Effects of combinations of maternal agents on the fetal cerebrum in rat—Ethanol or caffeine with X-irradiation in utero—

Effects of Combinations of Maternal Agents on the Fetal Cerebrum in Rat-Ethanol or Caffeine with X-Irradiation in UteroHarumi Tanaka, MD, Setsuo Iwasaki, PhD, Masataka Arima, MD and Kazuharu Nakazawa, MSc Fetal cerebral development influenced by maternal ethanol or caffeine either singly or in combination with X-irradiation was investigated in rat. Female Wistar rats were given 20% ethanol, 0.04% caffeine and water during the premating period and pregnancy, and 0.03% vitamin E only during pregnancy. Pregnant rats were X-irradiated with 100R or sham-irradiated on gestational day 13. Ethanol-treatment alone much reduced the fetal body and cerebral weights, and X-irradiation alone resulted in great reductions in weight and DNA concentration in the fetal cerebrum. The reduction in body weight with ethanol exceeded that with X-irradiation, therefore, the addition of X-irradiation had no effect on that of ethanol. The reduction in cerebral weight on X-irradiation exceeded that with ethanol, thus the addition of ethanol had only a slight effect on that with Xirradiation. The decrease in body and cerebral weights and the increase in lipid peroxide (LP) formation on caffeine-treatment and the decrease in cerebral weight and the increase in LP on vitamin E-treatment were inhibited by X-irradiation as compared to the combined effects of the other drink treatments. The increase in placental weight and the decrease in cerebral weight on ethanol-treatment and the decrease in placental, body and cerebral weights on caffeine-treatment, which findings were covered by the addition of X-irradiation, became much clearer on single drink treatment. Independently of X-irradiation, ethanol-treatment resulted in increased fetal mortality and LP, and decreased body weight. Th ese results suggest that the combined effects of maternal agents on live fetuses should be investigated as to whether they act independently of or dependently with each other and how the effects appear either singly or mixed. Tanaka H, Iwasaki S, Arima M, Nakazawa K. Effects of combinations of maternal agents on the fetal cerebrum in rat ethanol or caffein with X-irradiation in utero. BrainDev 1985;7:10-20

As preventable causes of central nervous system dysfunctions in offspring, maternal environmental factors have been more frequently From the Division of Mental Retardation and Birth Defect Research, National Center for Nervous, Mental and Muscular Disorders, Kodaira, Tokyo. Received for pUblication: November 15, 1984. Accepted for pUblication: January 18, 1985.

Key words: Ethanol, caffeine, X-irradiation, lipid peroxide, DNA, pregnancy, fetal rat, cerebrum, vitaminE. Correspondence address: Dr. Harumi Tanaka, Division of Mental Retardation and Birth Defect Research, National Center for Nervous, Mental and Muscular Disorders, Ogawa-Higashi-Machi, Kodaira, Tokyo 187, Japan.

recognized during the past decade. Many clinical and animal studies have shown that maternal ethanol consumption may have toxic effects on the fetus [1- 4] . The possible harmful effect of caffeine ingestion on embryofetal development in humans and rat cannot be neglected [5-7]. It is well known that X-irradiation of the mammalian embryo causes a variety of brain malformations including microcephaly [8,9]. In Japan, children exposed in utero to the atomic bombs of Hiroshima and Nagasaki have experienced mental retardation associated with microcephaly [8, 10] . Pregnant women are exposed to a wide variety of environmental agents, some of which have harmful effects on their offspring. Since

the majority of mental retardation in man has

an unknown yet a likely multifactorial etiology, much remains to be learned about the embryo-fetal toxic effects of environmental agents acting in combination. Epidemiological studies on the relation between maternal environmental agents and the offspring were conducted after controlling the effects of other risk factors [11-13]. Experimental studies on the embryo-fetal toxic effects of combinations of several maternal agents have been reported in mouse and rat [14-17]. However, there have been few studies on combined effects for clarifying the mechanism of fetal brain development [15]. The present study was undertaken to determine whether or not, and if so how the fetal brain dysfunction induced by maternal ethanol or caffeine can be modified by X-irradiation in utero in the rat.

Materials and Methods The experimental procedures for preparation of fetal models are summarized in Table 1. These procedures were essentially the same as those described previously with regard to ethanoltreatment [2,4] , caffeine-treatment [6, 7] and X-irradiation [9]. One hundred and forty-nine virgin female albino rats of the Wistar strain were treated in the following two experiments. In Exp 1, after a premating period of 146 days from 8 wks of age with treatment with 20% ethanol or 0.04% caffeine or water as drinking Table 1 Methods Premating period

Mating period

Gestational period 0

13 20 21 d I

I

I

E:X-I00R E: S

~E1

tE-#.......j--.j E-II-+---i

C:X-100R C: S

~Cl

~C-#-+-l

V:X-100R V: S W:X-100R W: S

rWl

W

C-II--+-i

~V-#----i V-II~

~W-#-+--i W-II-t---1

E: 20% ethanol, C: 0.04% caffeine, V: 0.03% dl-a-tocopherol acetate, W: water. #, X-I00R: X-irradiation with 100R, II, S: sham-irradiation.

fluid ad libitum, females of three groups were mated with male rats of 11 wks of age of the same strain. The day on which a sperm-positive vaginal smear was obtained was considered as gestational day(g d) o. During pregnancy the three groups received the same drink as during the premating period. On g d 13 at 10:00 am, pregnant rats of each group were divided into two groups; they were X-irradiated with 100R and sham-irradiated, respectively, under the same conditions as described previously [9]. Pregnancies were terminated on g d 20 at 11 :00 am by cesarean sectioning after chloroform anesthesia for 45-60 secs. Pregnant rats were examined as to body weight and weights of solid diet (MM-3, Funabashi Corp, Chiba, Japan) and drink consumed during pregnancy. Fetuses were examined as to litter size, fetal viability, placental weight, body weight, cerebral weight and level of lipid peroxide (LP) in the fetal cerebrum 1 hr after cesarean sectioning. In Exp 2, female rats at 9 wks of age were pretreated for 113 days with ethanol or water (Exp 2A), and for 146 days with caffeine or water (Exp 2B), respectively. Seven male rats from the same litter were used for mating at 7 mos of age in Exp 2A and 8 mos of age in Exp 2B. During pregnancy, water-treated rats in Exp 2A were divided into two treatment groups; 0.03% dl-a-tocopherol acetate (vitamin E; using Juvela, Eisai Co, Tokyo, Japan) and water as drinking fluid ad libitum. On g d 13, pregnant rats were X-irradiated with 100R or sham-irradiated at 10:30 am in Exp 2A and 10:00 am in Exp 2B, respectively. Pregnancies were terminated on g d 21 at 10:00 am in both Exp 2A and 2B by cesarean sectioning. Pregnant rats and fetuses were examined in Exp 2A and 2B for the same factors as in Exp 1 with the exception of LP levels in the fetal cerebrum 1 hr 45 mins after cesarean sectioning and for DNA, RNA and protein concentrations in the fetal cerebrum 1 hr after cesarean sectioning. For determination of cerebral LP, a 10% homogenate was prepared using 1.15% KCl. Assaying of LP was performed by the thiobarbituric acid reaction described by Ohkawa et al [18], and fluorometric measurement was conducted with excitation at 515 nm and emission at 553 nm. The LP levels were expressed in terms of nmoles malodialdehyde (MDA) per gm wet weight using tetraethoxypropane as an external standard. Cerebral

Tanaka et al: Maternal agents and fetuses

11

homogenate , which was frozen at -SO°C in Exp 1 and preserved at 0-4°C in Exp 2A and 2B, was incubated at 37°C for 1 hr with continuous agitation and then assayed for LP again, immediately after a fresh homogenate was assayed. Furthermore, a cerebral homogenate was frozen at -SO°C for about 1 wk in Exp 1, for about 7 wks in Exp 2A and for about 2 wks in Exp 2B, and then the LP assay was performed using samples from the same drink-treatment group at the same time. Extraction of DNA and RNA was performed by the method of Schmidt-Thannhauser-Schneider [191 , and determination by the diphenylamine reaction [201 and orcinol reaction [21] , respecti"ely. Protein was detennined by the method of Lowry et al [22] . All chemicals were obtained from the commercial sources indicated. 2-Thiobarbituric acid and sodium lauryl sulfate were specially prepared reagents (Nakarai Chemicals Ltd , Kyoto, Japan); 1, 1,3,3-tetraethoxypropane (Tokyo Kasei Kogyo Co , Ltd , Tokyo , Japan), and DNA from calf thymus and RNA from baker's yeast (Sigma Chemical Co , St Louis, Mo, USA). Other reagents were of special grade and super special grade or specially prepared reagents of ultraviolet grade.

Statistical analysis in this study was perfonned by Student's t test between X-irradiated and sham-irradiated groups and between different drink-treatment groups with X-irradiation and those with sham-irradiation. Results 1. Maternal and Fetal Aspects As to maternal body weight, maternal intake of food and drink, litter size, placental weight and fetal viability , there was essentially no conclusive difference between Exp 1 on g d 20 and Exp 2 on g d 21 . Therefore, the results for only Exp 2 are shown below. Consumption of the solid diet during pregnancy was increased in caffeine-treated mothers and decreased in ethanol-treated mothers compared to the other groups, but there was no difference in the consumption between the X-irradiated and sham-irradiated groups. Mean litter size was not different among all the groups treated with ethanol, vitamin E, caffeine and water , Singly or in combination with X-irradiation . Mean maternal body weight, mean placental weight , and mean mortality 1.5 hrs after cesarean sectioning are presented in Table 2. No significant difference between all the groups was seen

Table 2 Maternal body weight, placental weight and fetal mortality Maternal body weightongd21 (g) Exp 2A { ~-lOOR

335±19[5]}

V

{S

388 ± 28 [4]

W

{S

359±12(4)} 368± 6(4)

E

316 ± 16 [4] X-lOOR X-100R

-:L I

-.J

}--.J

395 ± 56 [3]

***

W

{ ~-100R

429 ± 15 [3] } 450124(3)

{ X-lOOR S

0.63' 0.09 1 5 1 m 0.69 ± 0 0. 5 [4] ** 0.48 ± 0 .02 [4].J * 0.58 ± 0.10 (3)~ ***

{ 12% 19%

0.49 ± 0.05 (4) ± 0.04 [4]

{

8%

{

5%

{

6% 4%

0.57

Exp 2B C

Mean mortality 1.5 hrs after cesarean sectioning (%)

Placental weight (g)

l**

418±17[3]}J 391 ± 23 [4]

0.58 ± 0 .18 [3] 0.52 ± 0.05 (3)l 0.53 ± 0.09

[3]j

0.64 ± 0.07 [4]

Data expressed as means ± SD [no of dams]. Significant difference between groups; ***: p < 0.01, **: p < 0.02, *: p < 0.05.

12 Brain & Development, Vol 7, No 1,1985

I

{ 0% 0% 2%

5%

in the mean maternal body weight on g d 0, but several differences were seen on g d 21. Although the mean maternal body weight on g d 21 was not different between the X-irradiated and sham-irradiated groups, that in the ethanoltreated groups was lower and that in the caffeine-treated group was higher than in the other groups. Mean placental weight was higher in the ethanol-treated group and tended to be low in the caffeine-treated group. In general, lower fetal viability was observed in the ethanoltreated group. However, X-irradiation on g d 13 did not reduce the fetal viability in each drink-treatment group compared to with sham-irradiation.

2. Fetal Body Weight Body weights of live birth fetuses excluding the stillbirths in ethanol-treated groups of both Exp 1 and 2A are shown in Table 3. Although no

significant difference between X-irradiation and sham-irradiation was found in fetal body weight on g d 20, X-irradiated fetuses on g d 21, for which the body weight gain from g d 20 was greatest , showed a significantly reduced body weight compared to sham-irradiated fetuses in each drink group except for the ethanol group. It was found on comparison between groups treated with each drink combined with X-irradiation that fetal body weight of the ethanol group was significantly lower than that of the other drink groups; caffeine and water on g d 20 and vitamin E and water on g d 21. On the other hand, fetal body weight of the caffeine group on g d 21 was significantly higher than that of the water group , when combined with X-irradiation. Comparison between groups treated with each drink combined with shamirradiation showed that fetal body weight of the ethanol group was significantly reduced on

Table 3 Effects of combined treatment with ethanol or caffeine or vitamin E and X-irradiation on body weight of live birth fetuses

Body weight (g) Exp 1

{

X-looR

2.89 ± 0.34 (43)

S

2.97 ± 0.32 (30)

c

{

X-1OoR

3.37 ± 0.32 (27)

S

3.23

W

{

X-10oR

3.47 ± 0.46 (37)

S

3.61

{

X-I00R

4.33 ± 0.58 (50) 4.35 ± 0.53 (43)

V

{

X-I00R

w

{

X-100R

C

{

X-100R

W

{

X-I00R

E

±

±

I

***

I

0.44 (27)

.j. + .I. I I ***

I

0.41 (54)

I

Exp 2A E

S S S

4.82 ± 0.49 (52)--; *** 5.24 ± 0.61 (31)--'

I

*** I

4.69 ± 0.44 (43), *** 5.12 ± 0.5 7 (44)--'

I I

***

1

***

I

I

***

I

Exp 2B S S

4.92 ± 0.43 (28), *** 5.33 ± 0.40 (39)....1 4.59 ± 0.44 (37), *** 5.36 ± 0.62 (30)....1

Data expressed as means ± SD (no of fetuses). Significant difference between groups; ***: p < 0.01, **: p

***

I

< 0.02.

Tanaka et al: Maternal agents and fetuses 13

g d 20 and 21 with the same values as for ethanol combined with X-irradiation, and on g d 20 the reduction in body weight of fetuses treated with caffeine became clearer compared to that with water. These results demonstrate; 1) Ethanol alone causes significant reduction of fetal body weight, therefore, ethanol combined with X-irradiation does not show further reduction of fetal body weight. 2) Caffeine or vitamin E or water singly does not cause any changes in fe tal body weight on g d 21, and so these drinks combined with X-irradiation result in significant reduction of fetal body weight. 3) Compared to water-treatment, caffeine singly causes reduction in fetal body weight on g d 20. However, caffeine combined with X-irradiation results in a relatively high fetal body weight.

21 because the decrease in cerebral weight with vitamin E or caffeine-treatment was inhibited by X-irradiation. On the other hand , when each drink treatment was combined with shamirradiation, reduction in fetal cerebral weight became clearer with ethanol on g d 20 and 21, and with caffeine on g d 20. These results demonstrate; 1) X-irradiation causes significant reduction in fetal cerebral weight in all drink groups. 2) The effect of combination of each drink treatment and X-irradiation on fetal

3. Fetal Cerebral Weight

Exp 1

All fetal cerebral weights examined are shown in Tables 4 and 5. There was a very significant reduction in cerebral weight on g d 20 and 21 with X-irradiation on g d 13 in all drink groups as compared to with sham-irradiation. When each drink treatment was combined with Xirradiation , significant differences were observed in fetal cerebral weight between ethanol and water on g d 20, and between vitamin E and ethanol or water and caffeine and water on g d

Table 4 Effects of combined treatment with ethanol or caffeine and X-irradiation on fetal cerebral weight on g d 20 Cerebral weight (mg) X-100R E{ S

93 ± 7 ( 4 3 ) : 1 - i *** 114 ± 7 (29)--'

C { ~-100R

94 ± 10 (31)~* *** 109 ± 10 (27)--'

w{ ~-100R

0

***

(37)~***

97 ± 10 *** 122± 8(54)=='

1

Data expressed as means ± SD (no of fetuses). Significant difference between groups; ***: p < 0.01, *: p < 0.05.

Table 5 Effects of combined treatment with ethanol or caffeine or vitamin E and X-irradiation on fetal cerebral weight on g d 21 Cerebral weight (mg) Exp 2A E {~-100R

112± 6 [51---. 134 ± 9[4]~*

V { ~-100R

123 ± 5 [4]---, 151 ± 9 [3]~*

w{ ~-100R

..I

I

117± 5 [41---. *** 151 ± 5 [4]---'

113 ± 9 (50)-----;J * * 133 ± 11 (43)-

I

122 148

I

116 ± 10 (43)----, 149 ± 11 (44)....2!*

**

± ±

12 (52)-----. *** 9 (31)--'

Exp 2B C { ~-100R W {~-100R

118 ± 4 [3]---. *** 143 ± 5 [3]---'

119 144

±

111 ± 6 [3]--. *** 142 ± 10 [4]----1

111 141

±

±

±

10 (28)---, 8 (39)--.:1 '" 8 (37)--, 12 (30)---.!1*

Data expressed as means ± SD [no of dams] or (no of fetuses). Significant difference between groups; ***: p < 0.01, **: p < 0.02, *: p < 0.05 .

14 Brain & Development, Vol 7, No 1, 1985

I

***

I I

***

I

I

***

I

I

***

I

I

***

I

cerebral weight results in a slight decrease on ethanol-treatment and a relative increase on vitamin E or caffeine-treatment. 3) The reduced effect of drink agents on cerebral weight becomes clear on single treatment without X-irradiation as with ethanol-treatment and sometimes with caffeine-treatment. As another way of demonstrating these

effects on the fetal cerebrum, three histograms of cerebral weight on g d 21 are shown in Fig 1. Each histogram shows a single peak. 4. Lipid Peroxide Formation The mean levels of LP in fetal cerebrum under several conditions are shown in Tables 6 and 7. Only a few differences in LP level were observ-

Table 6 Effects of combined treatment with ethanol or caffeine and X-irradiation on lipid peroxide formation in the fetal cerebrum on g d 20 Sample A

Lipid peroxide (nmol MDA /g wet wt) Sample B

Sample C

Exp 1 E { ~-100R C { ~-100R

w{ ~- 100R

41.1 ± 12.4 (11) 33.2 ± 6.1 ( 9)

208 ± 37 (11) 213 ± 18 ( 9)

***

32.1 ± 7.8(10) 38.3 ± 8.1 ( 6)

126 ± 27 (10)~ 158 ± 27 ( 6)....--J'

***

37.8 ± 8.3 ( 8) 39.0 ± 8.2 (10)

190 ± 32 (10) 204 ± 44 (12)

65.5 ± 14.8 ( l l ) " - J

I

I

I

I ***

I

* J

63.9 ± 12.1 ( 9)

***

48 .1 ± 8.8 (l0)-.-J

54 .2 ± 12.8 ( 6) ~ *** 59.1 ± 8.1 (10)----1 58.5 ± 11.5 (12)

Data expressed as means ± SD (no of samples). Significant difference between groups; ***: p < 0 .01, *: p < 0.05 . Fetuses were obtained from 2 to 5 dams in each group and one sample was from 2 to 3 fetuses. Sample A: fresh sample with no treatment, Sample B: frozen sample with treatment at 37°C for 1 hr, Sample C: frozen sample with no treatment.

Table 7 Effects of combined treatment with ethanol or caffeine or vitamin E and X-irradiation on lipid peroxide formation in the fetal cerebrum on g d 21

Sample A Exp 2A E { ~-100R

Lipid peroxide (nmol MDA/g wet wt) Sample B

Sample C

I

40.8 43.2

± 7.1 (13)

190 8.1 (10)====,--] 202

±

±

±

19 (13)---, 27 ( l O ) T I i

X-lOOR V{ S

36.1

±

5.8 ( 9)-------, *j*

±

22 ( 9)~ *j*

w{ ~-100R

41.4 ± 3.1 (ll)~ *** 34.0 ± 5.1 (10)-'

181 ± 22 (ll)-.J 176 ± 20 (10)

68.9 ± 8.3 (11) --.J *j* 63 .2 ± 8.6 ( 1 0 ) - - - - - '

39.3±7.3( 7) 36.4±7 .6 ( 9)

156 ± 19 ( 7) 185 ± 33 ( 9)

56 .3 ±6.5 ( 7) 58.5 ± 9.4 ( 9)

35 .6 ± 7.6 ( 9) 36.8 ± 4.8 ( 9)

169 ± 21 ( 9) 176 ±26 ( 9)

64.3 ± 8.2 ( 9) 60.7 ± 8.1 ( 9)

33.2 ± 3.7 (7)~* *** 154 ±23 (

Exp 2B C { ~-100R

w{ ~-100R

I 140

I

7).....L....J~***

62.5 62 .3

±

±

6.5 (13)=:1 9.1 (10) ***

46 .5

±

5.3 (9)--.J

*

I ***

49 .9 ±6.9 ( 7 ) m

Data expressed as means ± SD (no of samples). Significant difference between groups; ***: p < 0.01, **: p < 0.02 , *: p < 0.05 . Fetuses were obtained from 3 to 5 dams in each group and one sample was from 2 fetuses. Sample A: fresh sample with no treatment, Sample B: fresh sample with treatment at 37°C for 1hr, Sample C: frozen sample with no treatment.

Tanaka et al: Maternal agents and fetuses

15

501

120

160 mg

120

160 mg

2.

80 501

3.

80

Cerebral weIgh t

Fig 1 Histograms of fetal cerebral weight on g d 21. 1. Ethanol-treatment, 2. Water-treatment in Exp 2A, and 3. Caffeine-treatment in Exp 2B. Key; ~ : X-irradiation with 100R on g d 13, c::J : shamirradiation on g d 13.

ed inconstantly between X-irradiated and shamirradiated groups with each drink treatment; X-irradiation reduced LP at 37°C for 1 hr on g d 20 on caffeine-treatment and enhanced LP with a fresh sample on g d 21 in Exp 2A on water-treatment. When each drink treatment was combined with X-irradiation, several significant differences were observed in LP levels; reduced LP levels with caffeine-treatment on g d 20 and with vitamin E-treatment on g d 21. On the other hand, single drink treatment without X-irradiation caused a reduction of LP at 37°C for 1 hr with caffeine-treatment on g d 20, and a reduction of LP with a frozen sample with vitamin E-treatment and enhancement of LP with ethanol-treatment on g d 21. These results demonstrate; 1) There is no constant enhancement of LP by X-irradiation. 2) Xirradiation causes a reduction in LP on caffeine or vitamin E-treatment. 3) Among single drink treatments without X-irradiation enhanced LP on ethanol-treatment becomes clear and reduced LP on caffeine or vitamin E-treatment is also observed.

5. DNA, RNA and Protein Cerebral DNA, RNA and protein concentrations on g d 21 are shown in Table 8. The

Table 8 Effects of combined treatment with ethanol or caffeine or vitamin E and X-irradiation on DNA, RNA and protein concentrations in the fetal cerebrum on g d 21 RNA (j.Ig/g wet wt)

Protein (mg/g wet wt)

4.65 ± 0.30 (12)----, *** 5.08 ± 0.41 (10)----1

4.27 ±0.49 (12) 4.38 ± 0.52 (10)

lOO± 7 (12) 101 ± 11 (10)

DNA (j.Ig/g wet wtJ Exp 2A E

{

V

{ X-100R S

4.52 ± 0.25 ( 9)----, ** 4.89 ± 0.26 ( 6)---l

4.57 ± 0.23 ( 9)----, ** 4.22 ±0.27 ( 6)----1

99 ± 7 ( 9) 97 ± 6 ( 6)

W

{ X-100R S

4.53 ± 0.26 (11)----, *** 4.93 ± 0.23 ( 7)----1

4.4 7 ±0.36 (11) 4.46 ± 0.25 ( 7)

98 ± 6 (11) 99 ± 9 ( 7)

C

{

X-lOOR

4.08

S

4.52±0.17( 9)~*

4.01

±

0.10 ( 9)

w

{

X-I00R

4.18 ± 0.09 ( 9)----;].* 4.61 ± 0.10 ( 7)---l

4.13

±

0.16 ( 9)

4.12

±

0.13 ( 7)

X-100R S

Exp 2B

S

±

0.14 ( 7)----,

3.95 ± 0.37 ( 7)

100 ± 6( 7)~ 107 ± 6 ( 9)---' 107 ± 4 ( 9) !l0± 5 ( 7)

Data expressed as means ± SD (no of samples). Significant difference between groups; ***: p
16 Brain & Development, Vol 7, No 1,1985

I

*

I

live fetus, or in other words, that there is some limitation to the action of combined maternal agents in live fetuses. In a practical human study, the effect of a single maternal agent such as alcohol on the offspring is analyzed with multiple regression techniques for adjustment for effects of other maternal agents such as nicotine and caffeine [11] . This study might provide evidence that the fetal alcohol syndrome is induced by maternal alcohol alone even in the presence of other toxic agents, but fetal alcohol effects might be related not only Discussion to maternal alcohol but also to other toxic There have been several reports concerning the agents in combination with weak alcohol. combined effects of maternal environmental When the findings for drink treatment agents at relatively low doses on embryo-fetal combined with X-irradiation were compared development in mouse and rat [14, 15]. with those for drink treatment without XHowever, studies on those at doses which irradiation, little but constant changes on Xcause adverse effects have not been reported. irradiation combined with caffeine or vitamin Therefore, in our present study, the conditions E-treatment were observed, which might show which cause adverse effects on fetal develop- the some function of brain. With caffeinement to some extent were employed singly or treatment , both fetal body and cerebral weights in combination for experimental rat models; were relatively increased on X-irradiation; 1) 1) Chronic 20% ethanol ingestion showing Single caffeine-treatment causes lower weights 50-100 mgjdl of ethanol in maternal blood [2, than single water-treatment, but caffeine and 4], 2) chronic 0.04% caffeine ingestion show- X-irradiation do not cause lower weights than ing 5-10 ~gjml of caffeine in maternal serum those with water and X-irradiation on g d 20. [6, 7], and 3) X-irradiation with 100R on 2) On g d 21 , caffeine alone does not cause lower weights than water alone, but caffeine gd 13 [9]. In our live fetuses, the effects of ethanol and X-irradiation show much higher weights alone were reductions in both fetal body and than water and X-irradiation. Furthermore, cerebral weights since the effects of ethanol with caffeine-treatment on g d 20, LP formais characterized by hypoplasia [4, 15] . On the tion in the fetal cerebrum was reduced by other hand , single X-irradiation with 100R X-irradiation; LP levels ranged as follows with caused decreases in fetal cerebral weight and treatment , caffeine with X-irradiation < caffetal cerebral DNA, because the target of feine alone < water or ethanol alone or that X-irradiation on g d 13 is cerebral DNA as combined with X-irradiation. However , on mentioned previously [9]. When reduction in g d 21 there were no more differences in LP fetal cerebral weight on X-irradiation alone levels between all these treatments. These disexceeded that with ethanol alone, there was a crepancies between on g d 20 and on g d 21 significant difference in cerebral weight be- at term delivery may depend on the dose of tween X-irradiation and sham-irradiation in- maternal caffeine consumed ad libitum, which dependently of ethanol as well as caffeine or might show that the interaction between cafvitamin E or water. On the contrary, when the feine and X-irradiation manifests only in the reduction in fetal body weight with ethanol presence of caffeine. The reduction in protein alone exceeded that on X-irradiation alone, concentration in the fetal cerebrum was also there was no difference in body weight between enhanced by caffeine with X·irradiation, com· X-irradiation and sham-irradiation in combina- pared to caffeine alone or water with X·irradiation with ethanol, and also not in combination tion. Several studies in vitro on the effect of with caffeine or vitamin E or water. These a combination of X-ray and caffeine were data suggest that the effect of a relatively reported in embryos [16] and cells [23,24]. strong agent on the target organ covers or in- Caffeine markedly enhanced the radiation risk hibits the other combined weak agents in the for embryonal development but the risk en-

DNA concentration with all drink treatments was significantly reduced by X-irradiation independently of the drink agents. The RNA concentration on vitamin E-treatment was significantly increased by X-irradiation. The protein concentration on caffeine-treatment was decreased by X-irradiation and this reduction was significantly different compared to that on water-treatment.

Tanaka et al: Maternal agents and fetuses 17

hancement was not so great for the proliferation of cells in the preimplantation mouse embryo as reported by Muller et al [16]. On the other hand, caffeine prevented the inhibition of replicon initiation normally caused by X-irradiation in Chinese hamster and human cells [23]. In general, postirradiation incubation in the presence of caffeine appeared to result in inhibition of repair of cells from a rat brain tumor [24] . These data suggest that there is not only an enhanced radiation risk after application of caffeine, but also an unchanged or even a diminished one in living organisms. In our present rat model, the combined effects of caffeine and X-irradiation on increases in body and cerebral weights may be partly related to decreases in LP levels, which may indicate a susceptibility to the damaging effects of free radical reactions [9, 25]. The inhibition of LP formation of liver microsomes by drugs undergoing oxidative demethylation such as caffeine has been reported by Orrenius et al [26] . Furthermore, this reaction has been indicated in rat brain microsomes [27]. Therefore, it is possible to consider that LP formation in the brain is prevented in the presence of caffeine. However, we have no evidence now at present as to how this prevented reaction is enhanced by X-irradiation which produces many free radicals. Although caffeine prevents the reduction in body and brain weights and the increase in LP formation induced by X-irradiation, we cannot conclude that combined caffeine and X-irradiation are rather more desirable for fetal development than X-irradiation alone because of the decrease in protein concentration in the cerebrum. Studies on the mechanisms through which caffeine and X-rays act are needed. In the case of treatment with vitamin E for its antioxidant capacity and lipophilic character, fetal cerebral weight was increased by X-irradiation; vitamin E-treatment alone does not cause any change in fetal cerebral weight compared to water alone, but combined vitamin E and X-irradiation causes a significant increase in cerebral weight compared to combined water and X-irradiation. Furthermore, on the treatment with vitamin E, LP formation in the fetal cerebrum was reduced by X-irradiation; LP levels ranged as follows with treatment, vitamin E with X-irradiation < vitamin E alone
18 Brain & Development, Vol 7, No 1, 1985

mice that received a vitamin E-deficient diet were more sensitive to X-irradiation than were normal mice, and that vitamin E-deficiency favored peroxidation in vitro as did X-irradiation. They suggested that the in vitro peroxidation capacity may be a reflection of tissue antioxidant capacity. On the other hand, several studies have been done on the protective effect of vitamin E against drug-induced peroxidative damage in rat [29,30]. From these reports it is possible to consider that vitamin E prevents X-irradiation-induced LP formation and damage to brain DNA. However, there has been no report on the effect of maternal Xirradiation and vitamin E on fetal cerebral LP formation and DNA. Therefore, our present study provides interesting evidence that maternal vitamin E at about 3.5 mg/kg body weight via the diet and about 35 mg/kg via the drinking water per day prevent the X-irradiationinduced reduction in fetal cerebral weight and X-irradiation-induced LP formation of fetal cerebrum even in fresh, non-treated samples. Summerfield et al [30] showed that a higher percentage of larger RNA was formed from the DNA damaged by peroxide in vitamin E-treated rats than from that in non-treated ones. Therefore, as compared to the RNA concentration in the fetal cerebrum from a mother treated with vitamin E alone, a higher one from a mother with combined treatment of vitamin E and X-irradiation may reflect the repair mechanism for irradiated DNA involving vitamin E. In addition to the above mentioned findings on the interrelation between maternal environmental agents, the present results might also show two interesting findings in view of the embryo-fetal toxicity. One is dependent on X-irradiation and the other appears to be independent. When the adverse effects of the embryo-fetal toxicity of ethanol or caffeine alone or its combination with X-irradiation were compared, the following findings became much clearer with single drink treatment than with combined treatment with X-irradiation; an increase in placental weight and a decreased in cerebral weight on g d 20 and 21 with ethanol-treatment, and a decrease in placental weight and body and cerebral weight on g d 20 with caffeine-treatment. These results show that the placenta is also influenced by X-irradiation, and that the reduction in body weight with caffeine alone is slight compared to that

with X-irradiation alone, so it becomes clear with caffeine alone. An ethanol-induced increase in placental weight has been reported by Henderson et al [15]. A caffeine-induced decrease in placental weight coincides with that in our previous report [6] . Independent factors on X-irradiation were observed especially with ethanol. Reductions in maternal and fetal body weights and increases in fetal mortality and LP formation in the fetal cerebrum were seen as the effect of ethanol alone. On the other hand, the effect of caffeine alone was an increase in maternal body weight. It is reasonable to consider that X-irradiation with 100R on g d 13 does not induce any reduction in maternal body weight at term except for a few days after irradiation. Reductions in maternal and fetal body weights and fetal viability on ethanol-treatment coincide with our previous reports [2,4,31]. Generation of LP stimulated by ethanol ingestion has been investigated both in experimental animals and in man [32-34], although such a mechanism in biological systems has not yet been fully resolved. However, no information is available on stimulated LP formation in the fetal cerebrum on maternal ethanol ingestion. Therefore, it might be an interesting mechanism of cerebral damage that increased LP formation caused by maternal ethanol in a fresh sample occurs in the fetal cerebrum. LP formation is influenced by complicated factors such as polyunsaturated fatty acids, radical-agents and radical-scavengers in tissues and that in vivo is not resolved in whole body experiments [35,36]. However, many investi-

gations have been done because of its serious consequences in vivo such as leakage of lysosomal enzymes and the toxic effect on vital cells. In our LP assay, the thiobarbituric acid reaction in vitro under aerobic conditions was employed in cerebral homogenates, in which, we consider, in vivo conditions are reflected much clearer. The inconstant increases in LP level on X-irradiation seen in this study at the end of pregnancy differ from the results in a previous study [9] . However, the prevention of LP formation by vitamin E combined with X-irradiation could not rule out changes in LP formation on X-irradiation during the early fetal period after irradiation as mentioned by Zicha et al [35]. The results reported here suggest that chemical and functional inter-

actions of maternal combined agents which occur during embryonic and fetal life may play an important role in embryo-fetal development. Acknowledgments This study was partly and 84-11 from the Mental and Muscular Ministry of Health and

supported by Grant Nos 84-01 National Center for Nervous, Disorders (NCNMMD) of the Welfare, Japan.

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