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A histological study on acrylamide and cadmium chloride altered chick

embryonic liver

Dr. Thyaga Raju Kedam 1 , Ruxana Begum Sheik 2 , Malekar Meena Bai 3 ,

SK HaseenaBhanu 4

1 ~ 4 (Department of Biochemistry, SVU, Tirupati, India)

Abstract

Histology is the study of the microscopic anatomy of cells and tissues. The present study was made on control and treated
chick embryonic liver tissues using toxic compounds like acrylamide (AC) & cadmium chloride (Cd), on the morphological
modification of embryonic liver and hepatic cells. The metal ion, Cd, had severe damaging effect on chick embryonic liver
than AC and however our study has revealed that these two are causing damage to the embryos.

Keywords: Acrylamide, Cadmium chloride, Chick embryonic liver tissues

1. Introduction

It is obvious that any chemical insult could cause injury to cells in animal if it is consumed beyond the safe doses.
Susceptibility to chemicals, exhibits variation among the tissues and cells. The extent of severity of tissue damage is a
function of the concentration and potentiality of the toxic compound [1].

The cyto architectural changes produced during chemical toxicity can be identified by microscopic examinations of the
tissues and also explains the extent of tissue specificity to the chemical action. So histology is the study of tissues and it gives
the insight into the functioning of tissues and organs. It is the study of changes in cell environment which envisage the cell
anatomy. These studies pave a way to understand the pathological conditions of the animal organs. Histological analysis
gives a clear picture to understand how the drugs cause injury to the tissue. The histopathology refers to the microscopic
examination of tissue in order to study the manifestations of disease. In the 11 th day old chick embryo, histological changes
were studied in the liver after Acrylamide and Cadmium chloride treatment.

1.1. Acrylamide: Acrylamide (AC) is an a, P-unsaturated carbonyl compound fig 1A with a significantly high chemical
activity. The International Agency for Research on Cancer (IARC) has classified acrylamide as "probably carcinogenic to
humans" [2]. Acrylamide, a monomer, from which polyacrylamides are synthesized, shall be used in the treatment of water,
cosmetics and paper packaging.

Acrylamide is largely oxidized in mice, rats and humans to glycidamide (GA) figl B by its oxidating agent Cyp p450 2EI [3].
In humans, at relatively low doses of acrylamide, glycidamide is formed at higher extent than in rats, because of the higher
levels of CYP2EI. Both compounds, AC and GA, are detoxified by glutathione conjugation and to some extent glycidamide
is detoxified by hydrolysis.

During detoxification acrylamide reacts rapidly with SH groups, and also with proteins SH groups and amino groups. The
most important reaction of acrylamide with proteins is the adduction to hemoglobin (Hb), SH groups of proteins and the NH 2
groups of the N-terminal valines of cytoskeletal proteins and protamines [4,5].

Acrylamide is clearly a direct-acting clastogen in mammalian cells in which it induces at lower extent chromosomal
aberrations, micronuclei, sister chromatid exchanges (SCE), polyploidy, aneuploidy and other mitotic disturbances in the
absence of metabolic activation [6].

The standard bioassays conducted on rats by giving dissolved Acrylamide in drinking water, observed a number of benign
and malignant tumors in a variety of sites (e.g. thyroid, adrenals, and testis). However, the clear cut genotoxicity of
acrylamide in vivo in somatic and germ cells are able to form DNA adducts strongly suggest that a genotoxic mechanism for
the carcinogenicity of Acrylamide.

1.2. Cadmium chloride: Cadmium, a non-essential transition heavy metal is commonly regarded as a pollutant of world wide
concern. Cadmium and its salts have been widely used by lead, copper and zinc smelteries, alkaline accumulators, paint and

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plastic industries, causing increase in environmental contamination and water pollution. The use of Cadmium containing
fertilizers, agricultural chemicals and pesticides might also contribute to contamination [7].

Cadmium is a potent human carcinogen and occupational exposure to it has been associated with cancers of lung, prostate,
pancreas and kidney, because of its characteristics as a category I carcinogen (human carcinogen) by the IARC and the
National Toxicology program of the USA [8,9].

The classification of Cadmium as a human carcinogen is supported by strong evidence from animal experiments. In rodents,
Cadmium induced tumors in various organs. Various Cadmium compounds have produced adenocarcinomas of the lung in
rats after inhalation [10, 11]. Tumors of prostate and pancreas were evoked by subcutaneous injection of Cadmium chloride
in rats, tumors of the testis were induced by oral exposure and Cadmium produced local tumors at various sites of injection,
typically sarcomas, in rats and mice [12].

Liver is one of the main target organs of environmental pollutants and xenobiotics. Thus the investigation on the capacity of
these environmental pollutants to alter the activation and detoxication balance has great importance. The current study was
designed to observe the effect of Acrylamide and Cadmium chloride in chick embryonic liver.

O
P a ||
H 2 C = CH-C-NH 2
A

O

H 2 C-CH- C -NH 2

B 1^^

Fig. 1. Structure of A. Acrylamide and B. Glycidamide

2. Materials and methods

2.1. Source of Fertilized Eggs and Incubation Conditions: Freshly laid Bobcock strain zero day old fertilized eggs were
purchased from Sri Venkateswara Veterinary University, Tirupati, Andhra Pradesh. They were incubated horizontally at
37.0±0.5°C with a relative humidity of 65% in an egg incubator, we considered dayl (dl) as an incubation period of 24h. The
humidity of the incubator was maintained by keeping the tray full of water inside. The water was replaced every alternate day
and the water level was maintained to keep the same percentage of humidity throughout the incubation. Eggs were rotated
manually four times a day and were examined through the candler every day for the proper growth and viability. The dead
eggs were removed immediately from the incubator. During all experiments, the embryos were maintained at 37.0±0.5°C
except for brief intervals (60-120 seconds) required during the different treatment conditions. During this interval embryos
experienced ambient room temperature (29-30°C).

2.2. Acrylamide Treatment: A group of six eggs (n=6) were maintained for each time point and dose. 0.1, 0.2 and 0.3 mg of
Acrylamide in saline was administered as single dose separately to fertilized chick embryos on day8 (d8), day9 (9) and day 10
(dlO) of incubation.

2.3. Cadmium chloride Treatment: Cadmium chloride in saline in concentration of 0.01, 0.02 and 0.03 mg was administered
as single dose separately to fertilized chick embryos on day 8 (d8), day 9 (d9) and daylO (dlO) of incubation.

The egg shell was opened at the blunt end of the top to obtain access to the air cell, where the respective test substance (50
|il) was injected directly on to the inner shell membrane. Covering the hole by wax tape could ensure the embryos vitality for
the remaining time until blood sampling and dissection. Chick embryonic liver was collected on dll after 24hrs (dlO), 48hrs
(d9) & 72hrs (d8) initial administration of the test substance. The tissue was washed with normal saline to remove blood and
fat debris and stored at -20°C until further use.

2.4. Fixation and staining of control and treated samples: The liver tissues were isolated from control and treated dll chick
embryos and were gently rinsed with physiological saline to remove blood and debris adhering to them. They were fixed in

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Bouin's solution until processing. This solution acts as a fixative for embryonic studies due to its excellent preservation of
nuclei and chromosomes. The tissues were washed with running tap water, overnight to remove Bouin's solution. After
dehydrating through a graded series of alcohols, the tissues were cleared in methyl benzoate and embedded in extremely hot
liquid paraffin wax. Sections of liver were cut at 6|i thickness were stained with haematoxylin, a basic dye, stains nuclei blue
due to an affinity to nucleic acids in the cell nucleus; eosin, an acidic dye, stains the cytoplasm pink. After dehydration and
cleaning, sections were mounted in Canada balsam. Histological examinations of the tissues were followed according to
Humason 1972 [13] and the specimens were observed under the light microscope.

3. Results

The results of control and treated chick embryo liver histology studies were presented in the fig 2 to 12. In these results the
control liver showed normal architectural hepatocytes showing clear nucleus and cytoplasm fig. 2. The O.lmg AC (24, 48 &
72 hr) treated liver showed early hepatic changes with mild granulation and vacuolating changes in the hepatocytes. Nucleus
was pushed to the periphery of the cell fig. 3-5. The chick embryonic liver treated with 0.2mg AC (24, 48 & 72 hr) showed
pycnotic nuclei i.e., nucleus of a cell undergoing programmed cell death or apoptosis due to irreversible condensation of
chromatin. It was followed by fragmentation of the nucleus, sinusoidal hemorrhages, i.e., excessive discharge of blood from
the blood vessels; profused bleeding and necrotic hepatocytes fig. 6 to 8. Upon 0.3 mg AC (24 hr and 72 hr) treatment the
chick embryonic liver hepatocytes showed more fatty changes, proliferation of sinusoidal spaces, hemorrhages, fatty
infiltrations, necrosis i.e., the premature death of cells and living tissue, and complete loss of architectural details of the
hepatocytes fig. 9,10. 0.3 mg Acrylamide (48 hr) treated tissue showed similar histopathological changes as to that of 0.2 mg
Acrylamide (72 hr) treated tissue.

Cadmium chloride treatment with 0.0 lmg (24, 48 hr) in chick embryonic liver showed similar changes as that of 0.2 mg
Acrylamide (48 hr) treated hepatocytes with mild vacuolations, and moderate degenerative changes of cytoplasm. Cadmium
chloride treatment with 0.0 lmg (72hr) in chick embryonic liver showed vacuolations in sinusoidal spaces fig 11. 0.02mg
CdCl 2 (24, 48 and 72 hr) treated liver showed hepatocytes revealing various necrotic changes, pycnotic nuclei and
hemorrhages fig. 8, 9. 0.02mg Cadmium chloride (24 and 48 hrs) treated tissues showed similar changes as that of 0.2 mg
Acrylamide (72 hr) and 0.3 mg Acrylamide (48 hr) treated tissues fig. 8. 0.02 mg CdCl 2 (48hr) treated tissue showed similar
changes as that of 0.2 mg Acrylamide (72hr), 0.3 mg. Acrylamide (48 hr) and 0.02 mg CdCl 2 (24 hr) treated tissues fig. 8.
0.02 mg of CdCl 2 (72 hr) treated tissue showed similar changes as that of 0.2 mg acrylamide treated tissue fig. 9. 0.03mg
CdCl 2 (24 hr) treated liver showed similar changes as that of 0.2 mg Acrylamide (48 hr), 0.0 lmg CdCl 2 (24, 48 hr) treated
tissues fig. 7. 0.03mg CdCl 2 (48 hr) treated liver showed complete loss of architectural details of hepatic cells, pycnotic
nuclei and complete necrosis fig 12. 0.03 mg of CdCl 2 (72 hr) treated tissue showed similar changes as that of 0.3 mg
Acrylamide (24 hr) and 0.02 mg of CdCl 2 (72 hr) treated tissues fig. 9.

Fig. 2. Control liver (1— >a. Normal hepatocyte with clear nucleus) (H &E- 40x)

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Fig. 3. O.lmg Acrylamide (24 hr) treated (H &E- 40x) (1— ► sinusoidal spaces)

Fig. 4. O.lmg Acrylamide (48 hr) treated (H &E- 40x)

Fig. 5. O.lmg Acrylamide (72 hr) treated (H &E- 40x) (1 —►vacuolating changes in sinusoidal spaces)

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Fig. 6. 0.2mg Acrylamide (24 hr) treated (H &E- 40x)

Fig. 7. 0.2mg Acrylamide (48 hr) treated (H &E- 40x)

Fig. 8. 0.2mg Acrylamide (72 hr) treated (H &E- 40x) (1— ► pycnotic nuclei, 2— ► hemorrhages, 3— ► necrotic changes)

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Fig. 9. 0.3mg Acrylamide (24 hr) treated (H &E- 40x)

Fig. 10. 0.3mg Acrylamide (72 hr) treated (H &E- 40x) (1— ► complete loss of architectural details of hepatocytes,

2— ►infiltrations)

Fig. 11. 0.0 lmg Cadmium chloride (72 hr) treated (H &E- 40x) (1— ►variolations in sinusoidal

spaces)

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Fig. 12. 0.03mg Cadmium chloride (48 hr) treated (H &E- 40x) (1— ► necrotic changes, 2— ^pycnotic nuclei)

3. Discussion

Chick embryos have been used in the past for several years to investigate the effect of environmental chemicals and
radiations on developmental effects, morphogenesis, etc. Liver is the major metabolizing organ which detoxifies a number of
drugs and xenobiotics. The current study on genotoxicity of acrylamide and cadmium chloride in chick embryonic system
using micronucleus test as an end point reveals that there is a significant induction of MN-Es in AC and CdCl 2 treated
animals and it is dose and time dependent. The MN-test in chick embryo gave clearly positive and dose-dependent results for
acrylamide and cadmium chloride, which are both well-characterized but weak mutagens. There was a dose and time
dependent increase in the induction of MN in peripheral blood erythrocytes in chick embryos treated with CdCl 2 . Cadmium is
non-mutagenic in bacterial tests and only weak mutagenic in mammalian cells in vitro. Inhibition of DNA repair has been
identified as a critical mechanism contributing to the genotoxic potential of cadmium. The MNT in chick embryo is a reliable
alternative genotoxicity assay system, which is physiologically closer to in vivo conditions than conventional in vitro
genotoxicity tests, not conflicting with ethical aspects or regulatory issues of animal protection.

In the present investigation the Acrylamide and Cadmium chloride treatment to chick embryo induced several pathological
changes in the liver like, pycnotic nuclei, hemorrhages, fatty infiltrations, necrosis, pushing of nucleus to the periphery,
sinusoidal dilations etc depending upon dose and duration of incubation of compound in liver hepatocytes.

Nagao et al., 2007 & Vasundhara in 2005 [14,15], reported that Acrylamide treatment in the liver of rats showed frequent
necrosis and bleeding, indicating hypertrophy of nuclei, pycnotic nuclei, proliferation of sinusoidal bile ducts and
hemorrhages. Fatty infiltration in liver of rabbits on administration of Cadmium was reported by Subramanyam et al., 1992
[16]. Extensive histopathological lesions like hepatocytic enlargements, necrosis and fatty changes were observed in rat and
mice by Koller, 2001 [17] in Cadmium administration. From the present study it is clear that Acrylamide and Cadmium
chloride caused mild to severe hepatic changes in 1 1 th day old chick embryonic liver.

4. Conclusion

In conclusion the metal ion, Cd, had severe damaging effect on chick embryonic liver than AC and however our study has
revealed that these two are causing damage to the embryos. Therefore our study, based on two environment associated
compounds, Acrylamide of plastics, and cadmium of industrial wastes, suggests that exposure of embryonic systems
including pregnant are necessary to avoid to get damage from these chemicals at the time of embryonic growth.

5. Acknowledgments

We thank University Grants Commission and DRDO New Delhi, for providing financial support to carry out our research
work. Department of Biochemistry of Sri Venkateswara University is financially supported by DST-FIST and UGC BSR,
New Delhi.

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