LIPID PEROXIDATION IN BRAIN OF CHICK EMBRYOS
J.Sravani1, K.Padmaja2, P.Eswara Prasad3 and K.Jayasri4
1,2,3,4
Department of Veterinary Biochemistry, C.V.Sc., SVVU, Tirupati *Corresponding Author: J. Sravani1
Abstract
A study was conducted on effect of Bisphenol-A (BPA) toxicity in antioxidant enzymes, Thiobarbituric acid reacting substances (TBARS) and Glutathione (GSH) during embryonic development in chicks. Bisphenol-A dissolved in distilled water in three concentrations of 100µM, 250µM and 500µM injected into the aircell separately to 11th and 14th day old chick embryos. Embryos were sacrificed after 24 and 48 hours of BPA exposure to collect brain tissues for estimation of TBARS, GSH and antioxidant enzyme activities (Superoxide dismutase, Catalase, Glutathione peroxidise and Glutathione-S-transferase). The results showed a significant dose dependent increase in TBARS, GSH, GPx, GST and SOD in both 11th and 14th day BPA treated
embryos. A significant increase in CAT activity was noticed after 48 hours in brain of 11th and 14th day BPA treated chick embryos.
Keywords: Antioxidant enzymes, Bisphenol-A, Brain, Chick embryos
I. INTRODUCTION
There has been increasing scientific concern since last two decades regarding, the adverse effects of chemical pollutants in the environment which interferes with normal functioning of different organs in animals and humans. Among endocrine disrupting chemicals, BPA is one of the most studied because of its extensive use (Gioiosa et al., 2013). The chemical bonds between BPA molecules are unstable and the chemical leaches into materials with time of storage and use. Studies in animals have shown that BPA exposure is associated with early puberty in females, low sperm counts, increased susceptibility to reproductive tract cancers and altered brain development in males and females (Newbold et al., 2009).
Bisphenol-A induced mullerian duct malformation in female quail embryos and feminization of the left testis in male chicken embryos (Berg et al., 2001). Highest concentrations of BPA was found in adipose tissue and liver (100%) followed by brain (70%) (Geens et al., 2011). There is evidence that several teratogens affect the developing embryo by increasing its oxidative stress, because of its relatively weak anti-oxidant defense especially at early stages of organogenesis, resulting in severe embryonic damage (Ornoy, 2007). It was reported that BPA exposure during embryonic/fetal life and infancy induces tissue oxidative stress and peroxidation, ultimately leading to underdevelopment of the brain, kidney and testis (Kabuto et al., 2004).
II.MATERIAL AND METHODS
A. Source of Fertilized Eggs and Incubation Conditions:
The present study was conducted at the Department of Veterinary Biochemistry, College of Veterinary Science, Tirupati. Freshly laid wild Bobcock strain zero day old fertilized eggs were procured from Department of Poultry Science, College of Veterinary Science, Tirupati. They were incubated at 37.5±0.5˚C with a relative humidity of 65% in an egg incubator.
Experimental groups:
Group II : Bisphenol- A (100µM) Group III : Bisphenol -A (250µM) Group IV : Bisphenol -A (500µM)
The embryos were sacrificed after 24 and 48 hours from 11th and 14th day BPA treated embryos for the collection of brain samples.
B. Biochemical Analysis:
Thiobarbituric acid reacting substances in tissues were estimated by the method of Ohkawa et al. (1979).
C. Assay of antioxidant enzymes: Preparation of enzyme extract:
Brain tissues were blotted dry, thawed and homogenized at 4ºC in 3 volumes of 0.25M sucrose containing 0.07M phosphate buffer (pH 7.2), 10mM EDTA and 0.1% Triton X-100. Post mitochondrial supernatant was prepared by centrifuging at 12,000 g for 15 minutes at 4ºC using REMI refrigerated centrifuge.
Glutathione peroxidase activity was assayed by the method of Rotruck et al., 1973. Glutathione-S-transferase activity was determined by the method of Habig et al., 1974. Superoxide Dismutase activity was measured according to Misra and Fridovich (1972). Catalase activity was measured by the method of Beers and Sizer (1952). Glutathione content was determined according to the method of Ellman (1959).
D. Statistical Analysis:
Statistical significance between the groups was analysed by one way ANOVA followed by Tukey’s post–hoc test using statistical package for social sciences (SPSS 15.0 version).
III. RESULTS
Significant increase in TBARS was observed after 24 hours in 11th day compared to 14th day BPA treated embryos. It was found to be 45, 53 & 100% increase in 11th day compared to 20, 37 & 64% in 14th day in Group II, III & IV respectively. Decreased production of TBARS observed with increase in growth of the embryo (Figs. 1&2). A significant increase GPx activity was noticed in 14th day compared to 11th day of BPA exposed chick embryos. The activity was decreased with development of the embryo. Maximum increase was observed after 48 hours compared to 24 hours and It was found to be from 1.5 to 3 fold in 11th day and from 3 to 4 fold in 14th day after 24 and 48 hours of treatment in Group IV compared to Group I (Figs.3&4). Significant induction in GST activity was noticed in group IV after 24 hours in 11th day. Whereas, the enzyme activity altered significantly after 48 hours in 14th day BPA treated embryos. Maximum induction was observed in Group IV (2 fold) after 24 hours in 11th day and 3 fold after 48 hours in 14th day treated chick embryos (Table 1).
A significant increase in SOD activity was observed after 24 hours compared to 48 hours in both 11th and 14th day treated embryos. It was found to be nearly 2 & 4 fold in 11th day and 4 & 5 fold in 14th day after 24 hours in Group III & IV respectively. The activity was decreased after 48 hours compared to 24 hours in all the groups (Figs.5 &6). Catalase activity was increased after 48 hours compared to 24 hours in 11th and 14th day BPA treated embryos. Significant increase was observed in Group IV which was found to be nearly 3 fold and 2 fold in 11th and 14th day respectively (Table 2). Significant increase in GSH levels observed after 48 hours in 11th and 14th day BPA treated chick embryos. It was increased to 2, 3 and 4 fold after 24 hours in 11th day BPA treated embryos. Whereas, it was increased from 2 to 3 fold in 14th day after 24 and 48 hours of treatment respectively in Group III (Table 3).
Table 1: Mean values of GST activity (U/mg protein) in brain tissue
Treatment 11
th
day 14th day
Group-I 4.04 ± 0.01a 4.19 ± 0.04a 4.46 ± 0.06a 4.87 ± 0.06a
Group-II 4.20 ± 0.10a 4.68 ± 0.07b 5.34 ± 0.13b 7.07 ± 0.06b
Group-III 4.39 ± 0.15a 5.60 ± 0.25c 6.22 ± 0.13c 6.04 ± 0.09c
Group-IV 8.46 ± 0.18b 7.52 ± 0.15d 5.32 ± 0.05b 12.36 ± 0.16d
df (3, 20) (3, 20) (3, 20) (3, 20)
F 273.06 97.19 50.32 1035.11
Table 2: Mean values of CAT activity (U/mg protein) in brain tissue
Treatment
11th day 14th day
24 hours 48 hours 24 hours 48 hours
Group-I 6.68 ± 0.41d 6.44 ± 0.03a 6.37 ± 0.03a 6.19 ± 0.20a
Group-II 6.17 ± 0.03c 6.38 ± 0.13a 5.98 ± 0.12a 6.70 ± 0.05b
Group-III 6.34 ±0.03b 8.12 ± 0.22b 6.03 ± 0.16a 9.89 ± 0.19c
Group-IV 5.76 ± 0.03a 16.89 ± 0.16c 6.32 ± 0.10b 13.99 ± 0.18d
df (3, 20) (3, 20) (3, 20) (3, 20)
F 111.20 1055.61 3.00 455.83
Table 3: Mean values of Glutathione (mg/g tissue) in brain tissue
Treatment 11
th
day 14th day
24 hours 48 hours 24 hours 48 hours
Group-I 0.32 ± 0.01a 0.39 ± 0.02a 0.41 ± 0.004a 0.45 ± 0.01a
Group-II 0.66 ± 0.03b 0.56 ± 0.01b 0.66 ± 0.01b 0.73 ± 0.03b
Group-III 0.88 ± 0.02c 1.62 ± 0.03c 0.97 ± 0.04c 1.20 ± 0.01c
Group-IV 1.26 ± 0.06d 1.86 ± 0.06d 1.55 ± 0.03d 2.03 ± 0.02d
df (3, 20) (3, 20) (3, 20) (3, 20)
F 108.26 416.40 364.69 1170.13
Fig.1: TBARS in brain of 11th day treated chick embryos 0
50 100 150 200 250 300 350 400 450 500
24 hours 48 hours
n
m
o
le
s o
f M
D
A
/g
tis
su
e
Group I
Group II
Group III
Fig.2: TBARS in brain of 14th day treated chick embryos
Fig.3: Glutathione peroxidase activity in brain of 11th day treated chick embryos
Fig.4: Glutathione peroxidase activity in brain of 14th day treated chick embryos
Fig.5: SOD activity in brain of 11th day treated chick embryos 0 50 100 150 200 250 300 350
24 hours 48 hours
n m o le s o f M D A /g tis su e Group I Group II Group III Group IV 0 20 40 60 80 100 120
24 hours 48 hours
m o le s o f GSH /m in /m g p ro te in Group I Group II Group III Group IV 0 20 40 60 80 100 120
24 hours 48 hours
m o le s o f GSH /m in /m g p ro te in Group I Group II Group III Group IV 0 5 10 15 20 25
24 hours 48 hours
Fig.6: SOD activity in brain of 14th day treated chick embryos
IV. DISCUSSION
Significant increase in TBARS was observed with BPA treatment in 11th and 14th day of embryonic development. Significant increase observed after 24 hours compared to 48 hours in 11th day BPA injected embryos may be due to high susceptibility of brain to oxidative stress because of large amounts of polyunsaturated fatty acids and higher oxygen consumption (Bashir et al., 2006). These results are in agreement with Chen et al. (2002) who observed increase in MDA and GSH levels in the brain of male rats exposed to BPA. In response to elevated TBARS, significant increase in GPx activity was observed in 11th day compared to 14th day BPA injected embryos. The increased GPx activity after 24 hours of BPA treatment may be responsible for reduction in TBARS (due to removal of H2O2 by GPx) after 48 hours in brain tissue.
Glutathione-S-transferase protects cells or tissues against oxidative stress and damage by detoxifying various toxic substrates derived from cellular oxidative processes (Sharma et al., 2004). Glutathione-S-transferase conjugates GSH to electrophiles (Ketterer et al., 1982) and also breakdown products of lipid peroxidation to GSH (Stephenson et al., 2002). The results are in agreement with Padmaja and Ramamurthi (1997) where zinc treatment increased GST activity in response to induced oxidative stress.
Our results shows that the end product of the dismutation reaction catalyzed by SOD is H2O2 which only be effectively detoxified by increased GPx activity in BPA exposed chick embryos. The results in the present study are in accordance with Kabuto et al. (2004), who observed that BPA injection induces over production of H2O2 in mouse organs and H2O2 is readily converted to hydroxyl radicals.
In the present study the CAT activity was increased after 48 hours of BPA exposure showing its role in converting H2O2 radicals produced after 24 hours due to elevated SOD activity both in 11th and 14th day treated embryos. Catalase activity was not altered significantly after 24 hours, which shows the major role of GPx in detoxifying H2O2 radicals. The results showed elevated levels of GSH after 48 hours of BPA treatment. Halliwell (2006) reported that increase in glutathione levels is caused by hydroxyl radicals produced from H2O2. Similar results are noticed by Gualtieri et al. (2010) where high doses of BPA increase cell content of GSH owing to increased GSH synthesis.
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