Upsala J Med Sci 91: 29-36, 1986 

Induction of Skeletal Malformations 
in the Offspring of Rats Fed a 

Zinc Deficient Diet 
Johan Styrud, V. Elisabeth Dahlstrom and Ulf J. Eriksson 

Department of Medical Cell Biology, University of Uppsala, 
Uppsala, Sweden 

ABSTRACT 

P r e g n a n t  rats w e r e  subjected t o  a t r a c e  m e t a l  poor d i e t  (1.2 ppm zinc, 5.9 ppm 
copper, 40 ppm manganese) during t h e  e n t i r e  gestation. The r a t  mothers did not gain 
weight during pregnancy and showed d e c r e a s e d  liver weight and lowered s e r u m  glucose 
levels on g e s t a t i o n a l  day 20. The offspring exhibited d e c r e a s e d  body and p l a c e n t a l  
weights, delayed ossification of t h e  skeleton, and a n  increased resorption rate. We 
also found 4 % s k e l e t a l  malformations in t h e  offspring (0 % in t h e  controls), which 
closely resembled a t y p e  of malformation previously encountered in r a t s  when t h e  
m o t h e r  was manifest d i a b e t i c  (i..~. sacral dysgenesis). The z i n c  l e v e l s  w e r e  d e c r e a s e d  
and manganese levels increased t o  t h e  s a m e  e x t e n t  in offspring of trace m e t a l  r e s t r i c t e d  
( t h i s  study) and manifest d i a b e t i c  r a t s  (previous studies). F u r t h e r m o r e ,  when pregnant 
rats on t h e  trace m e t a l  r e s t r i c t e d  d i e t  w e r e  resupplemented with 75 ppm z i n c  in t h e  
drinking w a t e r  t h e  offspring largely normalized t h e i r  s o m a t i c  and p l a c e n t a l  growth, 
s k e l e t a l  maturation, as well as t h e i r  z i n c  and manganese levels. In addition, t h e  f e t u s e s  
of t h e  z i n c  resupplemented rats did not show a n y  malformations. The possibility of 
common t e r a t o l o g i c a l  mechanisms in m a t e r n a l  d i a b e t e s  and t r a c e  m e t a l  deficiency 
may t h e r e f o r e  be considered. 

INTRODUCTION 
The etiology behind disturbed f e t a l  growth and congenital malformations in t h e  

offspring of d i a b e t i c  m o t h e r s  i s  at p r e s e n t  n o t  clear (10,13,18,20,21). In previous exper- 
i m e n t a l  a t t e m p t s  t o  study t h i s  problem w e  have described a rat model f o r  d i a b e t i c  
pregnancy in which d e c r e a s e d  f e t a l  weight, delayed s k e l e t a l  ossification and a n  increased 
number of f e t a l  resorptions and s k e l e t a l  malformations w e r e  found (6,8). F u r t h e r m o r e ,  
t h e  t w o  types of skeletal malformations found in this r a t  model (micrognathia and 
sacral dysgenesis, cf. refs. no. 7-10) show a s t r o n g  g e n e t i c  dependence to t h e  rat s t r a i n  
used, s i n c e  w e  have been unable to induce t h e s e  malformations in t h e  offspring of 
manifest d i a b e t i c  rats f r o m  o t h e r  s t r a i n s  (11,121. In addition, w e  have shown t h a t  t h e  
offspring of t h e  d i a b e t i c  r a t s  in our model are zinc-deficient but have normal levels 
of copper and increased levels of manganese, thus establishing a link b e t w e e n  m a t e r n a l  

29 



d i a b e t e s  and f e t a l  t r a c e  m e t a l  disturbances (9). In t h e  p r e s e n t  s t u d y  w e  have used 
t h i s  malformation-prone rat s t r a i n ,  which is presently k e p t  in a colony in Uppsala (denoted 
TJ rats"), in a n  a t t e m p t  t o  e l u c i d a t e  t h e  t e r a t o g e n i c i t y  of trace m e t a l  disturbances 
in t h e  absence of m a t e r n a l  diabetes. 

MATERIALS AND METHODS 
R a t s  f r o m  our l o c a l  colony in Uppsala ("U rats") w e r e  used in t h e  p r e s e n t  investigation. 

This colony consists of a Sprague-Dawley derived s t r a i n  of albino rats in which t h e  
offspring consistently exhibit a 1 5 2 0 %  incidence of s k e l e t a l  malformations when t h e  
m a t e r n a l  rat has been m a d e  d i a b e t i c  with s t r e p t o z o t o c i n  before pregnancy (12). The 
total incidence of all t y p e s  of f e t a l  malformations is less t h a n  1 % in t h e  c o n t r o l  preg- 
nancies of this s t r a i n  (12). The (U) rats in t h e  p r e s e n t  study w e r e  all non-diabetic and 
kept in a light and t e m p e r a t u r e  controlled environment with a 1 2 + 1 2  h light-dark cycle. 
Two to t h r e e  f e m a l e  rats w e r e  housed per c a g e  and w e r e  f e d  s t a n d a r d  rat p e l l e t s  contain- 
ing 1 9 2  ppm zinc, 33 ppm copper and 1 3 2  ppm manganese (R 3, Ewos AB, Sodertalje, 

Sweden) and t a p  w a t e r  a d  libitum before t h e  o n s e t  of pregnancy. They w e r e  m a t e d  
overnight with male r a t s  from t h e  s a m e  colony and conception w a s  confirmed by examin- 
a t i o n  of vaginal s m e a r s  f o r  t h e  presence of sperm in t h e  morning. The d a y  when a 
positive vaginal s m e a r  w a s  obtained was denoted g e s t a t i o n a l  d a y  0 and f r o m  t h a t  day 

e a c h  pregnant animal was k e p t  in a stainless steel c a g e  (Stiilstandard AS, Norway) 
with a m e t a l  grid floor and without wood c u t t i n g s  in o r d e r  t o  p r e v e n t  t h e  rats from 
ingesting t r a c e  m e t a l s  from o t h e r  sources t h a n  t h e  food and drinking water. On gesta- 
tional day 0 t h e  s t a n d a r d  food was changed t o  a s p e c i f i c  trace m e t a l  poor diet: 1.2 
ppm zinc, 5.9 ppm copper, 40 ppm manganese (Ewos AB, Sodertalje, Sweden). About 
half t h e  pregnant animals received zinc supplemented drinking w a t e r  with a z i n c  concen- 
t r a t i o n  of 1 5  ppm (Solvezinc, Tika AB, Lund, Sweden), which w a s  markedly increased 

in comparison with ordinary t a p  w a t e r  (0.1 ppm zinc). T h e  amount of food and w a t e r  
consumed by t h e  pregnant rats was assessed t w i c e  a week. On g e s t a t i o n a l  days 0 and 
20 t h e  m a t e r n a l  s e r u m  glucose levels w e r e  e s t i m a t e d  (Glucose Analyzer 2 ,  Beckman 
Inc., Fullerton, CA, USA). T h e  c o n t r o l  pregnant rats on s t a n d a r d  d i e t  are d e n o t e d  N ,  
t h e  zinc deficient ZD, and t h e  z i n c  resupplemented rats are denoted ZDZ in t h e  following. 

On g e s t a t i o n a l  day 20, t h e  pregnant rats w e r e  weighed and killed by a blow to t h e  
neck. The m a t e r n a l  liver w a s  dissected o u t  a n d  weighed, t h e  f e t u s e s  w e r e  e x t e r i o r i z e d  

and examined with r e s p e c t  to weight, viability and o c c u r r e n c e  of f e t a l  malformations. 
From e a c h  u t e r i n e  horn o n e  f e t u s  was randomly chosen and processed f o r  whole body 
trace m e t a l  determination. These f e t u s e s  - and t h e  m a t e r n a l  livers - w e r e  placed in 
pre-weighed, acid-washed plastic beakers and c u t  u p  thoroughly with a p a i r  of s t a i n l e s s  
steel scissors and freeze-dried to c o n s t a n t  weight f o r  a b o u t  2 4  h (GT 2 F r e e z e  Drier, 
Leybold-Heraeus AG, West Germany), in o r d e r  to r e c o r d  t h e  specimens dry weights. 
They w e r e  t h e n  t r a n s f e r r e d  to a platinum crucible, ashed overnight in a t e m p e r a t u r e -  
programmed f u r n a c e  (Carbolite, Sheffield, U.K.) and analyzed by a t o m i c  absorption 

30 



spectrophotometry. Zinc, copper and manganese w e r e  d e t e r m i n e d  on a Varian AA-6 
i n s t r u m e n t  in a n  oxidizing air-acetylene f l a m e  at wavelengths of 213.9 nm, 324.7 nm 
and 279.5 nm, respectively. 

One non-malformed (chosen a t  random) and all t h e  malformed f e t u s e s  per individual 
horn w e r e  processed f o r  skeletal staining (24). These f e t u s e s  w e r e  e v i s c e r a t e d ,  fixed 

in 7 0  % e t h a n o l  f o r  10-30 days, t r e a t e d  with 1 % K O H  and s t a i n e d  with Alizarin Red 

as described previously (7). Subsequent evaluation of s k e l e t a l  development was m a d e  
by counting t h e  visualized ossification c e n t e r s  in six d i f f e r e n t  locations in a c c o r d a n c e  

with Aliverti and coworkers (1). The probability (p) of a c h a n c e  d i f f e r e n c e  b e t w e e n  
means was e s t i m a t e d  by Student's t w o  tailed t-test. D a t a  are given as means k S.E.M. 

Table I. Body weight, body weight gain during pregnancy, liver weight and serum glucose 
c o n c e n t r a t i o n  of t h e  normal (N), zinc-deficient (ZD) and zinc-resupplemented 
(ZDZ) pregnant rats on g e s t a t i o n a l  day 20. Means * S.E.M. 

GROUP No.of Maternal Maternal Maternal Maternal 

(g) day 0-day 20 (g) (m m o W  
r a t s  body weight weight gain liver weight serum glucose 

(g) 

N 3 371 * 26 137 k 1 5  11.7 2 0.5 5.6 k 1.3 
ZD 11 242 k 8'" -1 k 5'" 7 . 7  ? 0.4"' 2.3 ? 0.3'" 

ZDZ 6 356k11 112+15++ 11.0+ 0.3 3.2 f 0.3" 
~ 

Significances: ++ p<0.01 vs. N rats; +++ ~ < O . O O I  vs. N rats. 

RESULTS 

During g e s t a t i o n a l  days 0-20 t h e  normal (N), zinc-deficient (ZD) and zinc-deficient 

and resupplemented r a t s  (ZDZ) consumed t h e  s a m e  a m o u n t  of food and w a t e r  ( d a t a  n o t  

shown). The body weight and body weight i n c r e a s e  during pregnancy d i f f e r e d  significantly 

between t h e  groups, t h e  ZD group did not gain any weight at all, whereas t h e  ZDZ rats 
showed normal body weights on g e s t a t i o n a l  day 20 (Table I). T h e  ZD m a t e r n a l  r a t s  exhi- 

bited d e c r e a s e d  liver weights on d a y  20 in c o n t r a s t  t o  t h e  ZDZ r a t s  whose m a t e r n a l  liver 

weight did n o t  d i f f e r  f r o m  t h e  normals (Table I). On g e s t a t i o n a l  day 0 t h e r e  w e r e  no dif- 

f e r e n c e s  in serum glucose levels b e t w e e n  t h e  groups ( d a t a  n o t  shown), but on pregnancy 

day 20, t h e  s e r u m  glucose levels of t h e  ZD and ZDZ r a t s  w e r e  both significantly d e c r e a s e d  

in comparison with t h e  N rats (Table I). 
The number of viable offspring was slightly lowered in t h e  ZDZ group (Table 11). The 

number of non-viable f e t u s e s  was increased in both t h e  ZD and ZDZ groups, t h i s  i n c r e a s e  

was most pronounced in t h e  ZD group. F e t a l  body and p l a c e n t a l  weights w e r e  slightly low- 

er t h a n  normal in t h e  ZDZ group, whereas t h e y  w e r e  f u r t h e r  d e c r e a s e d  in t h e  ZD group 
(Table 11). In four of t h e  eleven ZD l i t t e r s  a t o t a l  of f i v e  malformed f e t u s e s  w e r e  found. 

31 



Table 11. Number of viable and non-viable offspring (per u t e r i n e  horn), f e t a l  body weight 
and p l a c e n t a l  weight of t h e  offspring of normal (N), zinc-deficient (ZD) and 
zinc-resupplemented (ZDZ) rat m o t h e r s  on g e s t a t i o n a l  d a y  20. Means ? S.E.M., 
n d e n o t e  t h e  number of u t e r i n e  horns examined. 

GROUP n No. of No. of F e t a l  P l a c e n t a l  
viable non-viable body-weight weight 

offspring offspring (g) (g) 

N 6 6.5 * 0.6 0.2 * 0.2 4.6 2 0.3 0.50 ? 0.01 
ZD 2 2  5.7 ? 0.5 1.3 i 0.3"' 2.7 ? 0.1'" 0.39 ? 0.01+++ 

ZDZ 1 2  5.3 2 0.4' 0.6 t 0.1"' 3.8 ? 0.2"' 0.49 ? 0.01' 

Significances: + p<0.05 vs. N fetuses; ++ pC0.01 vs. N f e t u s e s ;  +++ p<O.OOl vs. N fetuses. 

All f i v e  f e t u s e s  had malformation of t h e  s a c r a l  dysgenesis t y p e  (Figure I, cf. refs. no. 6, 
9 , l l ) .  The rate of malformation was 3.9% (5/128) in t h e  ZD group, whereas i t  was z e r o  in 
t h e  N (0/39) and ZDZ (0/63) groups. 

The f i v e  malformed f e t u s e s  in t h e  ZD group weighed t h e  s a m e  (2.7 2 0.1 g) as t h e  non- 
malformed ones, whereas t h e  p l a c e n t a e  of t h e  malformed f e t u s e s  w e r e  slightly lighter 

(0.34 k 0.02 g) t h a n  t h e i r  non-malformed l i t t e r m a t e s  (p < 0.001, Table 11). 

Figure I. Alizarin R e d  s t a i n e d  day-20 f e t u s e s  f r o m  normal (N, l e f t ) ,  zinc-deficient (ZD, 
middle), and zinc-resupplemented (ZDZ, right) rats. The ZD f e t u s  shows s a c r a l  dysgenesis, 
(cf. refs. no. 7-10,121 t h e  o t h e r  t w o  f e t u s e s  show normal morphology. 

32 



Table 111. Maternal liver c o n c e n t r a t i o n s  of trace m e t a l s  in n o r m a l  (N), zinc-deficient (ZD) 
and zinc-resupplemented (ZDZ) rats on g e s t a t i o n a l  day 20. Means f S.E.M. 

GROUP No. of CONCENTRATION (mg/kg dry liver weight) 
rats 

ZINC COPPER MANGANESE 

N 

ZD 

ZDZ 

3 

11 

6 

104 ? 3 16.0 ? 0.4 8.1 2 0.3 

81 5 2'" 13.8 f 0.3'" 7.9 2 0.4 

104 f 2 16.0 2 0.2 7.2 ? 0.1"' 

Significances: + p<0.05 vs. N rats; ++ pC0.01 vs. N r a t s ;  +++ p<O.OOl vs. N r a t s .  

The m a t e r n a l  livers of t h e  ZD rats a c c u m u l a t e d  less zinc and copper during g e s t a t i o n  

(Table 111). The ZDZ rat showed normal z i n c  and copper levels, whereas t h e  accumulation 
of manganese w a s  lowered (Table 111). T h e  ZD f e t u s e s  exhibited g r e a t l y  diminished ac- 
cumulation of z i n c  and slightly increased l e v e l s  of manganese (Table IV).The ZDZ f e t u s e s  

showed less pronounced a l t e r a t i o n s  in t h e i r  accumulation of trace m e t a l s  t h a n  t h e  ZD 
offspring, but nevertheless had lowered zinc and increased manganese levels compared t o  

t h e  controls (Table IV). The copper l e v e l s  w e r e  similar in all groups of f e t u s e s  (Table IV). 
The sum of ossification c e n t e r s  in s i x  d i f f e r e n t  locations (sternum, m e t a c a r p u s ,  m e t a t a r -  

sus, a n t e r i o r  and posterior proximal phalanges, caudal v e r t e b r a e ,  cf. ref. no. 1 )  was de- 
c r e a s e d  in t h e  ZD f e t u s e s  and normal in t h e  ZDZ offspring (Figure 11). 

Table IV. T o t a l  body concentrations of trace m e t a l s  in f e t u s e s  of normal (N), zinc-deficient 
(ZD) and zinc-resupplemented (ZDZ) rats on g e s t a t i o n a l  day 20. Means f S.E.M. 

GROUP No. of CONCENTRATION (mg/kp d r y  body weight) 

ZINC COPPER MANGANESE f e t u s e s  

N 

ZD 

ZDZ 

6 1 3 1  2 1 10.920.4 

2 2  1 0 5  * 2"' 10.6 ? 0.4 
1 2  122 f 2+++ 10.7 f 0.3 

0.920 .O 2 

1.1 2 O.l+++ 

1.0 f 0.1+++ 

Significances: + p<0.05 vs. N fetuses; ++ pC0.01 vs. N fetuses; +++ p<O.OOl vs. N fetuses. 

3 -868571 33 



DISCUSSION 

The major finding in t h i s  s t u d y  was t h e  demonstration of fetal malformations in t h e  
t r a c e  metal-restricted pregnancies, of a t y p e  similar to those appearing in t h e  offspring 
of manifest diabetic rats (7,8). In addition, t h e  pregnant ( U )  r a t s  on t h e  t r a c e  m e t a l  poor 
d i e t  produced f e t u s e s  with s e v e r a l  c h a r a c t e r i s t i c s  i n  common with offspring of manifest 
d i a b e t i c  (U) rats (on s t a n d a r d  diet): decreased f e t a l  weight ( 6 , 7 ) ,  increased resorption rate 
( 6 , 7 ) ,  delayed s k e l e t a l  ossification (7,8), as well as d e c r e a s e d  z i n c  and increased manganese 
accumulation in t h e  f e t u s e s  (9). This suggests, t h a t  t h e  t e r a t o g e n i c  processes i n  t h e  t w o  
d i f f e r e n t  t y p e s  of (U) rat pregnancy -&g. m a t e r n a l  t r a c e  m e t a l  deficiency and m a t e r n a l  
d i a b e t e s  - may s h a r e  s o m e  etiological mechanisms. The i m p o r t a n c e  of zinc in t h i s  c o n t e x t  
is i l l u s t r a t e d  by t h e  e f f e c t s  of i t s  administration to t h e  trace m e t a l  r e s t r i c t e d  rat mothers. 
In t h e  ZDZ l i t t e r s  t h e r e  w e r e  no malformations, t h e  s k e l e t a l  ossification was normal and 
t h e  f e t a l  resorption r a t e ,  body and placental weights w e r e  also largely normalized. O t h e r  
a u t h o r s  have suggested t h a t  changes in m a t e r n a l  l e v e l s  of trace m e t a l s  may b e  involved 
in t h e  induction of congenital malformations both in humans (2,3,14,17,23) and animals 
(15,16). Diabetic individuals a r e  f u r t h e r m o r e  known to lose t r a c e  metals, via increased uri- 
nary output in relation to t h e i r  d e g r e e  of m e t a b o l i c  c o n t r o l  (19). A l t e r e d  levels of trace 
metals, in particular zinc, may t h e r e f o r e  b e  of significance f o r  t h e  induction of malforma- 
t i o n s  i n  d i a b e t i c  pregnancy. T h e  possible t e r a t o g e n i c  a c t i o n s  of a n  embryo-fetal zinc de- 

z 
a, 
t 
ar 
0 
C 
0 

a 
0 = 
v) 
v) 
0 

0 

Z 

4- 

.- 
c 

.- 

.c 

0 

30 

20 

10 

0 

I 

6 

N ZD ZD 
MALF 

L 

a 
ZDZ 

Figure 11. T o t a l  number of ossification c e n t e r s  in s i x  a n a t o m i c a l  l o c a t i o n s  (cf. ref. no. 1) 
in t h e  non-malformed and malformed (MALF) f e t u s e s  of n o r m a l  (N), zinc-deficient (ZD) 
and zinc-resupplemented (ZDZ) rats on g e s t a t i o n a l  day 20. Means i S.E.M. Significance: 
+++ p<O.OOl vs. N fetuses. 

34 



ficiency are several. This trace m e t a l  s e r v e s  as a co-factor of a number of enzymes, g.g. 
thymidine kinase, DNA-polymerase and superoxide dismutase, and a deficiency may t h e r e -  
f o r e  inhibit t h e  biosynthesis of DNA or affect t h e  defense a g a i n s t  f r e e  oxygen radicals in 
t h e  conceptus ( 4 , 5 , 2 2 ) .  The significance of t h e  concomitantly increased f e t a l  manganese 
levels in t h i s  s t u d y  as well as in f e t u s e s  of manifest d i a b e t i c  rats ( 9 )  remains t o  b e  eluci- 

dated. 

explain all t h e  malformations s e e n  in t h e  d i a b e t i c  r a t  pregnancy, s i n c e  t h e  frequency of 
s k e l e t a l  malformations was markedly lower in t h i s  investigation t h a n  in t h e  offspring of 
manifest d i a b e t i c  ( U )  r a t s  (4% versus 15-20%, cf. refs. no. 7 - 8 ) .  F u r t h e r m o r e ,  a l l  t h e  mal- 
f o r m a t i o n s  w e r e  of t h e  s a c r a l  dysgenesis t y p e  in t h e  offspring of t r a c e  m e t a l  r e s t r i c t e d  
r a t s ,  whereas in d i a b e t i c  rat pregnancy both micrognathia and sacral dysgenesis are en- 
c o u n t e r e d  (7-10). In addition, manifest d i a b e t i c  r a t s  of t h i s  malformation-prone s t r a i n  
a p p e a r  to give birth t o  a g r e a t e r  proportion of micrognathias, when t h e  m o t h e r  is zinc- 
supplemented (Eriksson, unpublished). A l t e r e d  f e t a l  zinc levels may t h e r e f o r e  play a role 
in t h e  induction of congenital malformations, and may b e  of s p e c i f i c  significance f o r  t h e  
etiology of t h e  s a c r a l  dysgenesis syndrome. This idea a w a i t s  f u r t h e r  investigation. 

A trace m e t a l  disturbance of t h e  t y p e  produced in t h e  p r e s e n t  s t u d y  may not, however, 

ACKNOWLEDGEMENTS 
The a u t h o r s  a r e  g r a t e f u l  to a s s o c i a t e  professor P e r  Mattsson, The Swedish National 

Food Administration f o r  help with t h e  analyses of trace metals, and to Lars J o r h e m ,  Arne 
P e t t e r s s o n  and Birgitta Sundstrom f o r  e x p e r t  t e c h n i c a l  a s s i s t a n c e  with a t o m i c  absorption 
spectrophotometry. Astrid Nordin, Olov Rosendal and P a r r i  Wentzel are acknowledged 
f o r  t h e i r  help in care of t h e  animals a n d  tissue sampling. This investigation w a s  supported 
by t h e  Swedish Medical R e s e a r c h  Council ( 1 2 X - 1 0 9 ,  Post-Doctoral Fellowship 12P-6346 
t o  UJE), t h e  "Expressen" P r e n a t a l  R e s e a r c h  Foundation, The Medical Faculty of t h e  Uni- 
v e r s i t y  of Uppsala, The Swedish D i a b e t e s  Association and The Nordic Insulin Fund. 

% REFERENCES 
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f e t a l  ossification as a n  index of delayed development in t e r a t o g e n i c  s t u d i e s  on t h e  
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Adress r e p r i n t  r e q u e s t s  to: 

Dr. Ulf J. Eriksson 
D e p a r t m e n t  of Medical C e l l  Biology 
University of Uppsala 
Biomedicum 
P.O. Box 571 
S-751 23 Uppsala 
Sweden 

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