M.M. El Mahady, Kawkab A. Ahmed, S.A. Badawy, Y.F. Ahmed ... · In Egypt El-Shahat et al. (2012) indicated that AFM1 was significantly higher in male and female patients with high

Middle East Journal of Applied Sciences ISSN 2077-4613

Volume : 05 | Issue : 04 | Oct.-Dec.| 2015 Pages: CC-CC

Corresponding Author: M. A. Aly, Animal Reproduction and A.I. Dept., Veterinary Research Division, National Research Center, Dokki, Egypt

E-mail: [email protected]

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Pathological and Hormonal Effects of Aflatoxins on Reproduction of Female Albino Rats 1M.M. El Mahady, 1Kawkab A. Ahmed, 2S.A. Badawy, 2Y.F. Ahmed and 2M. A. Aly 1Pathology Department, Faculty of Veterinary Medicine Cairo University, Giza, Egypt. 2Animal Reproduction and A. I. Department, National Research Center, Giza, Egypt.

ABSTRACT

Aflatoxins are one of the natural occurring contaminants of food and feed stuffs. The reproductive system especially in female suffers from lack of studies. The aim of this study is to investigate the deleterious effects of aflatoxins on the female reproduction and on ovarian oogenesis. A total number of forty mature cyclic female albino rats were used and randomly allocated into one of the four treatment groups: the control group received basal diet containing zero ppb aflatoxins and three aflaoxin treated groups (A, B and C) fed on basal diets containing 250, 500 and 1000 ppb aflatoxins/ kg diet, respectively for 45 days. Results showed delay and elongation of estrous cycle, decreased levels of estradiol, progesterone, and testosterone hormones; dose dependent histopathological changes in ovaries and uterus, where atrophy and necrosis of ovaries, decrease number of viable ovarian follicles and increased number of inactive corpous luteum. In conclusion, this study showed that aflatoxins are severely toxic to female reproductive system. Thus, at the end of females exposure to aflatoxins can bring about deterioration of female reproductive health.

Key words: Aflatoxins, Reproductive Pathology, Estrous Cycle, Vaginal Smearing, Female fertility, Estradiol,

Progesterone.

Introduction

Mycotoxins have become increasingly important worldwide problem in our understanding of food safety and food poisoning. Mycotoxins are abiotic hazards produced by certain fungi that can grow on a variety of crops; its concentration in finished products is usually lower than in raw materials (Marin et al., 2013, Ahmed and Jutta, 2015), also defined as molecules of low molecular weight produced by fungi that elicit a toxic response through a natural route of exposure both in humans and other vertebrate animals (Zain, 2011).

Mycotoxin contamination represents a worldwide problem for various agricultural commodities both pre and post-harvest (Bryden, 2012, Streit et al., 2012 and Muzaffer, 2015).

Aflatoxins are secondary metabolites produced by toxigenic strains of Aspergillus flavus (A. flavus), Aspergillus nomius, Aspergillus parasiticus and so on, which are known to be potent carcinogens (IARC 2012) and hepatotoxic agents and possess serious hazards to human and animal health (Sidhu et al., 2009, Rustemeyer et al., 2010 and Jamali et al., 2012). There are four major groups of aflatoxins: B1, B2, G1 and G2, the public health impact of aflatoxin exposure is widely pervasive in economically developing countries (Alim et al., 2014).

Aflatoxins M1 and M2 are hydroxylated products of aflatoxins B1 and B2, respectively in milk upon ingestion of B1 and B2 aflatoxins’ contaminated feed (Stroka and Anklam, 2002). In Egypt El-Shahat et al. (2012) indicated that AFM1 was significantly higher in male and female patients with high hepatitis C virus (HCV) titer compared to the normal healthy controls. Occurrence of aflatoxin M1 (AFM1) in infant formula milk powder (IFMP) and maternal breast milk (MBM) was investigated as a risk factor affects the health of newborns in Egypt (Wael et al., 2011).

Estrous cycles are characterized by morphological changes in ovaries, uterus and vagina which occur during different phases called proestrus, estrus, metestrus and diestrus (Goldman et al., 2007). Exposure to mycotoxins has been linked to adverse effects on female reproduction by interfering with the synthesis, metabolism or degradation of steroid hormones, interaction with steroid receptors or impairing oocyte maturation and competence. Mycotoxin exposure affects ovarian follicles at the early stage (primordial) to the most advanced (pre-ovulatory) stages (Regiane et al., 2013).

Various mycotoxins are known for their endocrine-like activity and have been identified as potential endocrine active substances. For example, aflatoxin B1 (AFB1) has been classified as a potential endocrine active substance, as it is metabolised into aflatoxicol in a human choriocarcinoma cell line (JEG-3 cells), resulting in the up-regulation of CYP19A1 expression after 96 h exposure to 1 µM AFB1 (Storvik et al., 2011).

The aim of this study is to investigate pathological and hormonal effects of aflatoxins on reproduction of female albino rats.

Middle East J. Appl. Sci., x(x): xx-xx, 2015 ISSN 2077-4613

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Material and Methods Aflatoxins

Aflatoxin production, extraction and quantification were performed in the Laboratory of Marine Toxins, Food Toxicology and Contaminants Department, National Research Centre as described by Conder et al. (1963) and Stubblefield et al. (1967). Animals and Diets

A total number of forty mature cyclic female Wistar Albino rats (Rattus norvergicus), three months old was used and randomly allocated into one of the four treatment groups (10 females per group): the control group received basal diet containing Zero ppb aflatoxins and three aflaoxin treated groups (A, B and C) that fed on basal diets containing 250, 500 and 1000 ppb aflatoxins/ kg diet, respectively for 45 days. Data Collection Techniques Identification of Vaginal Cytology

At the 30th day of the experiment estrous cycle phases were determined by vaginal smears for 15 day (Sahar and Abeer, 2007 and Parhizkar et al., 2011).

The estrous cycle was determined at 08:00–09:00 a. m using the method described in the Organisation for Economic Co-operation and Development (OECD) Guidance Document for Histologic Evaluation of Endocrine and Reproductive Test in Rodents (OECD 2008). The normal estrous cycle in the rat follows a 4-day pattern, the stages of rat estrous cycle were classified according to the presence, absence, or proportions of three cell types, cornified (keratinized) cells, epithelial cells, and leukocytes, in vaginal smears. Hormonal Assay

Blood samples were collected from the orbital sinus by eye puncture in sterile centrifuge tubes just before scarification, and centrifuged at 3000 rpm for 15 min. for serum collection and stored at -20C˚for further hormonal analysis. Histopathological Examination

At the end of the experimental period, all animals were sacrificed and subjected to post mortem examination and all post mortem observations were recorded. Specimens from ovaries and uteri were collected and fixed in formol saline 10% then washed, dehydrated, cleared and embedded in paraffin. The paraffin embedded blocks were sectioned at 4-5 micron thickness and stained with Haematoxylin and Eosin (Bancroft et al., 2012) for histopathological examination. Statistical analysis

Data were analyzed using SPSS (2007) software version 16.0. Data were presented as Mean ± SEM (Standard Error of Mean). Differences between experimental groups were analyzed using one-way analysis of variance. All differences were considered statistically significant at P < 0.05.

Results

A. Identification of Vaginal Cytology Results showed a delay and increase in the length of estrous circle of all aflatoxin treated groups compared

with the control group which showed normal cyclicity. Individual days and stages of the estrous cycle are presented in Fig. 1 & Fig. 2.

B. Hormonal Assay.

Treatment had no significant effect on estradiol concentrations (P>0.05). Estradiol concentrations were significantly low in animals of treatment C compared with animals of the control group. A slight decrease of estradiol concentrations was observed in animals of both treatment A and B compared to control (Table 1).

Progesterone concentrations were significantly influenced by the treatment (P < 0.05), whereas a significant decrease in progesterone concentrations compared with the control group. Insignificant decrease of progesterone concentrations was observed in animals of both B and C groups.

Testosterone concentrations are also significantly influenced by the aflatoxins treatment (P<0.05). Animals of group A have significantly low testosterone concentrations compared to those of the control group but animals of treatments B and C have insignificantly low testosterone concentrations compared to control one.


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Fig. 1: Vaginal cytology representing each stage of estrous and stained with Methylene blue stain. A: During estrus, clumps of anucleated acidophilic cells. B: During metestrus, the same proportion among leukocytes, cornified and nucleated epithelial cells. C: During Diestrus, primarily consisted of a predominance of leukocytes. D: During Proestrus, consisted of predominance of nucleated epithelial cells.

Fig. 2: Chart showing estrous cycle stages: proestrus, estrus, metestrus and diestrus; cycle number and cycle length.

0

2

4

6

8

10

12

14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Day

s

Proestrus Estrus Metesrus Diestrus Cycle number cycle length

Control Group A Group B Group C


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Table 1: Mean ± SE (Standard error of mean) of estradiol, progesterone and testosterone in control and aflatoxin treated female rats. Variables Estradiol

(pg/ml) Progesterone

(ng/ml) Testosterone

(ng/dl) Control 16.7 ± 2.2 20.7 ± 0.80b 0.63 ± 0.05b

A 15.3 ± 2.3 16.9 ± 0.80a 0.44 ± 0.04a B 15.9 ± 2.3 19.9 ± 0.80ab 0.53± 0.05ab C 12.2 ± 1.9 19.3± 0.154ab 0.53 ± 0.04ab

Sig. NS 0.05 0.04

C. Pathology. C.1. Gross pathology

In group B and C uterus showed slight congestion. Meanwhile, other groups showed no obvious gross pathological changes C.2. Histopathology Ovary:

Histopathological examination of ovarian tissues of group A showed mild histopathological changes manifested by desquamation of granulosa cells, associated with congestion and multifocal oedema in between congested blood vessels in the ovarian medulla (Fig. 3. A). Large numbers of luteal follicles compared to the number of Graffian follicles were observed. Some examined sections revealed the presence of vacuolar degeneration and necrosis within the corpous luteum (Fig.3. B), accompanied with hyperplasia of the interstitial cells in between lutein cells.

Microscopical examination of sections from group B revealed hyperplasia of interstitial cells associated with hypertrophy of the medulla. Congestion of blood vessels and capillaries were observed in most of the examined cases (Fig. 3. C). Presence of ovarian cyst (Fig. 3. D).

Fig. 3: Photomicrograph of ovarian tissue of aflatoxicated rat from A. group A showing congestion of ovarian

blood vessels and oedema of ovarian medulla (arrow) (H&E X 200) B. group A showing vacuolar degeneration and necrosis of lutein cells (arrow) (H&E X 200). C. group B showing hyperplasia of interstitial cells, hypertrophy of the medulla with congestion of blood vessels and capillaries (H&E X 100). D. group B showing ovarian cyst with congestion of ovarian blood vessels (H & E X 100).

Some cases showed marked atrophy of ovary characterized by the presence of one corpous luteum, other cases showed the presence of large number of corpora lutae associated with severe congestion of blood vessels at the periphery of corpous luteum. Degenerated and desquamated granulosa cells within ovarian follicle, severe


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degeneration of lutein cells and apoptosis in corpous luteum were noticed (Fig. 4. A). There was hypertrophic lutein cells with deeply eosinophilic cytoplasm.

In group C, most of the ovarian tissues were severely atrophied and necrosed (Fig. 4. B). there were decreased number of functional ovarian follicles compared with the presence of multiple corpora lutae. Diffuse severe congestion and hemorrhage in the stroma of ovarian tissue associated with the presence of multiple corpora lutae and necrosis. The multiple corpora lutae showed severe congestion and vacuolar degeneration of lutein cells with the presence of fatty cells, moderate pyknosis in some lutein cells leaving white hallow zone around them, apoptosis in the corpous luteum (Fig. 4. C). Some cases showed the presence of cystic Ovary (Fig. 4. d).

Fig. 4: A. group B showing apoptosis of some lutein cells (arrow) (H&E X 400). B. group C showing necrosis of

ovarian follicle and congestion of blood vessels (arrow) (H&E X 200). C. group C showing apoptosis of lutein cells, congestion of ovarian blood vessels and hyperplasia of interstitial cells (H&E X 200). D. group C showing different size of multiple ovarian cysts (H&E X 100).

Uterus:

Histopathological examination of uterine tissues of group A showed normal histological structures (Fig. 5. A). Some cases showed mild hyperplasia of epithelial cells lining endometrium and lining epithelium of endometrial glands. Mild focal aggregation of mononuclear inflammatory cells mainly neutrophils in the lamina propria in the submucosa of endometrium were seen.

Microscopical examination of the uteri in group B revealed marked necrosis of the lining epithelium of endometrium associated with congestion in some blood vessels. There were vacuolar degeneration of the lining epithelium of uterine glands with mild leucocytic cells infiltration in the submucosa and lamina propria was observed (Fig. 5. B) and focal necrosis of lamina epithelialis (Fig. 5. C). Congestion of the myometrial blood vessels and massive fibrosis of the lamina probria were noticed recorded in some cases.

Concerning group C the uterus showed severe thickening and increase in the height of the lining epithelium of endometrium associated with vacuolar degeneration of the endometrial lining cells (Fig. 5. D), the epithelial lining of the endometrium in some cases showed some degrees of hyperplasia and metaplasia (Fig. 5. E). Focal degenerative changes manifested by vacuolation of the endometrial epithelium with pyknosis of their nuclei and desquamation of some cells in the lumen associated with mild mononuclear cells infiltration in the submucosa


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were observed (Fig. 5. F). The uterine wall showed connective tissue proliferation with the formation of newly formed blood capillaries in some areas.

Fig. 5: Photomicrograph of uterine tissue sections. A. group A shows normal histological structure (H&E X 200).

B. group B shows vacuolation of lining epithelium of endometrium associated with few leucocytic cells infiltration (H&E X 400). C. group B shows focal necrosis of lamina epithelialis and leucocytic cells infiltration in lamina propria. (H&E X 400). D. group C shows hyperplasia, metaplasia and vacuolation of lining epithelium of endometrium (H&E X 200). E. group C shows mononuclear cells infiltration in the submucosa (H&E X 200). F. shows hyperplasia of endometrial glands and epithelium (H&E X 100).


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Discussion

In the literature, information reviewed for aflatoxicosis effects on the reproductive performance of females, were scanty. Female infertility accounts for approximately 40% of all infertility cases, ovulatory disorders are a predominant cause for women not being able to conceive and accounts for 25% of female infertility (Paul and Lauren, 2004).

AFB1 was reported to exert deleterious effects on the reproductive capacity of lab and domestic female animals (Oliveira et al., 2002; Ogido et al., 2004 and Abdelhamid et al., 2007).

The delay in estrous circle is probably due to the fact that aflatoxin treatment inhibits the hormonal function probably while compromising cellular integrity and function. Aflatoxicosis may impair reproductive efficiency including abnormal estrous cycle (too short and too long) (Cassel et al., 1988). Compounds that have been reported to destroy or impair the growth of ovarian follicles can have marked effects on cyclicity.

Aflatoxins are very potent toxins affecting the growth of all animals, delay in genital system growth (Hafez et al., 1982a & b), high disturbances in estrous cycle, reduced pregnancy rate and number of live new born, failure in nidation and intrauterine death of the foetus (Shapour and Saeedeh, 2013).

In this study aflatoxin administration led to significant decrease in the levels of some reproductive hormones such as estradiol, progesterone, testosterone. After prolonged AFB1 administration and also during the oestrus cycle in goats, decreased progesterone concentrations during the luteal phase and decreased oestradiol-17β concentrations during oestrous synchronization were also confirmed by Kourousekos et al. (2008) who also proposed that a direct effect of AFB1 either on ovarian secreting cells or on the hypothalamus–hypophysis–ovarian axis.

Most authors hypothesise that aflatoxin may affect the reproductive system by its toxic effect on the liver, where the cellular hepatic damage could inhibit enzyme synthesis and/or enzyme activity or inhibition of lipid metabolism or fatty acid synthesis, causing decreased synthesis of precursor molecules for gonadal as well as gonadotropic hormones, e.g. FSH, luteinizing hormone (LH) oestradiol, testosterone and progesterone (Handan and Güleray 2005).

Aflatoxin may also affect the reproductive system by causing lysis of germ cells, as it was known that aflatoxin at graded doses induced severe oxidative damage in the testis and accessories that promoting their apoptosis (Kawkab et al., 2012).In vitro evidence from animal model and clinical studies suggests that reactive oxygen species (ROS) plays a role in the aetiology of adverse reproductive events (Walsh et al., 2000; Acevedo et al., 2001; Sikka, 2001) causing the generation of radical species exceeds scavenging by antioxidants as a result of excessive production of ROS and/or inadequate intakes or increased utilization of antioxidants. Increased ROS is associated with decreased steroidogenesis and their cyclic productions contribute to a decline ovarian function (Kodaman and Behrman 2001).

Also changes develop during the transport of these hormones in the bloodstream. An example for that is a significant reduction of plasma protein content by AF, Aflatoxins targets protein synthesis pathways especially the DNA template, RNA template (mRNA, tRNA, rRNA), proteins, transcription, translation and cellular metabolic reactions (Hossam El-Din, 2013).

In the current work, the severity of the pathological alterations observed in the Ovaries and uteri of aflatoxins treated female rats were dose dependent, since the ovaries showed severe atrophy in most of examined cases, decreased number of functional ovarian follicles compared to the presence of multiple corpra lutae, these lesions agreed with that mentioned by Shapour and Saeedeh, (2013).

Uterus showed dose dependent histopathological changes summarized as severe thickening and increase in the height of the lining epithelium of endometrium, associated with vacuolar degeneration of the endometrial lining cells, the endometrial lining cells in some cases showed some degrees of hyperplasia and metaplasia, with focal degenerative changes and pyknosis of their nuclei with desquamation of some cells in the lumen associated with mild mononuclear cells infiltration in the submucosa, these changes are parallel with the result of Abd El-Wahhab (1996) and Panchakshari et al., (2010). Conclusion

The investigations led to the conclusion that aflatoxins are severely toxic to female reproductive system. The manifestations include severe histopathological changes in the ovaries and uterus associated with decrease in the levels of some reproductive hormones such as estradiol, progesterone, and testosterone. Thus, chronic exposure of female rats to aflatoxins can bring about deterioration of female reproductive health.

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M.M. El Mahady, Kawkab A. Ahmed, S.A. Badawy, Y.F. Ahmed ... · In Egypt El-Shahat et al. (2012) indicated that AFM1 was significantly higher in male and female patients with high