Protection Against Cadmium-Induced Abnormalities and Hepatotoxicity in ovo by Allium sativum

| Cadmium (Cd), a toxic heavy metal, is a potential carcinogen, mutagen and teratogen through bioaccumulation in tissues. Aim of present study was to analyze various developmental abnormalities by a sub-lethal dose of cadmium chloride and protective role of garlic (Allium sativum), to minimize the intensity of these toxicities. For this purpose, fertilized eggs of Gallus domesticus were randomly divided into four groups of forty eggs each. Control group was intact and untreated. Eggs of one group were injected with a sub-lethal dose of cadmium chloride (1.5 μg/egg) in albumin on 7th day of incubation. In another group, eggs were treated with cadmium chloride and fresh garlic juice (0.2 μg / egg) after a short interval of 10 minutes. Antidote group was injected with fresh garlic juice (0.2 μg / egg). Eggs of each group were incubated at 37 ± 0.5 °C and at 50-60% relative humidity till hatching. Natural hatching was obtained as 92.5, 32.5, 72.5, and 87.5% in control, dose, dose+antidote, and anitodote groups respectively with delayed hatching in some cases in dose and dose+antidote groups. Chicks and dead embryos were studied for morphological and morphometric analysis. Liver tissues were dissected out from each group for histological analysis. Cadmium chloride induced significant developmental abnormalities such as reduced body weight and crown rump length, exencephaly, ablepharia, crossed beak, gastroschisis, crooked toes, non-alcoholic fatty liver and pyknosis. Fresh garlic juice has successfully attenuated these toxicities and increased the body weight, crown rump length. Results of present study determine that garlic is a potential antidote to ameliorate the cadmium induced teratogenic and hepatotoxic defects in developing Gallus domesticus. Article History Received: January 19, 2018 Revised: March 16, 2018 Accepted: March 23, 2018 Published: May 10, 2018 Authors’ Contributions SA conceived the concept and executed the research. SS performed the experimental work. CA helped to study samples and wrote the manuscript.


Introduction
H eavy metals including cadmium owing to human ac- tivities have become common environmental pollutants and bio accumulated in human through food chain ( Jan et al., 2015).Cadmium is known as group 1 carcinogen (Kim et al., 2015).Prenatal exposure in mice has resulted into deleterious changes on the behavioral activities, neurotransmitters, oxidative stress, and brain neurons morphology (Allam et al., 2016), and altered thymocyte over induces hepatotoxicity and DNA methylation (Castillo et al., 2012;Sanders et al., 2014;Vilahur et al., 2015).Neurulation is found to be most vulnerable state (Robinson et al., 2010) leading to neural tube defects in mice (Robinson et al., 2011).
Garlic is well known for its protective role against cancer and so many other diseases (Setiawan et al., 2005) for its therapeutic and prophylactic effects (Ugwuja et al., 2016) owing to biologically active antioxidant substances like alliinase, allicin, alliin, and S-allylcysteine (Cruz et al., 2007;Majewski, 2014).Administration of garlic juice during pregnancy and lactation is known to protect from apoptosis in rat offspring's eye retina (Khordad et al., 2013).Aged garlic extract (AGE), even can reduce side effects of anticancer drugs (Nasr, 2014).Keeping in view the potential ameliorative role of garlic, the present study is designed to find its protective activity in developing chicks against cadmium.

Chemical used and dose preparation
Cadmium chloride (SIGMA-ALDRICH), was used as source of cadmium in the study.A sublethal dose of cadmium (Dżugan et al., 2011) was prepared in sterilized 0.7% avian saline (Pawlak et al., 2013) in such a way that each 0.05ml contained 1.5 µg of cadmium.

Preparation of antidote
Fresh garlic, Purple Glazer (Allium sativum) was used as antidote.Fresh garlic bulbs were purchased from local market in order to prepare fresh garlic juice.For this purpose, garlic cloves (2g) were peeled, washed, chopped and ground in pastel, mixed with distilled water and filtered afterwards and filtrate was used to prepare the desired concentration of 0.2 µg/0.05ml.

Experimental animal
Embryos of Gallus domesticus (domestic fowl, Comb Leghorn) were used as experimental animal.Fresh fertilized eggs (n=160) were purchased from a local hatchery at Pasrur road, Narowal.Eggs were cleaned, labelled and incubated horizontally at standard conditions (incubation days: D0-D21, temperature: 37 ± 0.5 °C, relative humidity: 50-60%) in a rolling egg incubator (24̋ × 30̋ × 17).Humidity was maintained by keeping the water filled tray inside the incubator which was replaced after every 24 hours and its level was maintained to provide eggs with equal percentage of humidity throughout the incubation period.Eggs were observed through the candler on 4 th day (D4) of incubation to check the embryonic development to remove the unfertilized eggs.

Experimental grouping and drug administration
On 7 th day (D7) of incubation, all the fertilized eggs were randomly divided into four groups of 40 eggs each as follows: Control group: untreated Dose group: CdCl 2 1.5 µg /0.05 ml/egg Dose + Antidote group: CdCl 2 1.5µg/0.05ml/egg+ Garlic juice 0.2 µg/0.05ml/ egg (after a short interval of 10 minutes) Antidote group: Garlic juice 0.2 µg/0.05ml/eggDose administration was carried out through a hole in the egg shell into albumen at blunt end using a sterile needle in sterilized environment of laminar air flow.Following injections, holes were sealed with molten paraffin wax immediately to avoid contamination.The remaining incubation period was continued safely until hatching.

Hatching of chicks
Fertilized eggs of each group were allowed to hatch naturally on 21 st day of incubation.Immediately after hatching chicks were kept separately in an environmentally controlled room at 27-30 °C with a photocycle of 14 hours light and 10 hours dark for two days.Chicks were constantly supplied with pearl millet and fresh tap water for drinking during this time period.

Morphological analysis and macrophotography
Un-hatched embryos, hatched chicks of all groups were studied morphologically for developmental abnormalities of skull, beak, eyes, limbs, tail, vertebral column and abdomen.

Morphometric analysis
Body weight and crown rump length of each embryo was recorded for each group.

Histological preparation
Liver tissues were dissected out and subsequently chopped into small pieces with sharp cutter and preserved in Bouin's fluid for fixation for 48 hours and processed for histological analysis using paraffin wax and hematoxylin-eosin staining technique.

Microphotography and histological study
Histological sections of liver were observed for various anomalies using microscope SWIFT (M4000-D) and microphotographed with the help of digital camera BESTSCOPE (BUC2-500C).

Statistical analysis
Data were analyzed using one-way analysis of variance (ANOVA) Tukey test using GraphPad Prism (Version 5.01) to find out the significant difference (p< 0.05) among various groups.

Morphological analysis
Chick hatchlings as well as embryos of control group were quite healthy and uniform in appearance and in other anatomical details.The body was well differentiated into head, neck and trunk regions.They had completely developed morphological features, including head crown, beak, eyes, fore limbs, hind limbs and digits.In dose group, 32.5% of embryos hatched naturally at day 21.These embryos showed minor morphological abnormalities, retarded growth and weight loss as compared to control group.Among rest in this group, 48.14% dead embryos were recovered on 23rd day.Embryos recovered on D23 showed various anomalies.Exencephaly, ablepharia, crossed beak, gastroschisis, and crooked toes was observed in 7.14, 21.43, 57.1 and 35.71 of embryos respectively in dose group, while crooking of toes was also observed in 21.43% of chicks in dose +antidote group (Table 1 and Figure 1 A-D).Hence, overall percentage of resorbed embryos in dose group was 35% and malformed embryos was 42.5%.
In dose+antidote group, 72.5% embryos hatched naturally at 21 st day of incubation.A total of 10% delayed hatching was recorded in dose and dose+antidote group on 23 rd day of incubation.All the hatchlings were subjected to morphological studies (Table 1).With the exception of some malformed hatchlings, all were quite healthy.They had completely developed morphological features, including head crown, beak, eyes, fore limbs and hind limbs.In this experimental group, 15% were malformed while 22.5% embryos were resorbed during incubation.A total of 87.5% naturally hatched chicks of antidote group were similar to control group with only 12.5% embryonic resorption (Table 1).

Morphometric analysis
Significant difference in birth weight was observed among all groups except control and antidote as well as dose+antidote and antidote group (Figure 2).

Histological analysis
Histological analysis of liver of chicks from control group appeared with intact and normal association of sinusoids and hepatic cords.A cross section of liver of chick embryo treated with 1.5µg/0.05ml/egg cadmium chloride showed various abnormalities including steatosis leading to pyknosis.Section through liver of chicks treated with dose plus antidote shows increased number of dividing cells indicating recovery from toxicity of cadmium.Antidote group showed normal association of sinusoids and hepatocytes similar to control group (Figure 3A-D).

Discussion
Cadmium is notorious for its toxicity in adults as well as in prenatal exposure ( Jacobo-Estrada et al., 2017).In this study it has been found to reduce body weight (Figure 2)  and successful natural hatching, induce simultaneously the malformations, resorption and death as compared to control.Among deformities, crossed beak was found to be the most prevalent.Gastroschisis, the most lethal one was second major anomaly obtained.Crooked toes and ablepharia were third abundant malformation, while exencephaly was encountered with least incidences.Such craniofacial and skeletal malformations and retarded growth in form of exencephaly, ablephary have also been recorded in mice and in vitro studies (Paniagua-Castro et al., 2008;Arguelles-Velazquez et al., 2013).Inhalation of cadmium even is found toxic and causes cataractogenesis in chronic smokers (Ramakrishnan et al., 1995).Crooking of toes may result from decreased bone mineral density, as it is known to cause bone brittleness for such reason in rat (Bhattacharyya, 2009).In chick embryos, such anomalies have dose relationship (Rodriguez-Fragoso et al., 2012).Among 11 differentially expressed genes (DEGs) in chicken, which are linked with beak deformity in addition to biosynthesis of unsaturated fatty acids and glycerolipid metabolism (Bai et al., 2014), over-expression of LOC426217 in the beak is thought to be the actual responsible (Bai et al., 2016).Cadmium is found to induce delayed hatching in this study as also reported by Dzugan and Lis (2016).
Histological analysis in current study revealed nonalcoholic fatty liver (steatosis) leading to pyknotic cells with shrunk nuclei.Such results are also evidenced in human (Hyder et al., 2013;Go et al., 2015) that may be due to altered gene expression in human hepatocellular carcinoma (HepG2) cells (Cartularo et al., 2015, or decrease in hepatic enzymatic and non-enzymatic antioxidants reduced glutathione, catalase, superoxide dismutase (Oyinloye et al., 2016).
In the group, co-treated with garlic along with cadmium, increased body weight, least malformations and recovery of fatty liver with increased number of normal and dividing cells, possibly due to its anti-Cd properties, radical scavenging assay, ferric reducing ability power assay, chelating activities, superoxide, and hydroxyl scavenging assay (Poljsak and Fink, 2014;Boonpeng et al., 2014), complexation of Cd to glutathione (GSH) and metallothionein (MT), prevention of endoplasmic reticulum(ER) stress, mitophagy and metabolic stress, as well as expression of chaperones (Sandbichler and Hackner, 2016).Aged garlic extract (AGE) contains S-allylcysteine (SAC) activates Nrf2 factor and inhibits prooxidant enzymes, and chelating effects (Cola -N-Gonzalez et al., 2012).Thiacremonone, another constituent of garlic has potent anti-inflammatory and anti-arthritic properties through the inhibition of NF-ÎºB (Ban et al., 2009).
Cadmium toxicity is suggested to overcome by high water intake, chemical antidotes (Rafati et al., 2017), however, dietary strategies including plants is likely to be more cheaper to encounter such unseen hazards (Zhai et al., 2015), as the use of fresh garlic along with high-fat diet keeps safe from its hepatotoxicity (Qamar et al., 2016).Results of present study authenticate the potent ameliorative nature of garlic against cadmium and suggest its use in any way at regular basis to withstand the harmfulness of unseen toxins like cadmium through food and water.

Figure 2 :
Figure 2: A bar graph showing body weight of chicks of different dose groups in ovo exposure to cadmium at 7th day of incubation.Control: untreated ; Dose group: treated with 1.5µg/0.05ml/egg of CdCl2; Dose+antidote group: treated with 1.5µg/0.05ml/egg of CdCl2 and 0.2 µg/0.05ml/egg of garlic juice; antidote group: treated with 0.2 µg/0.05ml/egg of garlic juice (*** indicate significant difference at p<0.05).

Table I : Ameliorative effects of fresh garlic juice against cadmium induced teratogenicities in chick embryos against control.
Values not sharing common letters are significantly different from each other