Comparison of the teratogenic effects of the isomeric forms of aminophenol in the Syrian golden hamster

TOXICOLOGY

AND

APPLIED

Comparison

PHARMACOLOGY

63, 264-269

(1982)

of the Teratogenic Effects of the lsomeric Aminophenol in the Syrian Golden Hamster JOSEPHV. RUTKOWSKIANDVERGIL

Department

Forms of

H. FERM

of Pharmacology and Toxicology and Department of Anatomy and Cytology, Dartmouth Medical School, Hanover, New Hampshire 03755

Received

September

14, 1981;

accepted

December

3, 1981

Comparison of the Teratogenic Effects of the Isomeric Forms of Aminophenol in the Syrian Golden Hamster. RUTKOWSKI, J. V., AND FERM. V. H. (1982). Toxicol. Appl. Pharmacol. 63, 264-269. para-Aminophenol (p-AF’), recently demonstrated to be a metabolic product of the analgesic-antipyretic agent, acetaminophen, was evaluated for teratogenicity. Also included for evaluation were the other isomeric forms of aminophenol, meta and ortho-aminophenol (m-AP and o-AP, respectively). Timed pregnant Syrian golden hamsters (SGH; LKV strain) were used. On the morning of Day 8 of gestation (8 days following the evening of breeding), the SGH received a dose of 100 to 200 mg/kg (ip) of pAP, m-AP, or o-AP. The dams were killed on Day 13 of gestation. A significant teratogenic response was elicited by p-AP and o-AP. A dose-effect phenomenon was evident by a rise in the frequency of litters with one or more malformed fetuses. The number of fetuses with one or more malformations also increased with dose, as did the resorption response. Frequently observed malformations were exencephaly, encephalocele, eye defects, rib fusion, tail defects, spina bifida, limb defects, and umbilical hernia. Other rare malformations were also noted. Although conclusive evidence regarding the teratogenicity of m-AP was not obtained, the data support the conclusion that p-AP and o-AP are teratogenic in the SGH without compromising maternal health.

para-Aminophenol (p-AP) was employed as an analgesic agent during the late 18OOs, yet proved to be more toxic than its predecessor, acetanilid. Subsequent chemical modification of p-Ap produced acetaminophen (Nacetyl-p-aminophenol, APAP), which is now used extensively as an analgesic-antipyretic agent. Recently, p-AP ,has been demonstrated to be a metabolic product of APAP (Carpenter and Mudge, 198 1; Gemborys and Mudge, 198 1). p-AP is also utilized in the dyeing of furs and leathers and as an intermediate in the manufacture of certain sulfur and azo dyes. pAminopheno1 is a significantly toxic chemical. It produces methemoglobinemia (Heubner, 19 13; Bodansky, 195 1; Smith et al., 1967). Contact dermatitis and bronchial 0041-008X/82/050264-06%02.00/0 Qpyright 8 1982 by Academic Plgll. Inc. AU rights of rcpraluction

ia any form rcscmd.

264

asthma resulting from p-AP exposure have been attributed to the structural resemblance of p-AP to p-phenylenediamine (Sax, 1975). p-Aminophenol has long been known to be cytotoxic, an effect attributed to its action as a metabolic inhibitor (Bernheim et al., 1937), yet no report has been made concerning the teratogenic potential in a mammalian system. This study was undertaken to evaluate the teratogenicity of p-AP and the meta- and o&o-isomeric forms of aminophenol (m-AP, o-AP, respectively). METHODS Timed pregnant Syrian golden hamsters (LKV strain), purchased from Charles River Breeding Laboratories

AMINOPHENOL (N. Wilmington, Mass.), were used in this study. Day 1 of gestation is that day following the evening of breeding. The animals were housed in Plexiglas cages, with food (Purina Lab Chow, Ralston Purina Co., St. Louis, MO.) and water provided ad libitum. On the morning of Day 8 of gestation the pregnant hamsters received an ip injection of p-AP, o-AP, or m-AP. Each agent was administered on a milligram per kilogram basis with the concentration adjusted to provide an injection volume of 0.5 ml/100 g body wt. p-AP was also administered iv (via the sublingual vein) and po (via gavage tube) during preliminary experimentation to evaluate the various routes of administration. Three or more dosage levels were employed to demonstrate a possible dose-effect phenomenon, except with the po administration of p-AP where only two dose levels were tested. pAminopheno1 (Eastman Kodak Co.) and o-AP (K&K Laboratories, Inc.) were administered in acidified isotonic saline (0.5 mM HC!). m-AP (Aldrich Chemical Co., Inc.) was administered in a 10% aqueous solution of dimethyl sulfoxide (DMSO; Fisher Scientific Co.) because it was not soluble in the acidified saline. DMSO is not teratogenic in the hamster at this level (Ferm, 1966). The dams were killed on Day 13 of gestation by carbon dioxide-induced anoxia. The uteri were removed and the contents examined. The live fetuses were examined for any external gross malformations, and the dead fetuses were classified as resorptions. After fixation in Bouin’s solution, three fetuses from every litter demonstrating at least one malformation were examined by dissection for soft tissue anomalies of the abdomen. For the data on the number of resorptions and the number of malformed fetuses, a Freeman-Tukey arc sine transformation was performed (Mosteller and Youtz, 1961). These transformed data were analyzed by analysis of variance and the Newman-Keuls test. Linear regression analysis was employed to confirm the presence of a dose-effect relationship. x2 analysis was performed on the data expressed as a percentage of the total number of litters (Snedecor and Cochran, 1967).

TERATOGENICITY

265

fetuses presenting with one or more malformations (expressed as a percentage of the total number of live fetuses) also increased with the dose administered (p < 0.0 1 ), as did the fetal resorption response (p < 0.01). At high doses (250 mg/kg, iv), a total of only four malformations (two encephaloceles and two tail defects) was observed in three litters (36 total implantation sites); whereas, there were 33 resorptions. Although the incidence of malformations is notably low, the exceedingly high resorption rate (91.7%) probably reflects the teratogenic potential of p-AP at this particular dose since severely malformed hamster fetuses undergo resorption after death in uteru (Ferm, 1965). Table 1 also indicates that po doses of p-AP were not teratogenic in the doses employed (p > 0.1). The malformations produced by p-AP and their respective incidences (expressed per number of litters demonstrating the particular malformation) are listed in Table 2. p-Aminophenol was also tested for its teratogenicity after solutions had remained at room temperature and room air for 1, 2, and 4 weeks during which time autooxidation to the quinone (p-benzoquinoneimine, PBQI) and its subsequent polymerization occurred as evidenced by discoloration (Bernheim et al., 1937). No difference was found between the teratogenic response elicited by p-AP and that of the quinone and its polymerized form (Tables 3, 4). o-Aminophenol, when administered ip, evoked a teratogenic response similar to that of p-AP. A comparative dose-effect response RESULTS was elicited in the incidence of malformaIntraperitoneal and intravenous injection tions and resorptions (p -C 0.01) (Table 1). of p-AP produced a significant teratogenic The incidence of each malformation for the response in the LKV strain of the Syrian respective dosage levels is enumerated in golden hamster (Table 1). A dose-effect Table 2. phenomenon was evident by a rise in the frem-Aminophenol appears to possess some quency of litters with one or more malteratogenic potential (Table 1). The few formed fetuses (expressed as a percentage malformations produced were morphologiof the total number of litters with one or cally similar to those produced by both pAP and o-AP, although the response was not more live fetuses) which paralleled the insignificant (p > 0.5). crease in dose (p < 0.01). The number of

ip

m-AP

0 100 150 200

100 150 200

0 loo 200

0 loo 150 200 250

0 loo 150 200

’ Litters with one or more live fetuses. * Percentages are in parentheses.

ip

PO

iv

ip

o-AP

p-AP

Drug

Route of administration and dose hg/kg)

EFFECT OF DOSE AND ROUTE

314 l/2 216 2/S

2/7 617 7/7

3/6 l/2 l/3

317 7/12 6/8 515 3/3

416 IO/22 619 23/26

(75.0) (50.0) (33.3) (40.0)

(28.6) (85.7) (100.)

(50.0) (50.0) (33.3)

(42.9) (58.3) (75.0) (100.) (100.)

(66.7)b (45.5) (66.7) (88.5)

No. of litters with resorptions/ total No. of litters

OF ADMINISTRATION

1 IN THE LKV

3/51 2/28 3187 4154

5/85 20/88 56197

3/86 2124 2/39

4192 19/164 36/102 42170 33166

7/69 23/285 9/l 13 106/320

(5.9) (7.1) (3.4) (7.4)

(5.9) (22.7) (57.7)

(3.5) (8.3) (5.1)

(4.3) (11.6) (35.3) (60.0) (91.7)

(10.1) (8.1) (8.0) (33.1)

No. of resorptions/ total No. of implantations

OF THE AMINOPHENOLS

TABLE OF THE SYRIAN

(89.3) (100.)

25/28 3/3

l/6 o/5

o/2

Of4

l/7 617 7/7

(0) (0) (16.7) (0)

(14.3) (85.7) (100.)

(0)

6/84 O/50

O/26

Of48

7/80 44168 41/41

(0) (0) (7.1) (0)

(8.8) (64.7) (100.)

(0)

O/37

(57.1)

40/70

o/3

(1.1) (17.2)

(1.6) (7.3) (23.1) (60.8)

l/88 25/145

l/62 19/262 24/104 130/214

No. of malformed fetuses/total No. of fetuses

HAMSTER

O/83 (0) 0122 (0)

(14.3) (50.0) (87.5) (loa.) (100.)

(16.7) (36.4) (66.7) (92.3)

GOLDEN

O/6 (0) o/2 (0)

l/7 6/12 7/8 5/5 2/2

l/6 8/22 619 24126

No. of litters with one or more malformed fetuses/ total No. of litters”

STRAIN

E

d 8

ti

E

8

sz

E

ip

PO

iv

ip

100 150 200

0 100 200

0 100 150 200

0 100 150 200

8017 6817 4117

8316 2212 3713

8817 145112 9518 2815

6216 262 122 10419 214126

No. of fetuses/ No. of litters

211 2515 3717

demonstrating

211 2614 3117

o/o o/o o/o

the particular

211 2814 3417

o/o o/o o/o

513 1715 1713

o/o o/o o/o

o/o

o/o

312 412 74117

111

Limb defects

GOLDEN

1315 2417 1213

512 1114 75122

o/o

Eye defects

OF THE SYRIAN

‘I’

of litters

STRAIN

2

713 1516 1514

915 1113 95121

o/o

Neural tube defects”.*

IN THE LKV

a Neural tube defects include exencephaly, encephalocele, and spina bifida. b All defects are expressed in a ratio of the number of fetuses to the number

o-AP

p-AP

Drug

Route of administration and dose (mdkg)

EFFECT OF THE AMINOPHENOLS

TABLE

defect.

o/o 1514 2717

o/o o/o o/o

o/o 511 111 29111

Rib defects

HAMSTER

o/o 1115 2016

o/o o/o o/o

o/o 312 413 1115

o/o 513 413 2417

Tail defects

111 1414 1417

o/o o/o o/o

o/o 111 813 712

o/o 313 111 44114

Umbilical hernia

268

RUTKOWSKI

AND TABLE

EFFECT OF ~BENZOQUINONEIMINE

Treatment No.” 1

No. of litters with resorptions/ total No. of litters 3/3 3/3 3/6

2 3

(PBQI)

3

IN THE LKV

No. of resorptions/ total No. of implantations

(loo.) (100.) (50.0)

4/37 7/37 IS/75

FERM

STRAIN

OF THE SYRIAN

No. of litters with one or more malformed fetuses/ total No. of litter8

(10.8) (18.9) (20.0)

DISCUSSION

16/33 24/30 33/60

TABLE

Treatment No.” 1 2 3

(PBQI)

11, 2 weeks (treatment

(48.5) (80.0) (55.0)

2), and I

other than gastroschisis detected in those embryos examined by gross dissection. Maternal health was not compromised following treatment with increasing doses of pAP, o-AP, or m-AP. Normal levels of activity and appetite were maintained by the dams. There was no evidence of maternal disease, characterized by listlessness, hair loss, diarrhea, or overt weight loss, during the course of experimentation. The teratogenicity of p-AP may be related to the formation of a reactive quinone metabolite which can readily bind macromolecules. The oxidation of p-AP to PBQI is accomplished by renal tissue cytochrome P450 (McMurtry et al., 1976). The nephrotoxicity of p-AP is attributed to the subsequent binding of PBQI to cellular components after glutathione depletion (Calder et

It is evident from the data that both p-AP and o-AP are significantly teratogenic in the Syrian golden hamster (LKV strain). The more frequently induced malformations were encephalocele and limb, tail, and eye defects. Umbilical hernia, often to the extent of eventration of the abdominal viscera, rib defects, spina bifida, and exencephaly were also observed with notable frequency. Anterior limb defects occurred with greater frequency than hindlimb defects. Eye defects include microphthalmia and anophthalmia, both of which occurred with equal frequency. Rare malformations observed were ectopic heart (2), cleft palate ( 1 ), occult cranioschisis ( 1), and abnormal genitalia (1). There were no abdominal anomalies

OF ~BENZ~QUINONEIMINE

HAMSTER

No. of malformed fetuses/total No. of fetuses

3/3 (100.) 3/3 (100.) 516 (83.3)

“p-AP was allowed to undergo autooxidation to PBQI in room air for 1 week (treatment month (treatment 3). Each solution was administered ip at a dose of 200 mg/kg. ’ Litters with one or more live fetuses. ‘Percentages are in parentheses.

EFFECT

GOLDEN

4

IN THE LKV

STRAIN

No. of fetuses/ No. of litters

Neural tube defects”’

Eye defects

Limb defects

33/3 30/3 6016

13/3 1713 2315

1213 1613

513 1613 1714

915

OF THE SYRIAN Rib defects

212 513 1215

GOLDEN

HAMSTER

Tail defects

Umbilical hernia

o/o

312 312

o/o 814

“pAP was allowed to undergo autooxidation to PBQI in room air for 1 week (treatment I), 2 weeks (treatment month (treatment 3). Each solution was administered ip at a dose of 200 mg/kg. b Neural tube defects include exencephaly, encephalocele, and spina bifida. ‘All defects are expressed in a ratio of the number of fetuses to the number of litters demonstrating the particular

512 2). and 1

defect.

AMINOPHENOL

al., 1979; Crowe et al., 1979). As stated above, no difference was evident between the teratogenic response elicited by PBQI or its polymerized form and that of p-AP. p-AP, o-AP, and m-AP have been shown to lack any mutagenic activity in a number of bacterial assay systems (Garner and Nutman, 1977; Lavoie et al., 1979; Degawa et al., 1979). The results obtained with m-AP are inconsistent. A teratogenic response was demonstrated at a dose of 150 mg/kg in only one of six litters and teratogenicity was not evident at a dose of 200 mg/kg. Further investigation into the teratogenic potential of this particular isomer is warranted. ACKNOWLEDGMENTS We gratefully acknowledge the valuable assistance and advice of Dr. Gilbert H. Mudge and the generosity of Dr. Roger P. Smith for supplying the aminophenols. This study was supported by USPHS Grants ES00697 and ES07 104.

REFERENCES BERNHEIM, F., BERNHEIM, M. L. C., AND MICHEL, H. 0. (1937). The action of p-aminophenol on certain tissue oxidations. J. Pharmacol. Exp. Ther. 61, 311320. BODANSKY, 0. (1951). Methemoglobinemia and methemoglobin-producing compounds. Pharmacol. Rev. 3, 144-196. CALDER, I. C., YONG, A. C., WOODS, R. A., CROWE, C. A., HAM, K. N., AND TANGE, J. D. (1979). The nephrotoxicity of p-aminophenol. II. The effect of metabolic inhibitors and inducers. Chem.-Biol. Interact.

27, 245-254.

CARPENTER, H. M., AND MUDGE, G. H. (1981). Acet-

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aminophen nephrotoxicity: Studies on renal acetylation and deacetylation. J. Pharmacol. Exp. Ther. 218, 161-167. CROWE, C. A., YONG, A. C., CALDER, I. C., HAM, K. N., AND TANGE, J. D. (1979). The nephrotoxicity of p-aminophenol. I. The effect on microsomal cytochromes, glutathione and covalent binding in kidney and liver. Chem-Biol. Interact. 21, 235-243. DEGAWA, M., SHOJI, Y., MASUKO, K., AND HASHIMOTO, Y. (1979). Mutagenicity of metabolites of carcinogenic aminoazo dyes. Cancer Left. 8, 71-76. FERM, V. H. (1965). The rapid detection of teratogenic activity. Lab. Invest. 14, 1500-1505. FERM, V. H. (1966). Congenital malformations induced by dimethyl sulphoxide in the golden hamster. J. Embryol. Exp. Morphol. 16, 49-54. GARNER, R. C., AND NUTMAN, C. A. (1977). Testing of some azo dyes and their reduction products for mutagenicity using Salmonella typhimurium TA 1538. Muiat. Res. 4, 9-19. GEMBORYS, M. W., AND MUDGE, G. H. (1981). Formation and disposition of the minor metabolites of acetaminophen in the hamster. Drug Metab. Dispos. 9(4), 340-351. HEUBNER, W. (1913). Studien uber Methamoglobinbildung. Arch. Exp. Pathol. Pharmakol. 72,241-281. LAVOIE, E., TULLEY, L., Fow, E., AND HOFFMANN. D. (1979). Mutagenicity of aminophenyl and nitrophenyl ethers, sulfides, and disulfides. Mutat. Res. 67, 123-131. MCMURTRY, R. J., SNODGRASS, W. R., AND MIT(:HELL, J. R. (1976). Metabolic activation of acetaminophen, cephaloridine, and other chemically stable compounds to nephrotoxic metabolites. Clin. Res. 24, 407A. MOSTELLER, F., AND YOUTZ, C., (1961). Tables of the Freeman-Tukey transformations for the binomial and Poisson distributions. Biometrika 48, 433-440. SAX, N. I. (1975). Dangerous Properties of Industrial Materials, 4th ed., p. 383. Reinhold, New York, SMITH, R. P., ALKAITIS, A. A., AND SHAFER, P. R. (1967). Chemically induced methemoglobinemias in the mouse. Biochem. Pharmacol. 16, 3 17-328. SNEDECOR,G. W., AND COCHRAN, W. G. (1967). Sfatistical Methods. Iowa Univ. Press, Ames.