Gouda, Sahar G.; Khalil, Mahmoud S.; Naim, Magda M.
Acrylamide is a chemical used in many industries around the world and more recently has been found to form naturally in foods cooked at high temperatures, such as fried potato products, bread, and coffee . Acrylamide is shown to be a neurotoxicant, a reproductive toxicant, and a carcinogen in animal species . Exposure to acrylamide from foods is a growing concern because it causes cancer, such as mammary adenomas in rats . Furthermore, it is a possible human carcinogen with genotoxicity .
Reproductive toxicity of acrylamide has been tested in animals [5,6]. In addition to the occurrence of testicular damage, DNA breakage during specific germ cell stages was also reported . It was suggested that acrylamide toxicity affects the male reproductive organs, whereas female rodents seem to be resistant to this reproductive toxicity . Acrylamide also induced genotoxicity including micronuclei , chromosomal aberrations, sister chromatid exchanges, and mitotic disturbances in vitro . Chromosomal aberrations were detected in spermatocytes, and micronuclei were observed in spermatids .
Numerous studies have shown that the random amplified polymorphism DNA PCR (RAPD PCR) could be used as powerful tools to detect genomic instability in cancerous cells. RAPD PCR reaction uses short primers (10 bases long) to amplify random segments of DNA. Amplified products are separated on agarose gels and stained with ethidium bromide. Change in DNA sequence at the primer annealing sites produces different amplified segments (DNA bands). The amplified bands usually originate from unique sequences rather than from repetitive elements. Furthermore, the amplification is semiquantitative, in that the intensity of an amplified band is proportional to the concentration of its corresponding template sequence. The degree of aneuploidy of a tumor cell genome is reflected in the intensities of bands, compared with those from the normal diploid genome of the same individual. In this context, by carefully adjusting the concentration of the template DNA, it is possible to detect losses or gains in the number of copies of a sequence by changes in the intensity of a band. This property is very useful in many studies, such as the investigations of the genetic events that occur in the process of transformation from normal to cancerous cell. The strategy consists of comparing the RAPD profiles obtained from normal and cancerous cells obtained from the same individual .
Curcumin is a natural phenolic component of yellow curry spice. It has been reported to possess diverse pharmacological properties including antioxidant, anti-inflammatory, and antiproliferative activities . Curcumin has proven efficacy in protection against cisplatin-induced oxidative injury in rat testis , aflatoxin-induced biochemical changes in mice testis , and in protection of mice Leydig cells from the damage induced by chronic alcohol administration . It was also reported to be efficient in the suppression of cancer . However, its possible effects on suppression of the reproductive toxicity and genotoxicity produced by acrylamide are unknown.
This study was performed to evaluate the reproductive toxicity as well as the genotoxicity of acrylamide in male albino mice. It also aimed at testing the possible protective effects of curcumin against these induced toxicities.
Materials and methods
Twenty-eight adult male albino mice with average weight between 25 and 30 g were used in this study. After the acclimatization period, the animals were randomly divided into four groups of seven mice each. Group 1 served as the control and received distilled water through oral intubation for 5 days. Group 2 received curcumin (purchased commercially as a powder produced by Al Doha Company, Al Doha Company, 10th of Ramadan City, Egypt) at a dose of 80 mg/kg body weight/day  for 5 successive days. Curcumin was dissolved in distilled water and was given by oral intubation. Group 3 animals were treated with acrylamide (Winlab Ltd., UK) at a dose of 60 mg/kg body weight/day for 5 days . Acrylamide (supplied as a powder) was dissolved in distilled water and was also given by oral intubation. Animals of group 4 were given both curcumin and acrylamide at the same previous doses for 5 successive days. Each time, curcumin was given 1 h before the acrylamide.
Animals were kept on standard animal diet. After 72 h of the last administration, the mice were anesthetized by ether inhalation, blood samples were taken from the hearts into tubes containing EDTA for genetic analysis, and then they were killed. For histological studies, the right testis, from each animal, was used for light microscopy, the left was prepared for transmission electron microscopy (TEM; Jeol, JEM 1010, Japan), and the epididymis was used to prepare sperm smears. The right testes were fixed in Bouin's fixative, then processed to prepare 5-μm-thick paraffin sections, and stained with hematoxylin and eosin. The epididymis was minced (using a sharp blade) in 1 ml of 0.9% saline solution. The resulting sperm solution was filtered, to remove tissue fragments, and used to prepare sperm smears (five for each animal), which were stained with 1% Eosin-Y, and taken for sperm morphology examination.
Quantitative analysis was carried out where the sperm smears were examined with the light microscope to detect sperm abnormalities. Sperms were counted in 20 high power field in the 5 smears from each animal. Morphological abnormalities of sperm head and tail were recorded. The percentage of each abnormality was compared to that of the control group.
The left testis, from each animal, was immediately immersed in 2.5% glutaraldehyde solution. They were trimmed into small pieces, in the presence of glutaraldehyde, and then placed in the same fixative at 4°C for 24 h. The specimens were then washed overnight with several changes of 0.1 mol/l sodium phosphate buffer, pH 7.4 at 4°C. They were later postfixed in 2% osmium tetroxide in 0.1 mol/l sodium phosphate buffer, pH 7.4 at room temperature. After dehydration in ascending grades of cold ethanol and propylene oxide, the testicular specimens were embedded in Spurr's resin. Semithin sections were used for observation. Ultrathin sections stained with uranyl acetate and lead citrate were examined at 80 kV under the TEM in Al Azhar University, Cairo, Egypt.
For genetic analysis, a volume of 300 μl of the whole blood was poured into microcentrifuge tubes. The alcohol precipitation method was used for isolation of genomic DNA according to the methodology described by Wizard Genomic DNA Purification Kits (Promega, UK). The DNA purity and concentration were estimated by measuring the absorbance (optical density) of the extracted genomic DNA at 260 and 280 nm using the spectrophotometer (Promega). The genomic DNA purity was calculated by using the formula (A260/A280) ratio, which represents a high-quality DNA sample if the results were between 1.7 and 2.0. The genomic DNA concentration was also calculated by using the following formula: Concentration (microgram/milliliter)=(A260 reading−A320 reading)×dilution factor×50 μg/ml. The total yield is obtained by multiplying the DNA concentration by the final total purified sample volume using the following formula: DNA yield (microgram)=DNA concentration×total sample volume (milliliter).
RAPD analysis was carried out on the genomic DNA extracted from the white blood cells of the different groups of the studied mice. For this RAPD PCR, five oligodecamers  (Table 1) were used. For each DNA extracted per animal, five PCR reactions with same conditions were performed. Each reaction had one primer. The PCR amplification was conducted in 50 μl of reaction mixture containing 100 ng of genomic DNA, dNTPs (100 μmol/l), primer (40 nmol/l), MgCl2 (2.5 mmol/l), Taq DNA polymerase (2.5 U), and Promega 10× Taq DNA polymerase buffer (5 μl). The reactions were carried out in a thermocycler (Perkin-Elmer 9700, UK) programmed with a first denaturation of 5 min at 94°C, followed by 45 cycles of 0.5 min at 94°C, 1 min at 36°C, 2 min at 72°C, and finally one cycle at 72°C for 5 min. Negative controls were carried out. They included the mentioned mixture in which the amount of DNA was replaced by nanopure water.
Then, 25 μl from each PCR products was analyzed by agarose gel electrophoresis. For detection of PCR products, 2% agarose gel was prepared by adding 1 g of agarose to 50 ml of 1× TBE buffer. For each gel, 2 μl of ethidium bromide was added. A DNA marker (GeneRular DNA Ladder, Fermentas, Germany) was used in each gel to size the products. Electrophoresis was carried out at 80 V for an hour at room temperature. Photographs of the gels were taken by a Polaroid camera (Cole-Parmer, Illinois, USA) or by a digital camera .
The χ2-test or Fisher's exact test was used as appropriate to assess differences in sperm abnormalities between the groups. Significance was set at P value less than 0.05 for all comparisons. All statistical analyses were carried out with the aid of SPSS 15 (SPSS Inc., Chicago, Illinois, USA) software. Moreover, genomic template stability (GTS) is a qualitative measure reflecting changes in RAPD profiles. The GTS (%) was calculated as follows: GTS=[1−a/n]×100, where ‘a’ was the average number of polymorphic bands detected in each treated sample and ‘n’ is the number of total bands in the control. Polymorphism in RAPD profiles included disappearance of a normal band and appearance of a new PCR band in comparison with control RAPD profiles. All experiments were repeated at least two times .
Examination of hematoxylin and eosin-stained sections revealed normal histological structure of the testis. The parenchyma was formed of seminiferous tubules and interstitial cells of Leydig. The seminiferous tubules were lined by the spermatogenic epithelium and Sertoli cells. They appeared to be lying on the basement membrane (which was surrounded by myoid cell nuclei) and surrounding the central lumen. The spermatogenic epithelium included the spermatogonia, primary spermatocytes, early round spermatids, and late elongated spermatids. The tubules were separated by the interstitial tissue that contained interstitial cells of Leydig and small capillaries, in addition to fibroblasts and macrophages (Fig. 1).
Light microscopic examination of sperm smears, from this group, revealed that most (88.4%) of the sperms appeared with normal architecture; however, a small percentage showed abnormal heads (4.9%) and abnormal tails (6.7%; Table 2).
TEM examination revealed that the spermatogenic epithelium appeared to be resting on the regular basement membrane, which was surrounded by myoid cells with flat nuclei. The stratified spermatogenic epithelium was formed of spermatogonia, primary spermatocytes, and round early spermatids, which had many small peripherally arranged mitochondria. Sertoli cells were also seen (Fig. 2). Normal-shaped sperm heads were embedded in processes of Sertoli cells, which contained many mitochondria. There were junctional specializations between the heads of sperms and Sertoli cells, in addition to other specializations between Sertoli cell processes (Fig. 3). The interstitial tissue showed active interstitial cells of Leydig that had euchromatic nuclei, lipid droplets, mitochondria, smooth endoplasmic reticulum, and some microvilli. Adjacent small capillaries containing red blood cells and platelets were also seen (Fig. 4).
Light microscopic examination of hematoxylin and eosin-stained sections, from this group, revealed normal appearance of the testes. Moreover, the sperm morphology test revealed that the percentage of normal sperms was 86.4%, whereas that of the abnormal heads was 5.7% and abnormal tails was 7.9% (Table 2). The abnormalities showed no significant difference, when compared with the control group; however, it showed significant decrease when compared with the acrylamide group (Table 2).
Examination with TEM revealed normal appearance of the spermatogenic epithelium, which rested on the basement membrane that was surrounded by myoid cells. The interstitial space of the testis showed normal appearance of Leydig cells that contained many lipid droplets (Fig. 5).
Hematoxylin and eosin-stained sections, from this group, showed either atrophy or degeneration of the germinal epithelium of seminiferous tubules with appearance of abnormal vacuolations and thickened basement membranes. The basement membrane also showed irregularity and separation from the germinal epithelium in some parts. The lumen of the seminiferous tubules contained exfoliated germinal epithelium and abnormal-shaped spermatozoa, which had coiled tails. Congestion of the capsular blood vessels was also seen. In some regions, the interstitial tissues showed an increase in amount (Figs 6 and 7).
Results of the sperm morphology test revealed that 47.5% of the counted sperms showed abnormalities of which 13% showed abnormal heads and 34.5% showed abnormal tails (Fig. 8 and Table 2). These abnormalities showed a significant increase compared with the control group (P<0.001 for all; Table 2). The head abnormalities included amorphous head, head without hook, and double head; the most common was the amorphous head (12.2%). The tail abnormalities included coiled, angulated, broken, and double tail and the most common was the coiled tail (23.1%).
TEM examination confirmed the light microscopic results. There was irregularity of the basement membrane, which was surrounded by markedly thickened boundary tissue (the myoid cells). This was in addition to the presence of abnormal vacuolations and electron-dense bodies in the processes of Sertoli cells (Fig. 9). The early round spermatids showed abnormal vacuoles (Fig. 10) and the late elongated spermatids appeared abnormal in their shapes (Figs 10–12). The elongated spermatids were embedded in Sertoli cells, which showed disintegrated cytoplasm, abnormal vacuoles, dilated smooth endoplasmic reticulum, and less developed junctional specializations between the cells and sperm heads (Figs 11 and 12). The interstitial cells of Leydig also showed many dilated smooth endoplasmic reticulum, some lysosomes, and small glycogen particles, in addition to the lipid droplets. The microvilli on the cell surface appeared destructed (Fig. 13).
Light microscopic examination of hematoxylin and eosin-stained sections revealed the nearly normal appearance of the seminiferous tubules and the interstitial tissue. The tubules exhibited normal stratification of the spermatogenic epithelium (Fig. 14).
Results of the sperm morphology test revealed that most (76.7%) of the counted sperms appeared with normal architecture; however, a small percentage showed abnormal heads (7%) and abnormal tails (16.3%). All abnormalities were significantly decreased compared with the acrylamide group (P<0.001 for all; Table 2).
Examination with TEM revealed a more or less normal appearance of the spermatogenic epithelium. It showed early round spermatids in the developing stage in which the nucleus showed an acrosome and a head cap, whereas the cytoplasm showed the Golgi zone and mitochondria (Fig. 15). The late elongated spermatids appeared normal in shape (Figs 15 and 16). They were embedded in normal-shaped Sertoli cells, which contained mitochondria and showed junctional specializations with sperm head (Fig. 16). The interstitial cells of Leydig also appeared nearly normal and contained an increased number of lipid droplets (Fig. 17).
Genetic analysis results
The genomic DNA purity indicated that it was high-quality DNA as the results of the studied DNA were between 1.7 and 2.0. The genomic DNA concentration ranged from 50 to 150 μg/ml. The total yield obtained was 30–50 μg.
Gel electrophoresis showed PCR reactions (Figs 18 and 19): control (lanes 8–12; Fig. 18), curcumin (lanes 8–12; Fig. 19), acrylamide (lanes 3–7; Fig. 19), and curcumin+acrylamide (lanes 3–7; Fig. 18). Each lane showed the reaction with each of the used five primers.
Negative controls showed no PCR reactions (lane 1), which indicated that the PCR mixtures had no contamination with the genomic DNA (Figs 18 and 19).
Primers 2, 3, 4, and 5 only gave positive and detectable bands in the control group, where these primers amplified a total of five different bands, ranging from 3000 to 900 bp (Fig. 18; Table 3). In the curcumin group, eight different bands ranging from 2000 to 600 bp, in the acrylamide group, 17 different bands ranging from more than 3000 to 900 bp, and in the acrylamide+curcumin group, 11 different bands ranging from more than 3000 to 800 bp were amplified by using the different primers (Figs 18 and 19; Table 3).
The GTS was calculated according to the calculation given in the ‘Materials and methods’ section. The GTS was the highest in the curcumin-treated group (60%) compared with the acrylamide-treated (15%) and curcumin+acrylamide-treated groups (56%).
Acrylamide is a highly reactive and water-soluble polymer, which is commonly used in industries and laboratories . Individuals can be exposed to acrylamide either in their workplace  or in the environment . A previous study reported the presence of acrylamide in heat-treated food products . The formation of acrylamide is particularly associated with the high-temperature cooking process for certain carbohydrate-rich foods, especially when asparagine (one of the most common amino acids present in both animal and plant sources such as potatoes) reacts with sugars .
Acrylamide causes peripheral neurotoxicity, mutagenicity, male reproductive toxicity, prenatal lethality, and endocrine-related tumors in rodents . However, avoidance of foods containing acrylamide would result in worse health issues from an unbalanced diet or from pathogens in undercooked foods ; therefore, strategies to decrease its toxic effects are mandatory.
Previous studies reported that antioxidant agents reduced the acrylamide-induced reproductive toxicity . Curcumin exhibits a variety of biological activities such as anti-inflammatory , antioxidant [13,26], and antitumor . It was also reported that it is pharmacologically safe ; therefore, this study was carried out to investigate its protective effect against acrylamide-induced reproductive toxicity and genotoxicity.
In this study, the use of acrylamide (in group 3 animals) resulted in atrophy or degeneration of the germinal epithelium with appearance of abnormal vacuolations and thickened basement membrane. The lumen of the seminiferous tubules contained exfoliated germinal epithelium and abnormal-shaped spermatozoa. In some regions, the interstitial tissues showed an increase in amount. Both the sperm morphology test and the TEM examination confirmed the abnormality in sperm morphology, which showed a significant increase. The TEM also showed degenerative changes in the spermatogenic epithelium as well as in Leydig cells. These data were in agreement with results of previous studies [5,6,24]. Yang et al.  used different doses of acrylamide to test its reproductive toxicity on rats. They reported that the changes were dose-dependent and that they were marked with the highest dose (60 mg/kg/day). The investigators also emphasized that the number of sperms in cauda epididymidis decreased significantly; however, this was not observed in this study probably because of a technical fault as the mincing process of the epididymis, in the control group of our study, produced many cut sperms (heads or tails alone) and these were not included in the count. Tyl and Friedman  reported the significant increase in abnormal sperm count after the use of acrylamide.
The RAPD PCR analysis showed that the GTS, in the acrylamide group, was the lowest compared with the other studied groups. The decreased GTS percentage indicated a high polymorphic pattern as a result of amplification of new bands due to breakage and mutation of the DNA . These new bands could be considered as potential markers, which were attributed to acrylamide intake. This DNA fingerprinting assay indicated the mutagenic effect of acrylamide as RAPD primers displayed some changes in polymorphism band patterns of DNA, including loss of stable bands and acquisition of unstable bands in the acrylamide-treated group.
The mechanism of acrylamide action involves interference with the kinesin-related motor proteins in nerve cells and eventual cell death. Neurotoxicity and resulting behavioral changes, such as hind limb weakness, can affect the reproductive performance of acrylamide-exposed laboratory animals. Further, the kinesin motor proteins (also found in sperm flagellum) are important in sperm motility, which would be affected and could alter reproduction parameters. Affection of kinesin motor protein in sperm flagellum could also be responsible for the most common abnormality (coiled tail) in the sperm morphology in this study. It was also reported that acrylamide binds to spermatid protamines causing dominant lethality of gonadal cells and morphological abnormalities of sperms . Effects on kinesin proteins could also explain some of the genotoxic effects of acrylamide. These proteins form the spindle fibers in the nucleus that function in the separation of chromosomes during cell division and their affection could explain the clastogenic effects of acrylamide noted in a number of tests for genotoxicity and assays for germ cell damage .
Tyl and Friedman  also mentioned that acrylamide lowers testosterone hormone levels and this additional mechanism could be concluded in this study as there were degenerative changes in Leydig cells seen using TEM, which in turn affect the secretion of the hormone and the spermatogenesis process. The presence of increased amounts of Leydig cells that appeared in only some parts of hematoxylin and eosin-stained testicular sections, in this study, could reflect a compensatory mechanism for this decreased hormone level.
Regarding the atrophy of the spermatogenic cells, Creasy and Foster  stated that necrosis and loss of these cells are the most frequent manifestations of any testicular injury. The necrotic cells are rapidly sloughed and exfoliated into the lumen of seminiferous tubules, presumably due to loss of contact between Sertoli cells and spermatogenic cells, which could be related to the retraction of Sertoli cell lateral processes enclosing the spermatogenic cells.
Abnormal vacuolations of the spermatogenic epithelium and Leydig cells that were observed in this study by the use of light electron microscope and TEM examination have been attributed, in previous studies, to swelling of the cytoplasmic organelles . Thickening of the boundary tissue of the spermatogenic epithelium, which was also reported by Yang et al. , occurs most probably secondary to the toxic effect of acrylamide, which leads to inflammatory reaction and fibrosis. This toxic effect also stimulates the release of chemical mediators after tissue damage, resulting in vascular congestion .
The use of curcumin, in this study, (in group 4) resulted in protection of the testicular parenchyma, both the seminiferous tubules and the interstitial cells of Leydig. This was in addition to the significant decrease in sperm abnormality, compared with the acrylamide group as was evident with the use of sperm morphology test. These previous results were also confirmed by the use of TEM, which furthermore revealed an increase in lipid droplets in the Leydig cells. In genetic analysis results, GTS showed that curcumin increased the DNA stability as a result of prevention of genotoxicity compared with the acrylamide-treated group.
There are no reported studies about the use of curcumin against acrylamide-induced testicular damage; however, there are many studies that used it for protection against other injurious agents, for example, cisplatin , ischemia-reperfusion injury , chronic alcohol administration , and the lindane pesticide .
Curcumin exerts its protective effect through different mechanisms . It has a wide array of pharmacological and biological activities, that is, a potential anti-inflammatory , antioxidant , and antigenotoxic  with phytonutrient and bioprotective properties, which are again linked to its anti-inflammatory and antioxidant activities. Moreover, curcumin was also reported to have an antifibrotic effect, which could also be related to the anti-inflammatory property . This anti-inflammatory action was reported to be due to inhibition of inflammatory cytokine production in macrophages . An additionally postulated mechanism was the prevention of apoptosis . All these mentioned mechanisms were most probably responsible for the protection of the testicular structure and preservation of the normal sperm morphology observed in this study. The protective effect was evident in the interstitial cells of Leydig as well, which even exerts a compensatory mechanism and showed increased activity in the form of increased lipid droplets to release more testosterone, which is needed for normal spermatogenesis. The genoprotective effect was also evident in this study, in group 4 where there was increased percentage of GTS, which might be due to increased DNA stability and prevention of genotoxicity by using curcumin.
In conclusion, curcumin proved marked efficacy in amelioration of the reproductive toxicity and genotoxicity induced by acrylamide in male albino mice. Therefore, it is recommended to be used routinely by sprinkling it on any fried foods.
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