Teratogenic effects of benzo[a]pyrene in developing chick embryo

Toxicology Letters, 40 (1988)

195-201

195

Elsevier

‘I-XL 01931

Teratogenic developing

effects of benzo[a]pyrene chick embryo

Jaseem

and N.K. Mehrotra

Anwer

in

Industrial Toxicology Research Centre, Mahatma Gandhi Marg, Lucknow-226001, (Received

10 September

(Accepted

27 May 1986)

India

1985)

Key words: Benzo[a]pyrene;

Embryo;

Teratogenesis;

Oedema

syndrome;

(Toxicity;

Chick)

SUMMARY The effect vestigated.

of benzo[a]pyrene

The embryos

yolk sac route.

(BP), an established

were exposed

This resulted

in retarded

crown-rump

length and bill length.

of the bones,

abdominal

is a teratogen

when injected

teratogenic

effects

oedema,

in different growth,

Abnormal haematomas,

via this route,

in developing

carcinogen,

on developing

stages of development as reflected

survivors

by lower embryonic

showed

chick embryos

to various

remarkably

body weight.

chick embryos

syndrome’

when treated

reduced

twisted legs with shortening

blisters and a short neck. These findings

and the ‘oedema

was in-

doses of BP via the

is a possible

suggest that BP

mechanism

causing

with BP.

INTRODUCTION

After soiling with crude or refined mineral oil, the hatchability eggs of various avian species has been reported to be lowered

of neonates from [1,2]. This is par-

ticularly so during the breeding season, when sublethal amounts of environmental oil get transferred to the eggs from feathers, feet or nesting materials [3]. White et al. [4] have reported that small quantities (I-10 ~1) of various crude and refined oils applied to the surface of fertile eggs of different avian species resulted in a considerable reduction in hatching, an increased incidence of teratogenicity and stunted

Address

for correspondence:

Jaseem

Marg, Lucknow-226001, Abbreviations: BP,

India. benzo[a]pyrene;

aromatic

MFO,

hydrocarbon;

037%4274/88/$

03.50

0

Anwer,

DBMA,

mixed function

1988 Elsevier

Industrial

Science

Toxicology

7.12.dimethyl oxidase.

Publishers

Research

Centre,

benzanthracene;

B.V. (Biomedical

Division)

P.B. 80, M.G.

PAH,

polycyclic

196

growth of the embryos. petroleum hydrocarbon

In another study, a single application of a synthetic mixture in microlitre quantities to mallard duck eggs

significantly enhanced the mutagenic and embryotoxic effects of crude petroleum. This synthetic petroleum mixture contained polycyclic aromatic hydrocarbons (PAH) such as 7,12-dimethyl benzanthracene (DMBA), benzo[a]pyrene (BP) and chrysene [5]. Moreover, as BP has been proved to be naturally present in crude, refined and waste oil [6,7], the present study investigated the effect on chicklings of this well-known carcinogenic PAH, when directly injected into the yolk sac. MATERIALS

AND

METHODS

BP was obtained from Sigma Chemical Co., U.S.A. and ground-nut oil (commercial grade) was obtained locally. Fertile eggs from white Leghorn hens were procured from the State Poultry Farm, Ganjaria, Lucknow, India. Bioassay procedure White Leghorn eggs were kept in an incubator at 38°C and at a relative humidity of 60-65%. All the eggs were candled before treatment, only fertile ones, weighing 55-60 g, with live embryos were selected for the study. Each experimental group contained 40 eggs. BP, dissolved in ground-nut oil, was injected aseptically (on a lamellar flow table under a bacteria-free flow of air) into the yolk sac of developing embryos. BP was injected in doses of 5, 50 and 100 pg in 20 ~1 of ground-nut oil, from the fourth to the eighth day of incubation. Control eggs with similar specifications were injected an equivalent volume of ground-nut oil only. All the eggs were injected only once on the specified day and placed in the incubator again. In order to detect the viability of embryos, the eggs were finally opened after 20 days of incubation and examined. Study variables included body size, body weight, gross examination of various tissues and organs, anomalies, and subcutaneous oedema in the trunk,

abdomen,

girdles

and neck.

RESULTS

In the present study, an overall growth retardation in the BP-treated chick embryos was observed. This included reduced body size, body weight (Table 1) and length of the leg bones (Table 11). The tibia showed a mild degree of angulation and lateral twisting at its lower end and the metatarsus showed marked lateral twisting (by almost 180” in some cases) at the tibio-metatarsal joint, making the plantar surface of the foot face upwards and the dorsal surface downwards (Fig. 1). The cutis covering the metatarsus and foot also appeared to be thinned with very thin cuticular rugae; however, no abnormal projections, fractures, oedemas OI haematomas were observed. In the wings, no change in shape or skeletal configuration was noticed.

197

TABLE EFFECT

I OF BP INJECTION

INTO CHICK

EGGS” ON BODY WEIGHTS

OF EMBRYOS

ON THE

20th DAY OF INCUBATION Day of incubation

4

6

9

BP

Body weightb

(pg/egg)

(g)

0 (contro1)

25.0

5

25.2

+ 1.54

50

22.8

+ 1.83*

100

20.8

t- 1.63*

0 (control)

26.5

f

5

26.0

k 1.32

50

24.8

?

1.41*

100

23.9

?

1.24*

0 (control)

25.2

k 2.00

5

25.5

+ 1.84

100

b Mean

EFI-tCT

1.45

24.3

into the yolk sacs of chick embryos

on the specified

t

2.85*

day of incubation.

+ SE for six chick embryos.

*Significantly

TABLE

-

24.95 + 1.78

50

a BP was injected

~~~___ * I.82

different

from control

(P
II OF BP INJECTION

INTO CHICK

EGGS” ON THE LENGTH

OF LONG

BONES ON DAY

20 OF INCUBATION Mean length

BOIlC

(mm) of long bones at the indicated

dosage

of BP

Controth

5 Lcg/eg&

50 @/egg

100 fig/egg

Femur

17.1

17.2

15.8*

15.6*

Tibia

22.0

21.8

17.5*

17.2*

hletataraus

17.5

17.6

13.6*

13.4*

,’ BP wa$ injected

into the yolk sacs of chick embryos

” Each value represents

the mean

*Significantly

from control

different

I

on day 6 of incubation.

SE for six chick embryo,. (P
Of all defects noticed, the incidence of leg deformity was highest, followed by abdominal oedema. Increases in the dose of BP injected were found to be directly related to the rise in mortality and in the percent survivors with abnormally reduced weight, crown-rump length and bill length embryonic growth, e.g., embryonic (Tables III and IV). Other defects, such as a short neck, haematomas, blisters, a reduced muscle mass, a transparent thin membranous gizzard filled with water and an enlarged spleen, were also recorded in the chicklings obtained from BP-treated eggs.

“Values

significanily

diff‘erent

from

control

(P~rO.05)

The toxicity of BP (50 or 100 kg per egg) in terms of mortality of embryos ~vas high when BP was injected on day 4. However, administration of these doses of BP at later stages of development lowered the mortality rate. 5 fig of BP exerted no toxic effect on the chick embryos at any stage of growth.

199

TABLE SOME

IV EFFECTS

OF

BP” MEASURED

ON THE

20th

DAY

OF DEVELOPMENT

OF CHICK

EMBRYOS Measurement

Dose of BP (pg)

Crown-rump Bill length Percent

length

Abdominal

survivors (%)

oedema

” BP was injected

5

78.5

78.4 i

+ 6.34

10.1 * 0.53

abnormal

Leg deformation

* P 10.05;

(mm)

(mm)

0 (control)

(%I)

50 5.10

10.2 & 0.62

72.5

100 t

9.7 i

4.39* 0.70

68.5

5

7

42**

3

5

35**

50**

3

6

25**

34**

into the yolk sac in the specified

i

6.54*

8.1 * 0.50” 58’*

doses on the 6th day of incubation.

** P
DISCUSSION

The present study of the effects of BP administered via the yolk sac route on developing chick embryos resulted in several malformations. These were qualitatively somewhat different from the deformities reported to be produced after surface application of oil containing BP to the egg shells of mallard ducks. In the latter study duck embryos showed incomplete feather formation, eye and brain defects including anophthalmia and exencephaly. Some had incomplete liver development. These differences are probably due to the difference in species and route of exposure. As regards embryo toxicity and teratogenicity, BP exposure in the present experiment led to skeletal malformations in chick embryos which showed a dose-response relationship. The leg bones were deformed and this defect could be ascribed to a general growth retardation of all skeletal elements of the legs; however, the most notable effects were observed in the tibia and metatarsus. The nature of the malformations found in chick embryos was qualitatively similar, regardless of the day when they were exposed. There are reports that following embryonic exposure to mutagens and teratogens, both avian [S] and mammalian foetuses [9] develop ‘oedema syndrome’, characterised by tissue oedema, haematomas and blister formation followed by abnormal embryogenesis and developmental defects. This has been explained as due to the development of ‘osmolar imbalance’ in these embryos [lo]. ‘Oedema syndrome’ also occurred in our experimental embryos, possibly because of a similar underlying osmolar imbalance. Whether the latter was a primary reaction to BP or secondary to some preceding although unknown cause requires additional study. PAHs, including BP, dimethylbenzanthracene and methylcholanthrene, are known to be embryotoxic and teratogenic in rats and mice [11,12]. When pregnant rats were exposed to BP, intraperitoneally or through the diet, the pups delivered

200

were found to be underweight, have birth defects or be stillborn [13]. In another study BP at doses of 50 and 300 m&/kg body weight given at day 7 or 10 of gestation caused in utero toxicity and teratogenicity, more so in genetically responsive C57B4/6 than in nonresponsive AKR inbred mice [ 141. Carcinogenic PAHs probably act as major factors for embryotoxicity when chick embryos are continuously exposed to automotive exhaust gases [15,16]. These carcinogenic PAHs are metabolically activated to reactive epoxides by microsomal mixed function oxidases (MFO). These epoxide metabolites are more mutagenic and carcinogenic than the parent compounds [ 17,181. The microsomal MFO enzymes are present in embryos and foetuses also [19,20] and avian embryos have a greater capacity to metabolise the PAHs than mammals [21]. From this study it can thus be concluded that BP is a teratogen when injected through the yolk sac into chick eggs. To some extent the incidence of toxicity and teratogenicity is dose-dependent. The embryotoxic and teratogenic response is more severe when BP is injected into the yolk sac on day 4 than on day 9. Preliminary studies (unpublished data) conducted earlier showed no teratogenic effects if BP was injected after day 1 1, probably because organogenesis in chick embryos is complete by that time.

The authors are grateful to Dr. P.K. Ray, Director, I.T.R.C., Lucknow, lndia for his constant encouragement and guidance. The authors are thankful to Mr. Mohan Das, P.M. for his secretarial assistance. One of the authors (Jaseem Anwer) is also grateful to the Indian Council of Medical Research, New Delhi for awarding a Senior Research Fellowship to conduct this study. REFERENCES I Rittinghaus,

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