeine lenses is considerably lower as compared with the normal values.

Table 5 shows the results of the lactic acid determinations in the three groups of

Fig. 3 (Pitel and Lerman). Control eye. Fetus injected with saline on the 17th day of gestation and killed on the 21st day.

468 M A R T H A PITEL A N D SIDNEY L E R M A N

T A B L E 4

CONCENTRATION OF A T P

Material Injected

Day of Gestation Injected

ATP (mg./gm. protein)

18 3.83 Caffeine 19 6.60

21 3.00

18 S..30 Saline 19 5.70

21 3.80

17 3.90 Normal 19 3.40

21 6.80

Fig. 4 (Pitel and Lerman). Complete destruc­tion of the lens. Fetus injected with 10 gammas of nicotine preparation on the 17th day of gestation and killed on the 21st day.

lenses. At the 18th day of gestation the saline and caffeine lenses show a lactate level of 10.7 mg. /gm. lens protein and 10.5 mg. /gm. protein respectively. This compares with a concentration of 40 mg./gm. protein in the 17-day normal lens. The amount of lactic acid remains relatively constant in the caf­feine and saline groups but shows a marked

decline to 15.5 mg. /gm. protein by the 21st day in the normal group.

The results o f the incubation experiments involving the recovery of C^^Oa from lenses incubated with g lucose - l -C" or glucose-6-C** are shown in Table 6. In the normal group the recovery of C " 0 2 from the lenses incubated with glucose-l-C** remained rela­tively constant but there appeared to be an increase in the recovery of C " 0 2 from the glucose-6-C" incubates at day 21 as com­pared with days 18 and 19.

The caffeine group showed a decrease in C**02 recovery from glucose-6-C*'' at day 18, and by the 21st day o f gestation this recovery was less than 10 percent of the normal value. There was a similar but less marked fall in the C " 0 2 level from g lucose - l -C" which oc­curred at day 21. The saline group showed

Fig. 5 (Pitel and Lerman). Partial destruction of lens. Fetus injected with 50 gammas of caffeine preparation on the 17th day of gestation and killed on the 21st day.

T A B L E 5

LACTIC ACID DETERMINATIONS

Material Injected

Day of Gestation Injected

Lactate (mg./gm, protein)

18 10.5 Caffeine 19 11 .9

21 9.5

18 10.7 Saline 19 14.1

21 13.0

17 40.0 Normal 19 20.9

21 15.5

E F F E C T S O F I N T R A U T E R I N E V A S O C O N S T R I C T O R S 469

T A B L E 6

RESULTS OF INCUBATION EXPERIMENTS

Material Injected

Day of Gestation Injected

C"02 Recovered

from Glucose-l-C"*

C'Oj Recovered

from Glucose-6-C"*

18 7,000 1,500 Caffeine 19 11,300 1,500

21 5,000 ~ 4 0 0

18 19,800 2,300 Saline 19 9,400 2,100

21 10,300 2,800

18 11,400 3,000 Normal 19 12,400 2,400

21 13,500 4,500

Expressed as cpm./gm. protein.

a relatively constant level of C " 0 2 recovered from glucose-1 and glucose-6-C" during the period under observation except for an in-

.250 Γ

.200 h

5 .150

r ξ-fe .100

.050 h

17 18 19 20 21

AGE OF FETUS, days

Fig. 7 (Pitel and Lerman). Total protein levels in the normal and experimental fetal lenses.

creased recovery of Ο ' Ό ζ from glucose-1-C" at day 18.

DISCUSSION

Although the substances employed in this study have a profound vasoconstrictive effect in the adult animal, the fetus is apparently more resistant to the exogenous insult. The vasoconstrictors generally demonstrated a high degree of toxicity, as is evidenced by the high percentage of fetal deaths. But those fetuses which withstood the action of the vasoconstrictor did so without destruction of the target organ, the lens, which is so ex­tremely vulnerable to a number of known teratogenic agents.

.200 Γ

Fig. 6 (Pitel and Lerman). Partial destruction of lens. Fetus injected with 300 gammas of caffeine preparation on the 18th day of gestation and killed on the 21st day.

18 19 AGE OF FETUS, doys

Fig. 8 (Pitel and Lerman). Soluble and insoluble protein levels in the normal and experimental fetal lenses.

470 M A R T H A P I T E L A N D S I D N E Y L E R M A N

A n interesting feature is the high degree

of susceptibility of the Holtzman strain of

rats to exogenous environmental alterations;

whereas, the Carworth strain displays a

greater tolerance and resistance to environ­

mental modifications. Note in Table 1 the

quantitative response to the teratogens.

Since a dosage range for any substance is

very difficult to ascertain for the fetus, the

guide lines provided were those used in the

adrenalin-noradrenalin study.* These sub­

stances were most destructive to the lens in

the range of 25 to 50 gamma. With the pres­

ent studies, the specificity of adrenalin-nor­

adrenalin for the lens is further validated in

light of the negative teratogenic effect o f

some of the presently employed vasocon­

strictors.

The results of the biochemical studies on

the effects of the intrauterine administration

of caffeine indicate that this substance does

cause some derangement in the metabolism

of the fetal lens, as evidenced by a decrease

in A T P , lactate and soluble protein concen­

tration and by a marked fall in the recovery

of C " 0 2 from glucose-1 and glucose-6-C**.

However, the intrauterine injection o f saline

also gives rise to a similar but less pro­

nounced effect, except for the data concern­

ing the C^Oa recovery experiments. In these

studies saline does not appear to exert any

significant effect on the recovery of C ^ O s

from glucose-l-C**, but the recovery from

glucose-6-C*^ fails to increase from day 19

to day 21 as compared with the normal lens.

Further analysis of this data is also of in­

terest in that it tends to corroborate previous

obser\'ations'' that the Krebs cycle does not

appear to be functioning to any significant

extent until the 19th to 21st day of gestation.

It is noteworthy that the lactate levels in the

normal lens begin to decline after the 19th

day and there is a concurrent increase in the

recovery o f C " 0 2 from glucose-6-C". A

sharp rise in the concentration of A T P also

becomes apparent after day 19.

The data presented here together with

data previously reported^ would indicate that

the fetal lens oxidizes glucose by means of

the hexose monophosphate shunt and anaero­

bic glycolysis and derives its A T P from these

pathways until the 20th day of gestation. A t

this time the respiratory phase of glycolysis

appears and gives rise to a significant incre­

ment in high energy phosphate.

260 Crittenden Boulevard (20).

REFERENCES

1. Pitel, M., and Lerman, S.: Studies on the fetal rat lens. Invest. Ophth., 1:406-412, 1962. 2. Nishimura, H., and Nakai, K.: Developmental anomalies in offspring of pregnant mice. Science,

127:877-878, 1959. 3. : Congenital malformations in offspring of mice treated with caffeine. Proc. Soc. Exper. Biol.

&Med., 104:140-142, 1960. 4. Nakai, K. : Relation of occurrence between hematomas and malformation induced on mouse embryos

by administration of large doses of caffeine during gestation. Acta Anat. Nippon, 36:521-528, 1961. 5. Landauer, W . : Nicotine-induced malformations of chicken embryos and their bearing on the

phenocopy problem. J. Exper. Zool. 143:107-122, 1960. 6. Vassilev, L, Dabov, S., and Rankov, B.: Contribution å I'etude de Taction de I'ergotine sur les yeux.

Arch. Ophtal., 19 :524-528, 1959. 7. Fein, H . D. : Modern Drug Encyclopedia and Therapeutic Index. New York, Donnelley, 1961. 8. Goodman, L., and Oilman, A . : The Pharmacological Basis of Therapeutics, New York, Macmillan,

1%1. 9. Lerman, S., Donk, J., and Pitel, M . : Further studies on the metabolism of the fetal rat lens. Growth,

26:103-109, 1962.