Transplacental passage of nucleotides, nucleosides, and bases

‘I’.

TERRY

BERNARD Pittsburgh,

HAYASHI, I.

M.D.

GARVEY,

B.S.

Pennsylvania

When CWabeled nucleotides, nucleosides, and bases were administered intrauenously to near-term pregnant rats, the transfer of radioactive material across the placenta was observed with both purine aiid pyrimidine series. There was proportionately higher counts in the pyrimidine series; moreover, cytidine and or,otic acid showed the highest incorporation into maternal liver. Colchicine and 5-fhorouracil also displayed transplacental passage. The intraperitoneal administration of C~UabeEed precursors to near-term pregnant rats without anesthesia showed rapid and sizeable transfer of materials within few minutes followed by decline over period of hours. The phenomenon was observed in nucleotides, nucleosides, and bases. Analysis of acidinsoluble fraction with labeled erotic acid suggests that there has been incorporation of the material into maternal liver RNA, fetal RNA, and to a lesser degree into placental RNA..

day pregnant white albino Wister strain rats previously sedated with Biabital (1 ml. per 2 kilograms). An hour later the fetuses were removed by laparotomy, a 3.9 ml. sample of maternal blood collected by cardiac puncture, and maternal liver surgically extirpated. The whole fetuses and the maternal liver were homogenized separately in 10 ml. of Tris buffer, pH 7.2 in a Virtis homogenizer at top speed for 3 minutes. The preparation was deproteinized and extracted with 8.0 ml, of cold 10 per cent TCA (trichloroacetic acid). Radioactivity of the supernatant was determined in a liquid scintillation counter with Bray’s solution. The precipitate was dissolved in hot 10 per cent KOH and radioactivity measured. Similarly, maternal blood was treated with 3.0 ml. of cold 10 per cent TCA and the radioactivity of the supernatant determined again in Bray’s solution. In our second series 5.0 pc of labeled uridine, erotic acid, and thymidine was injected intraperitoneally without sedation into 19 to 20 day pregnant Sprague-Dawley rats. At intervals the rats were cervically decapitated and the various specimens, including

ALT H 0 u GH the injection Of l?” into pregnant rats showed incorporation of radioactivity into RNA fractions of fetal liver,l other experiments with different precursors, such as adenine2 and erotic acid,3 have produced negative results. In the particular study with erotic acid, radioactivity in the fetus could only be demonstrated when isotope was injected directly into the amniotic sac. Because such contrasting results suggested the possibility of differences in membrane transport rather than differences in RNA incorporation, the present study of measuring placental transfer of a va.riety of C4labeled nucleotides, nucleosides, and bases was undertaken in near-term pregnant rats.

Methods In the first series of experiments 2.5 pc of labeled purines and pyrimidines was injected intravenously via leg vein of 20 to 21 From the University 01 Pittsburgh Department of Obstetrics and Gynecology, Magee-Womens Hospital. This project was supported by Grant No. HD 01890 from the National Institutes of Health, United States Public Health Service. 1154

Volume Number

102 8

Transplacental

passage

placentas, were removed and processed for radioactive counting as previously described. In the third series of experiments 5 pc of C14-labeled erotic acid, uridine, and UMP (uridine monophosphate) was injected intraperitoneally in 19 to 20 day pregnant Sprague-Dawley rats and the animals were killed at specific intervals by cervical dislocation. Then 3.0 ml. of maternal blood was quickly removed by direct cardiac tap and mixed with equal volume of cold 10 per cent TCA. Precaution was taken to conduct all procedures at 4’ C. The mixture was centrifuged, the precipitate washed twice with cold 5 per cent TCA, and the radioactivity of the pooled supernatant determined in Bray’s solution. The precipitate was then placed in 3.0 ml. of 10 per cent TCA, heated for 15 minutes at 90’ C., centrifuged, and radioactivity of the supernatant measured. In addition, the fetuses, placentas, and maternal liver were immediately removed and placed separateIy in 20 ml. of cold O.lM sodium acetate buffer, pH 5.0, and homogenized at top speed in a Virtis for 3 minutes. One-tenth volume of cold 50 per cent TCA was added; the mixture stirred and centrifuged at 1.0,OOO x g for 10 minutes. The

of

nucleotides,

4

6

II. Radioactivity

after

intraperitoneal

injection

1155

The results of our intravenous injection of labeled purines and pyrimidines are shown in Tables I and II, respectively. The values recorded represent an average of 2 to 3 separate experiments. Since there were no

-0-0

fetus

- -a A- ---A l -

l

8

IO

12

placenta maternal maternal

14

liver blood

16

HOURS

Fig.

bases

Results

n

2

and

precipitate was washed twice with 10 ml. of cold 5 per cent TCA and the TCA supernatant pooled and radioactivity determined. Radioactivity of the acid-insoluble portion was measured by adding 10 ml. of 10 per cent TCA to the precipitate and heating for 15 minutes at 90’ C. The mixture was centrifuged and the radioactivity of the supernatant determined. From either the 60 or 120 minute samples, depending upon the maximum radioactivity recorded, supernatant liquids from both acidsoluble and acid-insoluble extractions were washed 5 times with equal volume of cold ether to remove TCA, lyophilized, and spotted on paper chromatogram with isobutyric of the acid-ammonia system. 4 Identification radioactive spot was determined by cochromatography with x-ray films or by cutting out appropriate spots and placing them in counting vials in Bray’s solution.

l

0

nucleosides,

of 5.0 pc of CWabeled

uridine.

18

1156

Hayashi

and

Decembel’ I& 1966

Garvey

9131. J. Obst. & Gym.

\

a-0-e

fetus

e---e

maternal

blood

\ \ \ \ \ \ -. -.

\ -. -.

I

2

4

\

-.

I

I

6

8

IO

12

14

16

18

HOURS

Fig. 2. Radioactivity

after

intraperitoneal

injection

measurable radioactivity in the precipitate in the first and second series the recorded total counts actually represent the radioactivity of the supernatant. There was, however, moderate radioactivity in the precipitate in our earlier experiments with thio’uraci1.5 The ratios of radioactivity in the fetus to maternal liver or to maternal blood were used to circumvent the possibility of incomplete intravenous injection of radioactive precursor when a smail thigh vein was used. In spite of variations in total counts administered, the ratios should still be reliable as an index of proportionate amount of material in the various organs and compartments. Figs. 1 and 2 illustrate the changes in radioactivity after intraperitoneal injection of uridine and thymidine. In both instances there was a rapid transfer of both isotopes followed by slow decrease over a span of 18 hours. The radioactive counts in acid-soluble and acid-insoluble counts in erotic acid, uridina, and UMP experiments are shown in Figs. 3, 4, and 5. The radioactivity in the acid-soluble portion represents the free base, nucleoside, or nucleotide and the acid-insol-

of 5.0 pc

of Cl*-labeled

thymidine.

uble portions presumably indicate lab&d RNA. Co-chromatography and radioactive analysis of the acid-insoluble preparation showed radioactivity in spots corresponding to uridine and UMP. Similar results were obtained with erotic acid studies where an additional system of N-butanol and 4 per cent boric acid4 was utilized to separate erotic acid from uridine. Comment

In our initial experiments with sedated animals and intravenous injection of isotopes (Tables I and II) the interval of one hour was chosen because previous work5 with amino acids and thiouracil under similar conditions had shown maximum transfer occurring in approximately 45 minutes. There was without exception radioactivity in the fetus in both purine and pyrimidine series including nucleotides, nucleosides, and bases. The integrity of the compound was not tested by co-chromatography in every instance, but nucleotide experiments usually showed radiochromatographic spots corresponding to both nucleosides and nucleotides, suggesting phosphatase or nucleotidase activity. It is possible that further pho’sphorylase and hydrolysis

Volume 102 Number 8

Table

Transplacental

passage

of

nucleotides,

nucleosides,

and

bases

1157

I. Purines

Purines

(total

ATP

GTP ADP AMP GMP Adenosine Inosine Guanosine Adenine Hypoxanthine Xanthine Guanine Uric acid Cyclic 5’-AMP

Table

II.

Fetus c&m.)

4,506 3,326 1,602 4,512 4,578 2,628 7,884 2,460 1,664 1,036 1,421 5,106 1,224 4,272

Maternal liver (tota! c.p.m.)

45,516 34,078 40,254 32,082 48,192 26,274 21,828 40,320 13,714 16,862 28,483 26,370 12,543 58,578

Maternal blood (c.p.m./

3.0 ml.) 15,319 10,550 20,206 9,185 16,080 22,800 6,749 35,562 34,814 2,803 10,729 12,825 49,214 18,189

maternal blood

Ratio: Maternal liver/maternal blood

0.10 0.10 0.04 0.14 0.10 0.10 0.36 0.06 0.12 0.06 0.05 0.19 0.10 0.07

0.29 0.31 0.08 0.49 0.29 0.12 0.12 0.07 0.05 0.37 0.13 0.40 0.25 0.24

3.0 3.2 2.0 3.5 3.0 1.2 3.2 1.1 0.4 5.9 2.6 2.1 2.6 3.2

Ratio:

Ratio: Fetus/ maternal blood

Ratio: Maternal liver/ maternal blood

Ratio: Fetus/ Ratio: Fetus/ maternal liver

Pyrimidines Maternal

Pyrimidines

TTP TMP UMP CMP Thymidine Uridine Cytidine Thymine Uracil Cytosine Orotic acid Thiouracil 5-Fluorouracil

Fetus (total c.p.m.)

2,589 9,620 10,404 6,216 12,125 13,198 3,607 10,164 19,109 4,682 1,554 39,264 11,064

liver (total c.p.m.)

Maternal blood (c.p.m./ 3.0 ml.)

Fetus/ maternal

liver

5,068 5,010 4,032 13,218 3,602 10,831 2,424 3,972 8,129 15,769 7,488 15,197 a,755

0.29 1.28 0.34 0.13 1 .oo 0.77 0.06 0.84 1.35 0.43 0.01 1.12 0.78

0.51 1.92 2.58 0.47 3.34 1.22 1.49 2.55 2.35 0.30 0.22 2.58 1.25

1.8 1.5 7.8 3.6 3.4 1.6 25.4 3.1 1.7 0.7 17.7 2.3 1.6

29,976

9,504

4,585 504 389

1.81 2.81 0.23 5.41

5.31 a.28 1.33 17.67

2.9

13,500 2,736 1,267

8,828 7,500 31,362 47,802 12,120 17.093 6(538 12,134 14,198 10,86 1 132,456 35,094 14,124

Miscellaneo,us

Glycine Phenylalanine Colchicine Actinomycin D*

50,472 37,920 672 6.854

3.0 5.4 3.3

“Tritium.

may have occurred but such reactions were not detected with the qualitative technique employed. The studies with nucleosides and bases usually showed corresponding radioactive spot. The entire purine series including cyclic 5’-AMP showed relatively low ratio of fetus to niaternai liver or maternal blood suggesting either poor incorporation or decreased

transfer. The pyrimidines, on the other hand, displayed much higher ratio of fetus to maternal liver, especially with uracil and thymine derivates. The unusually high ratio of maternal liver to maternal blood with erotic acid and cytidine is suggestive of rapid incorporation of these compounds into maternal liver. The antimetabolite 5-fluorouracil as well as DNA-dependent RNA polymerase

1158

Mayashi and Garvey

December Am. J. Obst.

ACID

SOLUBLE

COUNTS

.-*-. .---A a-.

\----

I,000 035

lo

20

30

fetus maternal placenta

. 40

50 TIME

A

15, 1968 & Gyxx.

liver

*

60 80 IN MINUTES

ACID INSOLUBLE

90

100

110

120

COUNTS

--I fetus A---A liver

-\‘\ ‘\

?l00,000 1 5 80,000 I

m-4 olacenta .--.

blood

‘1.

./

035

IO

20

30

B Fig. 3. lRadioactivity after intraperitoneai

40

___--50 60

60

90

100

110

TIME

injection

inhibitor actinomycin D displayed rapid transfer. Coichicine, commonly employed in the treatment of gout, likewise showed some transfer. The radioactivity plotted in Fig. 1 represents combined counts from both free nucleosides and from labeled RNA because of ribonuclease activity at room temperature. Fig. 1 does illustrate the rapid transfer of material followed by slow decrease over 18

of

5.0

pc of Cl&-labeled uridine.

hours. Similar experiments with erotic acid showed comparable results but higher radioactivity in the maternal liver. Fig, 2 with thymidine, a precursor for DNA, likewise showed the same ready transfer across the rat placenta. Previously, Atlas, Bond, and Cronkite6 injected tritiated thy&dine into mice earlier in pregnancy but were able to demonstrate Iabeling of DNA in the embryos studied only after 11 days of pregnancy;

Volume Number

102 0

Transplacental

passage

ACID 1.400,000

of nucleohdes,

SOLUBLE

nucleosides,

and

bases

1159

COUNTS

.

. IO

I 0

. 20

. 30

. 40

. . 50 60 TIME IN MINUTES

A

ACID INSOLUBLE

I 120

COUNTS

‘oomooo#

75,000

I

?1

i

I

s 50,0002

1

s

I

0s

40.000-

.--.

placenta

.-.-. A---. .-.

fetus maternal blood

liver

I I

-1 30,0002z

/. ,/

,.1X

‘*,/

,A

/ 20,00010,0005,000.

rI ! 1:. 1 I f $

I ,~~. .ooor

.k 0

-..+ j&w ~,“-‘-‘-‘-‘-‘-~ .v”’

after

--.__

_____ --__

.-• IO

.’ ;;.
--_.

. 20

30

40

intraperitoneal

injection

of 5.0

--. . 120

50 60 TIME IN MINUTES

B Fig. 4. Radioactivity

--Adm

PC of W4-labeled

UMP

(uridine

monophosphate).

they suggested that the development of embryonic blood circulation may be the important factor in DNA labeling. The resuIts of experiments with unanesthetized animals (Figs. 3 to 5) invariably showed a rapid and sizeable transfer of radioactive material across the placenta within a few minutes regardless of whether the substance was a nucleotide, a nucleoside, or a base. This is best illustrated in the acid-

soluble fraction (Figs. 3, A, 4, A, and 5, A). Fol.lowing the initial rise a period of equilibration occurred accompanied by varying degrees of RNA incorporation into fetus, placenta, and maternal liver. Thus, the initial transfer seemingly reflects variances in fluxes across the placental membranes rather than differences in regulatory mechanisms involved in RNA incorporation. Our initial experiments with sedated animals overlooked

1160

Hayashi

and

Garvey

Am.

ACiD SOLUBLE

2 & t-

60,000

;

December j. Ohst.

:j, !968 pr Gynec.

COUNTS

--

fstus

k--b r-. C.

liwr plocsnto noltrnol

btood

ti

1,000 !? Y

I

I

4 120

A

35

10

20

30

TIYEP#O NlNUTES ACtO INSOLUBLE COUNTS

soo,ooo*~

5,000

-i

1,000

- $\* 35

frtus liver

.--.

placsnta

.-.

blood

0

-.-• to

30 WE

0

Fig. 5. Radioactivity

C.-. b--A

after intraperitoneal

60 II MINUTES

injection

this initial rapid transfer and recorded radioactivity in the various components afttr a period of equilibration and possible RNA incorporation had occurred. Nevertheless, the presence of radioactivity in the fetks after one hour does suggest lthe early rapid transfer of such materials as a bolus across the plancenta, as observed in bur later studies.

I20

of 5.0 PC of CP4-labeled erotic acid.

Analysis of radioactivity in the acid-insoluble fraction after heat extraction is suggestive of incorporation into RNA with uridine, UMP, and erotic acid (Figs. 3, 23, 4, B, and 5, B). Since the extraction techniques employed in this study do not differentiate messenger RNA from ribosomal RNA or TRNA, the measured count is an approximation of total RNA. The maximum incorpora-

Volume

lo:!

Number

8

Transplacental

tion with uridine occurred at 60 minutes; whereas, it was 120 minutes with UMP. The latter may bse the result of phosphatase and nucleotidase enzymatic activity in the placenta. There was a slight decrease in radioactivity after 4 hours with UMP, but erotic acid showed the highest degrees of incorporation in maternal liver and fetus with only a measurable amount in the placenta. Moreover, the results of radiochromatography indicate that labeling was in the form of UMP, as reported earlier by Bresnick, Lanclos, and Gonzales.3 The different precursors, techniques, and isolation methods employed by various investigators make it difficult to compare the results of previous studies on pregnant rats. Smellie and associate? reported radioactive incorporatio,n after 2 hours in nuclear and whole cytoplasmic fractions. The study involved intr,amuscular injection of P32, homogenization, separation into cellular fractions by centrifugation, and finally extraction of RN.A from TCA-precipitated residue by NaCl at. 100’ C. with ethanol precipitation. In contrast, Dancis and Balis,2 inject-

passage

of

nucleotides,

nucleosides,

and

bases

1161

ing adenine-8-C l4 during the first half of gestation, could demonstrate radioactivity only in the maternal liver and none in the rat litter; RNA was extracted with sodium chloride and alkali. Similarly, Bresnick, Lanclos: and Gonzales,3 after intraperitoneal injection of C14-labeled erotic acid into 16 to 18 day pregnant rats, could not demonstrate significant labeling of fetal RNA. Radioactive incorporation of fetal RNA was effective only after direct intra-amniotic sac tap. The maternal liver RNA labeling was primarily as UMP and the ribonucleic acid in this case was extracted with sodium dodecyl sulfatephenol method. In our present work the entire fetuses, rather than fetal liver were analyzed and O.lM cold sodium acetate buffer, pH 5.0, was utilized to minimize degradation of RNA. By this approach there appears to be an appreciable amount of incorporation of uridine, UMP, and erotic acid into fetal and maternal ribonucleic acid; moreover, there is ample evidence to indicate the ready transfer of purine and pyrimidine nucleotides, nucleosides, and bases across the near-term rat placenta.

REFERENCES

1.

2. 3.

Smellie, R. M. S., McIndoe, W. M., Logan, R., and Davidson, J. N.: Biochem. J. 54: 280, 1953. Dancis, J., and Balis, M. E.: J. Biol. Chem. 207: 367, 1954. Bresnick, E., Lanclos, K., and Gonzales, E.: Biochim. Biophys. Acta 108: 568, 1965.

4.

Smith, I. : Chromatographic and Electrophoreted. 1, New York, 1960, Interic Techniques, science Publishers, Inc., vol. I., p. 234. 5. Hayashi, T. T., and Gilling, B. : Obst. & Gynec. 30: 736, 1967. 6. Atlas, M., Bond, V. P., and Cronkite, E. P.: J. Histochem. Cytochem. 8: 171, 1960.