teratogenic activity of several synthetic compounds structurally related to trypan blue

9
Teratogenic Activity of Several Synthetic Compounds Structurally Related to Trypan Blue' ALLAN R. BEAUDOIN AND MICHAEL J. PICKERINGZ Department of Anatomy, College of Medicine, University of Florida, Gainesville Gillman et al. ('48) were the first to re- port that trypan blue was teratogenic to the developing rat. This initial report has been followed by many others showing this disazo dye to be teratogenic to other mam- mals, e.g., mice, rabbits and hamsters (see review by Kalter and Warkany, '59). Tera- togenic activity has also been demonstrated in amphibians (Waddington and Perry, '56) and in chicks (Beaudoin and Wilson, '58). While the activity of this dye as a teratogeii is now well established, little is known of the site or nature of its action. Wilson ('55) has reported that the closely related disazo dye Evans blue is also tera- togenic in rats but to a lesser degree and that Niagara blue 4B and Niagara sky blue 6B are possibly active. Because of the close structural similarity of these dyes it has been postulated that a relationship exists between molecular structure and teratogenicity. The purpose of this paper is to present the results of an attempt to demonstrate a correlation between the structure of disazo dyes and their action as teratogens. MATERIALS AND METHODS Sixteen compounds were synthesized to resemble all or a part of the trypan blue molecule or one of the other closely related disazo dyes.3 The synthesized compounds were prepared as 2% solutions in distilled water and injected intraperitoneally into Sherman strain rats on the 8th day of preg- nancy. The dose was calculated to be 14 mg of dye per 100 gm rat body weight. Fetuses were recovered on the 20th day, weighed, examined for gross malforma- tions and fixed in Bouin's fluid. Subse- quently free-hand serial sections were made, with a razor blade, of the head to examine for brain and eye defects. The trunk was not sectioned. At autopsy repre- sentative tissues of the maternal macro- phage system were taken as well as kidney, placenta and yolk sac. These tissues were examined as stained and unstained sec- tions for presence of the injected dye. RESULTS Table 1 shows 5 closely related disazo dyes that have been analyzed for terato- genic activity by Wilson ('55) and by the present authors. Since the rats used in the present experiment ranged in weight be- tween 200-250 gm, the dose of dye re- ceived (28-35 mg) is comparable to the dose used in Wilson's study (30 mg/rat). All 5 of the disazo dyes tested were found to some extent in all of the tissues exam- ined histologically (table 3). The location of these dyes within the cells containing them was quite similar, except for Niagra sky blue 6B, and a single description will suffice for the other 4 dyes. The main var- iant was the amount of dye granules found in a given tissue. Lung, liver, uterus and ovary. The 4 dyes were detected in macrophage cells as distinct cytoplasmic granules (figs. 1-3). The size and number of the granules var- ied in a given cell; in some cells the nu- cleus was obscured by the dye. The re- ticulo-endothelial cells of the liver sinus- oids were made particularly prominent by these dyes (fig. 3). Spleen and lymph node. The spleen had very few dye-containing cells except in the capsule, where great numbers were found. The abdominal lymph node, on the ___ 1 Supported by research grants from the Na- tional Institute of Arthritis and Metabolic Di- seases, N.I.H., U.S.P.H.S. and from the Smith, Kline and French Company. 2 Medical Student Fellow. 3 Compounds prepared by Peninsular Chemical Company of Gainesville. 297

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Page 1: Teratogenic activity of several synthetic compounds structurally related to trypan blue

Teratogenic Activity of Several Synthetic Compounds Structurally Related to Trypan Blue'

ALLAN R. BEAUDOIN AND MICHAEL J. PICKERINGZ Department of A n a t o m y , College of Medicine, Universi ty of Florida, Gainesville

Gillman et al. ('48) were the first to re- port that trypan blue was teratogenic to the developing rat. This initial report has been followed by many others showing this disazo dye to be teratogenic to other mam- mals, e.g., mice, rabbits and hamsters (see review by Kalter and Warkany, '59). Tera- togenic activity has also been demonstrated in amphibians (Waddington and Perry, '56) and in chicks (Beaudoin and Wilson, '58). While the activity of this dye as a teratogeii is now well established, little is known of the site or nature of its action. Wilson ('55) has reported that the closely related disazo dye Evans blue is also tera- togenic in rats but to a lesser degree and that Niagara blue 4B and Niagara sky blue 6B are possibly active. Because of the close structural similarity of these dyes it has been postulated that a relationship exists between molecular structure and teratogenicity. The purpose of this paper is to present the results of an attempt to demonstrate a correlation between the structure of disazo dyes and their action as teratogens.

MATERIALS AND METHODS

Sixteen compounds were synthesized to resemble all or a part of the trypan blue molecule or one of the other closely related disazo dyes.3 The synthesized compounds were prepared as 2% solutions in distilled water and injected intraperitoneally into Sherman strain rats on the 8th day of preg- nancy. The dose was calculated to be 14 mg of dye per 100 gm rat body weight. Fetuses were recovered on the 20th day, weighed, examined for gross malforma- tions and fixed in Bouin's fluid. Subse- quently free-hand serial sections were made, with a razor blade, of the head to examine for brain and eye defects. The trunk was not sectioned. At autopsy repre-

sentative tissues of the maternal macro- phage system were taken as well as kidney, placenta and yolk sac. These tissues were examined as stained and unstained sec- tions for presence of the injected dye.

RESULTS

Table 1 shows 5 closely related disazo dyes that have been analyzed for terato- genic activity by Wilson ('55) and by the present authors. Since the rats used in the present experiment ranged in weight be- tween 200-250 gm, the dose of dye re- ceived (28-35 mg) is comparable to the dose used in Wilson's study (30 mg/rat). All 5 of the disazo dyes tested were found to some extent in all of the tissues exam- ined histologically (table 3). The location of these dyes within the cells containing them was quite similar, except for Niagra sky blue 6B, and a single description will suffice for the other 4 dyes. The main var- iant was the amount of dye granules found in a given tissue.

Lung, liver, uterus and ovary. The 4 dyes were detected in macrophage cells as distinct cytoplasmic granules (figs. 1-3). The size and number of the granules var- ied in a given cell; in some cells the nu- cleus was obscured by the dye. The re- ticulo-endothelial cells of the liver sinus- oids were made particularly prominent by these dyes (fig. 3 ) .

Spleen and l y m p h node. The spleen had very few dye-containing cells except in the capsule, where great numbers were found. The abdominal lymph node, on the ___

1 Supported by research grants from the Na- tional Institute of Arthritis and Metabolic Di- seases, N.I.H., U.S.P.H.S. and from the Smith, Kline and French Company.

2 Medical Student Fellow. 3 Compounds prepared by Peninsular Chemical

Company of Gainesville.

297

Page 2: Teratogenic activity of several synthetic compounds structurally related to trypan blue

298 ALLAN R. BEAUDOIN AND MICHAEL J. PICKERING

TABLE 1

Teratogenic act iv i ty of disazo dyes in the rat

Number Number of Embryos Survivors of mothers ~ ~ ~ & ~ - resorbed f$,$&

7; % TRYPAN BLUE

NIAGARA BLUE 28 ~

HO NH2

NaS a 0 3 N o

16 165 23 0

EVANS BLUE H2N OH HO NH2

S03No NaS03 -@" =N CH3 N=N @ 20' 162 23 14

S03No No SO3

NIAGARA BLUE 48 aN H2N OH = N B N = N a s o 3 N o

15l 126 15 4 O W J OCH3

No SO3 S03Na NO SO3

NIAGARA SKY BLUE 68 H2N OH HO NH2

S03Na N o S 0 3 a = "-"= OCH3 OCH3 @- 15l 145 8 3

S03Na NoS03

1 Data from Wilson. '55 .

other hand, had many dye-containing ma- crophages (fig. 4) . The dye appeared as granules in both of these tissues.

Kidney. The 4 dyes were found as clusters of particles in the basal portion of the majority of cells of the proximal tu- bules, but were not found in any of the other tubules (figs. 5-6). In most cases the dyes appeared as granules although in Niagara blue 2B and Niagara blue 4B spic- ules of dye were comomn (fig. 5). Macro- phage cells located between the tubules, in cortex and medulla, did contain dye.

Placenta. Small cytoplasmic granules of dye were seen in the giant cells and in cells of the connective tissue of the pla- cental free border.

Yolk sac. The yolk sac, together with the kidney, had accumulated the greatest amount of dye. The yolk sac epithelial cells were found to have various sized granules or spicules of dye within what very often appeared to be a single large vacuole at the apical end of the cell (figs. 8-10).

Niagara sky blue 6B, unlike the other 4 dyes, was found only sparsely distributed

Page 3: Teratogenic activity of several synthetic compounds structurally related to trypan blue

TERATOGENIC ACTION O F AZO COMPOUNDS 299

in the macrophages of the maternal tis- sues examined. In many cases this dye had stained the nuclei without appear- ing as granules in the cytoplasm. This was particularly noticeable in the kidney where all nuclei including those of glomeruli and Bowman’s capsule at times were stained. A few scattered granules were seen within vacuoles in some cells of the proximal tu- bules (fig. 7). The yolk sac epithelium had picked up fewer granules of Niagara sky blue 6B than of any other dye. Some cells were seen to contain only one discrete round granules of dye (fig. 11).

The 5 synthesized compounds most closely related to trypan blue are illustrated in table 2. The remaining 11, including 6 which simulated one-half a molecule of the teratogenic disazo dyes were all inef- fective as teratogens, nor was there any trace of these 11 compounds in the ma- ternal tissues examined. Of the 5 syn- thetic compounds examined in table 2 only two (8 and 10) were found in identical distribution in maternal tissues, although differing quantitatively, as the dyes in table 1, excluding Niagara sky blue 6B. It should be noted that compounds 8 and 10

TABLE 2 E f f e c t s of synthesized disazo compounds o n ru t gestat ion

Number Number of Survivors mal- of mothers resorbed forlned

HO OH

NoS03 03”’” S03Na

COMPOUND 6

C H 3

COMPOUND I H O

N = N- N = N&,.

C H 3 NoS03 a S03Na CH3 NaS03

NoS03

S03No

H2N OH

CON=

COMPOUND 8

NpqN=N~so3Na No SO3

N

COMPOUND 5

C H 3 I

S03Na NoS03

COMPOUND 10

N a S O 3 W N H2N OH = = Nao=No HO NHz

OCH3 OCH3 NaSO,

7

5

6

69

% %

22 0

48 4 2

76 14 3

53 0 0

60 7 0

Page 4: Teratogenic activity of several synthetic compounds structurally related to trypan blue

300 ALLAN R. BEAUDOIN AND MICHAEL J. PICKERING

TABLE 3 Tissue distribution of disazo dyes and selected synthetic compounds

Yolk sac epithelium

Maternal macrophage Maternal cells of liver, spleen, lymph node and lung kidney

Trypan blue Niagara blue 2B Evans blue Niagara blue 4B Niagara sky blue 6B Compound 6 Compound 1 Compound 8 Compound 5 Compound 10

++ ++ ++ ++ -+ + + +

- -

+++ +++ +++ +++ -+ - -

+++ ++ -

+++ +++ +++ ++ + +++ +++

- - -

represent combinations of 1 / 2 Evans blue with 1 / 2 trypan blue and 1/2 Niagara sky blue with 1 / 2 Niagara blue 4B re- spectively. This may somehow account for their uptake by macrophage cells and the kidney and yolk sac. The occurrence of an isolated malformation with compound 1 does not warrant its inclusion as a tera- togen. Its teratogenicity can be questioned because mild hydrocephalus is known to occur spontaneously (less than 1% ) in laboratory rats, and because only one ani- mal in a single litter was affected. On the other hand, compound 8 caused abnormal offspring in two of 8 litters (one case of hydrocephalus and one of gastroschisis). In addition 4 of the 8 litters showed evi- dence of resorptions, although no resorp- tions occurred in litters with malformed animals. The authors are reluctant to classify this compound as a teratogen at this time; however, further study seems indicated.

DISCUSSION

The tissue distribution of the 5 named disazo dyes and the two synthesized com- pounds (8 and 1 0 ) revealed that their up- take by the maternal macrophage system was very similar; however, there was no relation between this uptake and the tera- togenicity of the compound. These results of tissue distribution of the 5 named dis- azo dyes are somewhat at variance with the results reported by Wilson ( ' 5 5 ) in that he cited only trypan blue to be accum- ulated in the macrophages of various rna- ternal tissues and by the urinjferous tu- bules of the maternal kidney. He did re- port that all active (teratogenic) dyes were collected to some extent by the yolk s3c epi-

thelium and alluded to some macrophage accumulation of dyes other than trypan blue at higher concentrations than he used. In this connection it might be pointed out that while Wilson entertained some doubt as to the teratogenicity of Niagara blue 4B and Niagara sky blue 6B, the present auth- ors have accumulated evidence to show that these dyes can be teratogens at higher concentrations, e.g., 20 mg/100 gm rat body weight. On the other hand, Niagara blue 2B remains a non-teratogen even at concentrations as high as 30 mg per 100 gm rat body weight.

The present results further point to try- pan blue as being unique among the disazo dyes in its marked teratogenic ef- fect upon the development of the rat. Any alteration of its structure affects its tera- togenic activity. The substitution of-OCH., for -CH,, as in Niagara blue 4B, markedly reduced the teratogenic effect. When nei- ther -CH3 nor -OCH, groups were present, as in Niagara blue 2B, there was no tera- togenic effect. The NH, group likewise seems indispensable, for when it was re- placed by -OH (compound 6) or absent (compound 1 ) there was no activity. Sim- ilarly two NaSO, groups on each naphthol ring are necessary, for when one was ab- sent (compound 5) the compound was not a teratogen.

Substitution of groups on the trypan blue molecule is not the only way to affect its teratogenicity. Simple rearrangements of groups on the naphthol rings will also affect its teratogenic action, as when the NaSO, groups are shifted in Evans blue. If in addition to this shift of NaS03 groups the -CH, groups were replaced with -OCH3,

Page 5: Teratogenic activity of several synthetic compounds structurally related to trypan blue

TERATOGENIC ACTION OF AZO COMPOUNDS 30 1

as in Niagara sky blue 6B, there was a fur- ther decrease in activity. Symmetry of the compound is also important for when one- half molecules of two teratogenic disazo dyes were combined as in compound 8 ( 1/2 trypan blue + 1/2 Evans blue) and compound 10 (1/2 Niagra blue 4B + 1/2 Niagara sky blue 6B) activity was lost or diminished even though the tissue distri- bution of the compounds was like that of the teratogens.

It is apparent, then, that of all com- pounds tested only the intact trypan blue molecule is capable of such a marked ef- fect upon embryonic development in the rat and that any structural modification is likely to affect the teratogenic properties of the resultant compound. It is of interest to note that none of the changes made in the trypan blue molecule enhanced its teratogenicity.

SUMMARY

1. Sixteen compounds were synthesized to resemble all or a part of the trypan blue molecule or one of the other closely related disazo dyes.

2. Two per cent solutions of these com- pounds were injected intraperitoneally into

Sherman albino rats on the 8th day of pregnancy (14 mg/100 gm body wt).

3. Two of the synthesized compounds were found to be distributed in the ma- ternal tissues and yolk sac in the same pat- tern as trypan blue, although these com- pounds were of questionable teratogenicity.

4. It is concluded that structural modi- fications of the trypan blue molecule mark- edly affect the teratogenic activity of the resultant compound.

LITERATURE CITED

Beaudoin, A. R., and J. G. Wilson 1958 Tera- togenic effect of trypan blue on the developing chick. Proc. SOC. Exp. Biol. Med., 97: 85-90.

Gillman, J. C., C. Gilbert, T. Gillman and I. Spence 1948 A preliminary report of hydrocephalus, suina bifida and other conaenital anomalies in tGe rat produced by trypanblue. S. Afr. J. Med. Sci., 13: 47-90.

Kalter, K., and J. Warkany 1959 Experimental production of congenital malformations in mammals by metabolic procedure. Physiol. Rev., 39: 69-115.

Waddington, C. H., and M. M. Perry 1956 Ter- atogenic eEects of trypan blue on amphibian embryos. J. Embryol. exp. Morph., 4: 110-119.

Wilson, J. G. 1955 Teratogenic activity of sev- eral azo dyes chemically related to trypan blue. Anat. Rec., 123: 313-334.

Page 6: Teratogenic activity of several synthetic compounds structurally related to trypan blue

PLATE 1

EXPLANATION OF FIGURES

The first 4 figures show trypan blue in the macrophages of various maternal tissues. The tissues in all 6 figures were lightly stained with eosin only.

1 Lung. X 120.

2 Uterus. X 520.

3 Liver. x 520.

4 Abdominal lymph node. X 520.

5

6

Niagara blue 2B granules and spicules in the proximal tubules of the kidney. X 520.

Trypan blue granules in the proximal tubules of the kidney. X 520.

302

Page 7: Teratogenic activity of several synthetic compounds structurally related to trypan blue

TERATOGENIC ACTION O F AZO COMPOUNDS Allan R. Beaudoin and Michael J. Pickering

PLATE I

303

Page 8: Teratogenic activity of several synthetic compounds structurally related to trypan blue

PLATE 2

EXPLANATION OF FIGURES

The tissues of figures 7 and 9-12 were lightly stained with eosin only.

7 Niagara sky blue 6B in the proximal tubules of the kidney. Note arrows pointing to tubules with dye-containing cells. x 520.

The following figures show dye distribution in the yolk sac epithelial cells. All figures X 520.

8

9 Niagara blue 2B.

Whole mount of yolk sac with trypan blue granules.

10 Trypan blue.

11

12 Compound 8.

Niagara sky blue 6B. Note arrows pointing to dye-containing cells.

304

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TERATOGENIC ACTION OF AZO COMPOUNDS Allan R. Beaudoin and Michael J. Pickering

PLATE 2

305