The effect of acrylamide on sperm oxidative stress, total antioxidant levels, tyrosine phosphorylation, and carboxymethyl-lysine expression: A laboratory study

Abstract Background Acrylamide (AA) is a reactive molecule produced during food processing at temperatures above 120∘C. Objective To evaluate the impact of different concentrations of AA on human sperm parameters, oxidative stress and total antioxidant capacity (TAC). Materials and Methods In this laboratory study, semen samples were obtained from healthy donors referred to the Taleghani Hospital, Tehran, Iran between June and July 2019. Samples were divided into four groups (n = 10/each): one control and three treatment groups (0.5, 1, and 2 mM of AA). After 2 hr of exposure to AA, the superoxide dismutase and malondialdehyde levels were measured based on colorimetric methods. The TAC was determined by the ferric-reducing antioxidant power assay. Flow cytometry was performed to measure the intracellular reactive oxygen species generation. Also, immunohistochemistry was done to determine the effect of AA on tyrosine phosphorylation and carboxymethyl-lysine expression. Results Results of the study demonstrated that the motility and viability of spermatozoa were significantly decreased after AA exposure (p < 0.001). This decrease was also seen in the TAC and superoxide dismutase activity as well as in the phosphotyrosine percentage compared with the control (p < 0.01). However, the carboxymethyl-lysine and prooxidant activity including reactive oxygen species generation and lipid peroxidation level increased (p < 0.001). Conclusion Overall, the results confirmed the detrimental effect of AA on human spermatozoa which may be due to oxidative stress and decreased total antioxidant levels. AA may reduce fertility by reducing sperm capacitation and motility.


Introduction
The presence of acrylamide (AA) in many commonly consumed foods such as fried and oven-cooked foods was reported in 2002 by the Swedish Authorities for the first time, which attracted considerable concern worldwide (1). AA (CH = CH-CONH2) is a white, odorless, crystalline solid which has wide application in industry as well as in wastewater treatment and soil conditioner (2). The human daily uptake of AA is estimated at 1-4 µg/kg body weight (3). The main route for the formation of AA in high carbohydrate foods such as potato chips and cereal products is the Maillard reaction (a reaction between reducing carbohydrates and free amino groups of proteins) (4).
Genotoxicity and carcinogenicity of AA have been documented especially in hormonally regulated tissues such as the mammary gland, thyroid, and testis mesothelium (5). Therefore, it is considered a global concern by the World Health Organization and has come to be known as a reproductive toxin in recent years (6,7). Oral AA exposure induces testicular damage as well as a decrease in testosterone and epididymal sperm in rats (8). A toxicological impact was previously indicated in the male reproductive system of weaning rats when abnormal sperms and histopathological lesions appeared after AA treatment (9). Research about AA toxic effect on female mice indicated a major impact on oocyte quality, DNA methylation, reactive oxygen species (ROS) generation, and apoptosis induction (10). ROS in small amounts is essential for sperm physiological function including maturation, capacitation, and fertilization. However, when the balance between free radical activity and the body's antioxidant system is lost, oxidative stress occurs.
It is suggested that the reproductive toxicity of AA is mainly due to oxidative stress (11,12).
The sperm cell is particularly susceptible to oxidative attack due to the low volume of cytoplasm and therefore low levels of antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase. On the other hand, high levels of polyunsaturated fatty acids make this cell vulnerable to free radicals attack (13). Lipid peroxidation (LPO) caused by ROS leads to the formation of malondialdehyde (MDA) which is a stable peroxidation product in seminal plasma. MDA is a biomarker for measuring the level of oxidative stress in the cell and a well-known indicator of reduced fertility and sperm dysfunction (14).
Oxidative stress impairs sperm motility, the sperm membrane, and DNA integrity (15,16). AA is known to cause oxidative stress in the human body and numerous studies have indicated its toxic effect on the male reproductive system particularly in mice and rats (8,11). But the impact of AA on human spermatozoa ROS generation total antioxidant levels and sperm motility and capacity is not fully understood yet.
Tyrosine phosphorylation has been used as a hallmark to determine the influence of this substance on sperm capacitation (17). Also, it is considered that carboxymethylation of lysine residues in the sperm tail is involved in sperm International Journal of Reproductive BioMedicine Acrylamide's effect on human sperm factors movement regulation because of its crucial role in post-translational modification (15,18

MDA assay
Seminal MDA levels were determined using the Zell Bio colorimetrical kit (GmbH, Germany)

SOD activity assay
Seminal SOD activity levels were determined using the Zell Bio colorimetrical kit (GmbH,

TAC assay
To measure the TAC, the ferric-reducing

Phosphotyrosine
After treatment with AA, sperm samples (5

Ethical considerations
The

Impact of AA on human spermatozoa parameters
Exposure of human spermatozoa to AA caused a significant decrease in the percentage of progressive spermatozoa and the total motility at all doses of AA. Also, the percentage of immotile spermatozoa was significantly increased at high doses in comparison with the control (Tables I and II). In addition, the viability of the spermatozoa decreased significantly compared with the control.

Impact of AA on ROS levels
Results of flow cytometry revealed that after exposure of spermatozoa to different concentrations of AA, the ROS levels increased significantly ( Figure 1, and Table IV).  Figure 3 and Table VI

Discussion
So far, studies on the effect of AA on fertility are limited to rodent models (21,22). In our previous study, it was found that the mitochondrial membrane potential decreased after 2 hr of sperm incubation with AA (24).
On the other hand, AA could inhibit the activity of glotation s-transferase enzymes (a family of phase-II detoxification enzymes that catalyze the conjugation of glutathione) leading to cell membrane damage. These changes also appear to be due to an increase in the cellular ROS content which disrupts the process of oxidative phosphorylation in the mitochondria and the production of ATP in the cells (24,25). As a result, it is associated with reduced motility, inhibition of the acrosomal reaction, and reduced sperm ability for fertilization (26).
AA exerts its effects at least in some parts by weakening the antioxidant status in the sperm cells (22). It was reported that exposure to AA in mice caused reduced sperm motility and impaired fertility (27). As has been mentioned, LPO is one of the major mechanisms of cellular damage caused by ROS, as the sperm membrane is rich in unsaturated fatty acids that are very sensitive to free radicals. MDA is the main indicator of LPO identification (14). Our data also confirmed an increase in MDA levels following AA exposure.
The proposed mechanism for the effect of Tyrosine phosphorylated sperm proteins in the flagellum are related to hyperactivated motility which is initiated during in vitro capacitation (17).
As a result, abnormalities in sperm motility due to decreased tyrosine phosphorylation levels are not uncommon. The immunohistochemistry results of the present study also showed a decrease in the phosphotyrosine levels.

Conclusion
The results of this study indicated that different concentrations of AA had a negative effect on the motility and viability of human spermatozoa.
AA could reduce the sperm motility and ability for capacitation by increasing the ROS levels relative to the cell's antioxidant capacity. Therefore, given the rising male infertility rate and increasing consumption of deep-fried foods containing AA, more attention should be paid to the effects of AA on male infertility.