5-Fluorouracil-induced toxicity in both male and female reproductive systems: A narrative review

Abstract

The chemotherapeutic drug 5-fluorouracil (5FU) is frequently used to treat a wide range of solid malignant tumors, such as colorectal, pancreatic, gastric, breast, and head and neck cancers. Its antitumoral effects are achieved by interfering with the synthesis of RNA and DNA and by inhibiting thymidylate synthase in both malignant and non-malignant cells. Therefore, it can be responsible for severe toxicities in crucial body organs, including heart, liver, kidney, and reproductive system. Given the fact that 5FU-induced reproductive toxicity may limit the clinical application of this drug, in this study, we aimed to discuss the main locations and mechanisms of the 5FU-induced reproductive toxicity. Initially, we discussed the impact of 5FU on the male reproductive system, which leads to damage of the seminiferous epithelial cells and the development of vacuoles in Sertoli cells. Although no noticeable changes occur at the histopathological level, there is a decrease in the weight of the prostate. Additionally, 5FU causes significant abnormalities in spermatogenesis, including germ cell shedding, spermatid halo formation, polynucleated giant cells, and decreased sperm count. Finally, in females, 5FU-induced reproductive toxicity is characterized by the presence of atretic secondary and antral follicles with reduced numbers of growing follicles, ovarian weight, and maturity impairment.

Keywords

5-Fluorouracil toxicity reproductive systems male female

Introduction

5-fluorouracil (5FU) is a well-known chemotherapeutic agent commonly administered for treating a vast number of solid malignant tumors, including epithelial cell malignancies emerging from the genitourinary and gastrointestinal tracts (especially those of colon, pancreas, and stomach), breast, and also epithelial malignancies found in the head and neck. It is also used for treating carcinomas of the ovary, prostate, neck, bladder and oropharynx with less efficacy.^1,2 5FU acts by disruption of RNA and DNA synthesis and inhibition of thymidylate synthase in malignant and non-malignant cells.^2–4 Since it can interfere with division of noncancerous cells, it may cause severe toxicity in crucial body systems, including heart, liver, and kidney.^5–7 It has been also reported that 5FU could lead to notable defects in the reproductive system.⁸ Given the fact that 5FU-induced reproductive toxicity may limit the clinical application of this drug, in this study, we have explained the main locations and mechanisms of the 5FU-induced reproductive toxicity.

5FU-induced toxicity in male reproductive system

5FU-induced testicular damage

Drugs used to treat cancer can directly or indirectly damage DNA and interfere with it, causing chromosomal aberrations or strand breaks.^9–12 Research has shown that some chemotherapeutic drugs can also cross the blood-testis barrier, which can have negative effects on the testicles. These organs are sensitive to the toxic effects of chemotherapy, which can cause damage to germ cells or lead to and fibrosis of the interstitial tissue.^13–16 Previous studies indicate that men who have undergone the MOPP regimen (Mechlorethamine, Oncovin, Procarbazine, and Prednisone) to treat Hodgkin's lymphoma, may experience permanent infertility as a result.¹⁷

The testicular function involves an exocrine function by producing sperms in the seminiferous tubules that make up more than 75% of the testicular mass, and an endocrine function by secreting testosterone by Leydig cells in the interstitial space, and the rest of the testicular structure consists of the supporting and vascular tissues that play the role of nourishing and protecting sperms.^18–20 As mentioned, spermatogenesis, which takes place in the seminiferous tubules, which are made up of germ cells and Sertoli cells, is a process that can be divided into three stages: the proliferation of spermatogonia, spermatocyte meiosis, and finally spermiogenesis, which leads to spermatozoa production.¹⁸ This process of spermatogenesis is constantly taking place throughout the seminiferous tubules so that in each area of this tubule 5 to 6 generations of germ cells can be identified and the duration of this process in humans is 64 to 90 days until the release of adult sperms, in different species this time is different.^18,21 The process of spermatogenesis, as described, is a complex and always dynamic process that can be affected by cytotoxic agents in various ways, such as damage or loss of a specific cell type in the germinal epithelium or damage during the maturation of sperm and the development of dysfunctional sperm, another effect is to damage the Sertoli and Leydig cells or other supporting components to destroy the suitable environment for sperm production and thereby damaging the process.²²

Chemotherapeutic drugs affect both the functional aspects of the testicles and their fertility and androgen production potential. These drugs can also have an indirect effect on the reproductive system by affecting the hypothalamic-pituitary axis.¹³ The process of spermatogenesis is successful if there is a normal balance between the endocrine glands and the inhibition of the glands that can be caused by chemotherapy treatments, which has been found to be toxic to testicular tissue and to increase the risk of infertility.^23,24 Usually, this disorder occurs directly with the effect of the drug on stem cells because in later stages the germ cells are more resistant. The administration of alkylating drugs has been observed to cause thickening of the basement membrane, interstitial fibrosis and aplasia of the germinal epithelium, which ultimately leads to reduced fertility in men.²⁵ Findings from 5FU also suggest that the drug may cause genital damage by inhibiting thymidylate synthetase and its genetic pathways.^26–29

The impact of childhood chemotherapeutic drugs on testes remains largely unexplored. Given the physiological changes that occur during this period, including an increase in testicular volume and proliferation of immature Sertoli cells and Leydig cells, it is essential to consider the potential vulnerability of Sertoli cells to chemotherapeutic drugs.³⁰ Investigations on male childhood cancer survivors have revealed that alterations in Sertoli cells are indirectly reflected in variations in sperm count and follicle-stimulating hormone (FSH) levels.¹⁴ Since Sertoli cells are the primary producer of inhibin in the testis, the inhibin/FSH ratio can also be utilized to assess the abnormality in Sertoli cells due to the absence of quantifiable indicators.^31–33 However, it is challenging to utilize simply the inhibin/FSH index because of the strong contact between the gonadotropic system and Sertoli and Leydig cells, so it is preferable to investigate it by taking extra hormonal variables like testosterone and LH into account.³² Serum testosterone and LH are also used to assess the function of Leydig cells, and hormonal biomarkers such as FSH, LH and testosterone in blood samples are used to evaluate the testes in cases where semen analysis is not possible.^23,34,35 Also for detecting subclinical abnormalities of Leydig cells, LH-releasing hormone is used, which highly increases the serum LH level after administration if Leydig cells are abnormal.²⁰

Testicular atrophy often results from injury to the germinal epithelial cells since seminiferous tubules make up a significant portion of the testicles' bulk. On the other hand, decreased spermatogenesis is marked by a reduction in the quantity and mobility of sperms in each ejaculation.³⁶ Recent research has also revealed that men with germinal aplasia have lower levels of free testosterone and lower daily production of testosterone, which suggests that the mature testis's germinal epithelium is more vulnerable to chemotherapy than immature testicles are, as well as Leydig cells due to their higher mitotic rate.^17,36–39 These cases indicate that people undergoing chemotherapy may develop secondary sexual characteristics despite having oligospermia or azoospermia because the hormones secreted by Sertoli are responsible for their occurrence.^40,41 Exposure to chemotherapeutic agents can increase seminiferous tubules without germ cells, along with reduced tubule diameter and vacuolization, which results in decreased testicular weight in rodents, nevertheless, treated animals showed that impaired germ cells can escape abnormalities which cause testes to produce abnormal sperms.^42–44

Chemotherapeutic drugs can harm Sertoli cells and decrease their ability to produce two essential proteins, androgen-binding protein (ABP) and transferrin, which play a crucial role in spermatogenesis. Only one study has investigated the impact of doxorubicin exposure on myoid cell proliferation in testicular tissue, and no significant effects were observed.^45,46 In relation to Leydig, although it has been observed in pediatric cancer survivors that their function is impaired which subsequently leads to androgen deficiency, there is little information in animal studies and in a study which exposed the testes of premature mice to doxorubicin for 48 h in which there was no change in function or morphology, and in another in vitro study, 24 h of cisplatin, cyclophosphamide, and doxorubicin exposure did not affect Leydig cell density.^47–52

Other chemotherapeutic drugs such as paclitaxel and cisplatin have been shown to have minimal effects on germ cell and myoid cell proliferation. However, studies have shown that 6-Mercaptopurine, an anti-metabolite similar to 5FU, can lead to the death of Leydig cells in mice through caspase-3 activation, suggesting that 5FU can also have an impact on testicular function and reproduction.

While the impact of 5FU on male reproduction is not well understood, the effects of other chemotherapeutic medications, such as paclitaxel, a taxane-based chemotherapeutic drug that reduces testicular volume, or cisplatin, which result in a dramatic decrease in the number of germ cells in both the fetal testis and the pre-pubertal testis is negligible. In the case of 6-Mercaptopurine, which, like 5FU, is an anti-metabolite that activates caspase-3 to eventually kill Lydig cells in mice, which makes it possible to consider 5FU as a factor affecting reproduction and testes.^53–55 Mechanisms that have been reported for the pharmacological effects of 5FU in cytotoxicity include inhibition of thymidylate synthetase, which leads to a decrease in thymidine and ultimately reduced DNA production, or direct incorporation into nuclear RNA, which disrupts the formation of cytoplasmic RNA and also damages DNA through the attachment of residues.^56–59 And according to the mentioned cases that lead to toxic changes in germ cells, it has been observed that cells in S phase are very sensitive to 5FU toxicity, and this potential 5FU damage to DNA has also been reported in 6-MP, which increases the likelihood of the reproductive system toxicity.^60,61 In one study, the tissue of 5FU-treated mice showed destruction of the seminiferous epithelial cells and Sertoli cells vacuolization, while another study found Sertoli cells vacuolization (Figure 1) in the epididymal ducts but no histopathologic changes in the seminal vesicle. Additionally, some studies have reported weight loss in the testicles and defects in spermatogenesis after treatment with 5FU.^28,62–65 This morphological effect on Sertoli cells and the observed weight loss can be due to a hormonal imbalance in the serum; as seen in a study in mice, levels of pro-alpha C and GnRH were significantly elevated, while levels of inhibin B, prolactin, activin A, and testosterone faced significant decrease.²⁶ Also tubular contraction and atrophic cells and abnormal sperms have also been observed, which has ultimately reduced the number of spermatocytes, as well as swelling and shrinkage of the tubules following 5FU administration.^66,67

Figure 1.

The effect of 5FU on male reproduction. Sclerosis, vacuolization and polynucleated giant cells (PGCs) were observed in seminiferous tubules that are affected by 5FU.

5FU-induced prostate damage

There is not much information about the effect of 5FU on the prostate. In a study by Takizawa et al., It was found that 5FU did not cause any histopathological changes, although it did reduce the relative weight of the prostate.²⁶ Furthermore, Al-Asmari et al. conducted a study that revealed shrinkage of the prostate gland and the presence of nuclear stratification in the group treated with 5FU. This group also showed an increase in prostate gland secretion.⁶² In this study, it was also found that these effects were modified if Tau was administered. In weight loss of the prostate, the role of androgens in regulating its growth can be considered effective, As decreased serum testosterone levels can cause severe weight loss in this organ and it has been observed that in these patients serum levels of inhibin B, prolactin, activin A, and testosterone are significantly reduced, in contrast to serum levels of pro-alpha C and GnRH which showed a significant increase.⁶⁸ Nonetheless, in 5FU-treated patients, there were no noteworthy alterations in serum LH and FSH levels observed, although they showed an increase in serum GnRH levels.²⁶

5FU-induced sperm count and shape abnormalities

5FU treatment affects sperm count and causes lack of spermatozoa.⁶⁹ At 3, 7, and 11 days after receiving a single dosage of 250 mg/kg of 5FU through the tail vein, Mao et al. (2009) saw a decrease in the quantity of spermatocytes and spermatids within the mice's seminiferous tubules. During the 11th day, they also noticed edema. However, on day 14, the edema had almost completely subsided, and the spermatocyte population had increased, but the effects of 5FU on the germinal epithelium and the absence of mature sperms remained (Table 1).⁷⁰ In another study by D'Souza (2003), in which three different doses of 5FU (10, 20 and 30 mg/kg of body weight) were injected, a significant decrease in the sperm count was observed. Among the different studies at the time of sampling, samples taken on the 35^th day showed the maximum toxic effect. The reason could be explained by the fact that the cells exposed at this stage belong to the spermatogonial cell population which are more sensitive to 5FU at this stage. In the abovementioned study, decreasing sperm count was dose and time-dependent (Table 1).⁶⁶

Table 1.

Summary of studies with their findings on the effect of 5-FU on male reproductive system.

Studies	Specific tissue and species	Dose levels	Duration	Effect
Takizawa et al. (2002)²⁶	The sex hormones of SD-Slc male rats (12 males at 5 weeks old for the 4-weeks study and 12 males at 7 weeks old for the 2-weeks study)	0, 20, 30 mg/kg/day orally	2 and 4 weeks	Testosterone trend reduction
D'Souza (2003)⁶⁶	The spermatogonial cells of male albino rats of wistar strain weighing 150-200g (11–13-week-old)	10, 20 and 30 mg/kg of body weight	35th day (maximum toxic effect)	Decrease in the sperm count
Mao et al. (2009)⁷⁰	The germinal epithelium of mice	Single dosage of 250 mg/kg	3, 7, and 11 days	Decrease in the quantity of spermatocytes and spermatids
Gad et al. (2017)⁷¹	The sperms of male adult albino mice (Mus musculus) with age between (7 to 10 weeks) and weighing between 20 and 30g	5, 10 and 15 mg/kg	Five consecutive days	Irregular sperm production due to doses increased
Fahmy et al. (2020)⁷²	The germline cells of mature male albino mice of Swiss strain (Mus musculus L.) weighing between 20 and 30 g	5, 10, and 15 mg/kg	4 weeks	Chromosome aberrations
Fahmy et al. (2020)⁷²		5, 10, and 15 mg/kg	8 weeks	Insignificant increase in gamete morphology

Additionally, the percentage of abnormal sperms was significantly affected by 5FU treatment at different doses (5, 10 and 15 mg/kg, five consecutive days) (p < .05), and abnormalities in sperm shape, such as semi-normal or massively headed sperms, angular midpiece sperms, and bent tailed sperms, were noted. As medication doses increased, irregular sperm production was rised. The gonads of the treatment group showed signs of undeveloped germ cells shedding, spermatid halo appearance, and polynucleated giant cells (PGCs) (Figure 1) (Table 1).⁷¹

5FU-induced chromosomal & DNA damage

In a recent study, Fahmy et al. (2020) observed a noteworthy proportion of chromosome aberrations in spermatocytes following the injection of 5FU with different doses; most of them univalent forms.⁷² Further, in previous research, the effects of 3 distinct dosages of 5FU—5, 10, and 15 mg/kg—on the male germline cells of Swiss mice were evaluated. The results showed that 4 weeks after the treatment, 5FU significantly increased the number of genetic abnormalities. During week 8 after therapy, gamete morphology analysis did not reveal a statistically significant increase. This showed that there is a progressive reduction in the transfer of 5FU-related chromosomal aberrations from spermatogonia to sperm. This may be because the severely damaged spermatogonial cells are eventually eliminated during the spermatogenesis process.⁷³

In the seminiferous tubules, 5FU caused PGCs and spermatids with a halo-like appearance.^71,74 One of the possible mechanisms for the formation of PGCs can be karyokinesis without subsequent cytokinesis.⁷⁵

The genomic expression of the degree of laddering (total DNA fragmentation) and the tail length, was increased in testicular cells after 5FU treatment in comparison with control manifesting single strand DNA damage, while increasing 8-hydroxy-2-deoxyguanosine (8- OHdG) and heat shock protein 70 (HSP-70) were observed.⁷⁶

5FU-induced sex hormonal changes

Testosterone is the major sex hormone in males. In Takizawa et al. study, they examined the effects of 5FU on sex hormones, and a decreasing trend in testosterone was observed in the treated-group with 5FU (0, 20, 30 mg/kg/day orally for 2 and 4 weeks). The serum level of GnRH which controls the production of LH and FSH had a statistically significant increase in the 5FU group. Inhibin B is a hormone that inhibits the synthesis of FSH which 5FU treatment lowers its level. The rats with vacuolation of Sertoli cells, had the lowest level of this hormone. In this study, 5FU caused no significant changes in serum levels of LH and FSH. Nonetheless, Activin A and Prolactin were decreased in treated groups. Contrarily, Pro-alpha C, a subunit of inhibin B was increased in the group treated with 20-mg/kg/day dose of 5-FU for 4 weeks (Table 1).²⁶

In general, 5-FU effects can be divided into two categories: primary effects (direct effects on gonadal cells) and secondary effects (indirect effects on gonadal cells and the hypothalamic-pituitary-gonadal (HPS) system). Gonadal cells are affected by inhibition of DNA synthesis. This results in decreased cellular proliferation, decreased mitotic activity, and reduced sperm production. The indirect effects on gonadal cells include changes in the HPS system, such as reduced levels of testosterone and estradiol, as well as changes in the hypothalamic-pituitary-testicular axis (Table 2).

Table 2.

Primary and secondary effects of 5-FU on the hypothalamic-pituitary-gonadal (HPG) axis.

Reproductive system*	Primary effects in gonads	Secondary effects in gonads	Secondary effects in other HPG axis
Male	Lyding cells abnormality²⁶	Decrease testosterone level and reduction of stimulation of Sertoli cells^26,32	LH level and GnRh elevation^3,26
Male	Sertoli cells abnormality²⁶	Decrease inhibin hormone, ABP and transferrin proteins level^4,5	FSH level and GnRh elevation^6,7,32
Female	Elevated atresia in the growing follicles (secondary and antral)^8,9	Decrease estrogen level and aberrations of ovulation¹⁰	FSH level and GnRh elevation with negative/positive effect on LH^10,26

ABP, Androgen-binding protein; FSH, Follicle-stimulating hormone; GnRh, Gonadotropin hormone-releasing hormone; HPG, Hypothalamic-pituitary-gonadal; LH, Luteinizing hormone.

*This table summarizes the cases mentioned in the studies and the opinions of the authors, but many of the reactions are not presented due to the many hormonal interactions.

5FU-induced female reproductive toxicity

Effect of 5FU on ovarian size

Ovarian size can be considered as a good measure for ovarian competency. The reduced ovarian size could be due to elevated atresia in the growing follicles.^77,78 Different chemotherapeutic drugs have been reported to cause this condition as a negative side effect.^77,79–81 Previous experiments showed that after intraperitoneal administration of 50 mg/kg/day 5FU for 4 days, the ovarian size was remarkably diminished compared to the control group (Table 3).⁸ In another research, 5FU caused significant reduction of body weight and ovarian wet weight to body weight ratio in female rats.⁸²

Table 3.

Summary of studies with their findings on the effect of 5-FU on female reproductive system.

Studies	Specific tissue and species	Dose levels	Duration	Effect
Lambouras et al. (2018)⁸³	The ovary of healthy female C57BL/6 mice, 6–8 weeks of age	Single dose of 150 mg/kg	7 days	Reduction in corpora lutea
Naren et al. (2021)⁸	The ovary of female ICR mice aged 8 weeks	50 mg/kg/day (intraperitoneal administration)	4 days	Diminished the ovarian size
Naren et al. (2021)⁸	The ovary of female ICR mice aged 8 weeks	50 mg/kg/day (intraperitoneal administration)	4 days	Ovarian dysfunction

ICR: Institute of Cancer Research.

Effect of 5FU on ovarian function and secondary follicles

In order to examine the ovarian function, Naren et al. (2021) made both of their 5FU-treated and control groups super-ovulated by hormonal stimulation at the end of the 4th day of 5FU injection. The collected ovaries of the control group showed enlarged ampullas and a significant number of MII oocytes after hormone-induced super-ovulation. Unlike the control ovaries, no enlarged ampulla and mature oocyte was seen in the ovaries of 5FU-treated mice (Table 3).⁸

As secondary and antral follicles have rapidly dividing cells, including oocytes and granulosa cells, they are more vulnerable to 5FU-induced toxicity. Although observing growing follicles being atretic in the ovary is a normal physiological phenomenon, in a prior intervention, the 5FU-treated ovaries demonstrated a considerable elevation in the amount of morphologically atretic secondary and antral follicles (Figure 2).⁸³ At last, according to almost all the previous studies, it is allegedly true that 5FU leads to a significant reduction in the amount and a notable deficiency in the maturation of growing follicles.^8,78,84

Figure 2.

The effect of 5FU on female reproduction. The amount of morphologically atretic secondary and antral follicles was significantly increased in ovaries treated with 5FU. 5FU causes a considerable decrease in the number of growing follicles, ovarian weight and, a notable impairment in their maturity.

Effect of 5FU on primordial and primary follicles

It should be stated that, in some studies, the count of primordial and primary follicles had no remarkable differences between 5FU-treated and control ovaries which meant that 5FU did not significantly affect primordial and primary follicles.^78,83 However, Almeida et al. (2021) observed a significant depletion (p < .05) in the amount of morphologically regular primordial, transition and primary follicles in 5FU-treated ovaries in comparison with the control group.⁸⁵ Since there is a scientific gap between the aforementioned studies, it is recommended that further research be focused on evaluating the effect of 5FU on primordial and primary follicles.

Effect of 5FU on ovulation

In former research, corpus luteum quantification was performed in 5FU and saline treated mice, in order to distinguish whether 5FU treatment interfered with ovulation or not. The count of corpora lutea faced a considerable depletion in all 5FU-treated ovaries.⁷⁸ In the study of Lambouras et al. (2018), there were not any notable changes in the amount of corpora lutea between saline and 5FU-treated mice in 12-h-treated groups, however, outstandingly less corpora lutea were detected on the 7-th day after 5FU treatment in 5FU-treated ovaries, compared to controls⁸³ (Table 3).

The mechanism of 5FU-induced reproductive toxicity

Although the exact mechanism of 5FU-induced reproductive toxicity, in female, has not been completely revealed, there are a few reactions that could explain this phenomenon to some extent.

5FU induces cell apoptosis

5FU can terminate the proliferation of cancer cells and induce apoptosis. This is thought to be accompanied by mitochondrial state changes and increased caspase-9 activity.⁸⁶ Moreover, it has been reported that 5FU induces apoptosis through upregulation of some proapoptotic proteins, like FAS and Bax, and activation of caspase-3.⁸⁷ Furthermore, the 5FU-induced apoptosis was detected to be accompanied by DNA degradation and disruption of mitochondrial membrane capacity, and overexpression of p21 protein and caspase-8 and caspase-9.⁸⁸

5FU causes DNA and RNA damage

As mentioned previously, 5FU is a potent antimetabolite and performs its main inhibitory function through inhibition of thymidylate synthase (TS). However, enzymatic changes on the nucleotide (including ribosylation and phosphorylation) are needed to perform the cytostatic activity of 5FU.⁸⁹ Among all the derivatives of this chemotherapeutic agent, fluorodeoxyuridine monophosphate (FdUMP) is the most active one which creates a covalent ternary complex in combination with TS, thus, disrupting its ordinary activity by blocking the binding of its natural substrate.⁹⁰ 5-fluorouridine-50 -triphosphate (FUTP) and 5-fluoro-20 -deoxyuridine-50 -triphosphate (FdUTP) are the other major metabolites of 5FU that are respectively inserted into RNA and DNA and lead to the disruption of RNA processing and function, and inhibition of DNA synthesis and function (Figure 3).^90–92

Figure 3.

Damage to DNA and RNA is aggravated by 5FU. Thymidylate synthase (TS) is inhibited by 5FU. The cytostatic action of 5FU depends on its enzymatic conversion to the nucleotide, including ribosylation and phosphorylation. Combining with TS, fluorodeoxyuridine monophosphate (FdUMP) forms a covalent ternary complex. The other two primary metabolites of 5FU are 5-fluorouridine-50-triphosphate (FUTP) and 5-fluoro-20-deoxyuridine-50-triphosphate (FdUTP), which respectively impair RNA and DNA production and function.

5FU disrupts cellular function by oxidative stress

Although, reactive oxygen species (ROS) have a fundamental role in main cellular activities, including signal transduction and bactericidal effect of phagocytes, apoptosis and cellular injury can result from their accumulation.^93–95 Besides, a significant part of the chemotherapy-related oxidative damage has been reported to be played by an increase in ROS.^96–98 5FU, as a widely used chemotherapeutic agent, can also produce mitochondrial ROS through the p53-dependent route, and accordingly, it can cause neoplastic cell death.⁹⁹ But, 5FU can also bring about some serious adverse events in which ROS overproduction and oxidative stress play a crucial role (Figure 3).¹⁰⁰ It could be predicted that these mechanisms might be of vital importance in 5FU-induced reproductive toxicity in females. Undoubtedly, future studies should aim to determine the uncertain mechanism behind this phenomenon.

Conclusion

Despite being widely used in various types of solid tumors, 5FU can result in severe forms of multi organ toxicities. Importantly, reproductive toxicity is one of the major complications that may limit the clinical application of this drug. In the current review, we explained and discussed 5FU-induced reproductive toxicity in an organ-based method. First, we explained 5FU-induced toxicity in male reproductive system, which, in summary, showed destruction of the seminiferous epithelial cells and Sertoli cells vacuolization. Although, 5FU does not cause any histopathological changes, it reduces the relative weight of the prostate. The chemotherapy agent also causes severe sperm abnormalities such as germ cells shedding, spermatid halo appearance, and polynucleated giant cells along with reduction in sperm count. 5FU-induced female reproductive toxicity is mainly characterized by morphologically atretic secondary and antral follicles, along with decrease in the number of growing follicles, ovarian weight and, a notable impairment in their maturity.

Footnotes

Acknowledgments

All figures are created using

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Correction (December 2023):

Article updated online to correct “5-flourouracil” to “5-fluorouracil” throughout the article.

ORCID iDs

Ali Tavakoli Pirzaman

Pouyan Ebrahimi

Sohrab Kazemi

Data Availability Statement

The data that supports the findings of this study are available from the corresponding author upon reasonable request.

References

Alvarez

Marchal

Boulaiz

, et al. 5-Fluorouracil derivatives: a patent review. Expert Opin Ther Pat 2012; 22: 107–123.

Tavakoli Pirzaman

Aghajanian

Mansoori

, et al. Interaction of quercetin and 5-fluorouracil: cellular and pharmacokinetic study. Toxicol Mech Methods 2023; 33: 502–511. (accessed 14 March 2023). DOI: 10.1080/15376516.2023.2188928

Longley

Harkin

Johnston

. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer 2003; 3: 330–338.

Vodenkova

Buchler

Cervena

, et al. 5-fluorouracil and other fluoropyrimidines in colorectal cancer: past, present and future. Pharmacol Ther 2020; 206: 107447.

Sorrentino

Kim

Foderaro

, et al. 5-fluorouracil induced cardiotoxicity: review of the literature. Cardiol J 2012; 19: 453–457.

Gelen

Şengül

Yıldırım

, et al. The protective effects of naringin against 5-fluorouracil-induced hepatotoxicity and nephrotoxicity in rats. Iranian journal of basic medical sciences 2018; 21: 404.

Liu

X-Y

Zhang

F-R

Shang

J-Y

, et al. Renal inhibition of miR-181a ameliorates 5-fluorouracil-induced mesangial cell apoptosis and nephrotoxicity. Cell Death Dis 2018; 9: 1–14.

Naren

Wang

Zhang

, et al. The reversible reproductive toxicity of 5-fluorouracil in mice. Reprod Toxicol 2021; 101: 1–8.

Parrella

Lavorgna

Criscuolo

, et al. Acute and chronic toxicity of six anticancer drugs on rotifers and crustaceans. Chemosphere 2014; 115: 59–66.

10.

Gajski

Gerić

Žegura

, et al. Genotoxic potential of selected cytostatic drugs in human and zebrafish cells. Environ Sci Pollut Control Ser 2016; 23: 14739–14750.

11.

Kovács

Csenki

Bakos

, et al. Assessment of toxicity and genotoxicity of low doses of 5-fluorouracil in zebrafish (Danio rerio) two-generation study. Water Res 2015; 77: 201–212.

12.

Tavakoli Pirzaman

Ebrahimzadeh Pirshahid

Babajani

, et al. The role of microRNAs in regulating cancer cell response to oxaliplatin-containing regimens. Technol Cancer Res Treat 2023; 22: 15330338231206003. (accessed 18 October 2023). DOI: 10.1177/15330338231206003

13.

Giwercman

Petersen

. Cancer and male infertility. Baillieres Best Pract Res Clin Endocrinol Metab 2000; 14: 453–471. (accessed 01 December 2023). DOI: 10.1053/beem.2000.0091

14.

van Casteren

van der Linden

Hakvoort‐Cammel

, et al. Effect of childhood cancer treatment on fertility markers in adult male long‐term survivors. Pediatr Blood Cancer 2009; 52: 108–112.

15.

Kenney

Laufer

Grant

, et al. High risk of infertility and long term gonadal damage in males treated with high dose cyclophosphamide for sarcoma during childhood. Cancer 2001; 91: 613–621.

16.

Müller

Skakkebæk

. Quantification of germ cells and seminiferous tubules by stereological examination of testicles from 50 boys who suffered from sudden death. Int J Androl 1983; 6: 143–156.

17.

Brydoy

Fossa

Dahl

, et al. Gonadal dysfunction and fertility problems in cancer survivors. Acta Oncol 2007; 46: 480–489. (accessed 15 May 2007). DOI: 10.1080/02841860601166958

18.

Clermont

. Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev 1972; 52: 198–236.

19.

Heinrich

DeFalco

. Essential roles of interstitial cells in testicular development and function. Andrology 2020; 8: 903–914.

20.

Gradishar

Schilsky

. Effects of cancer treatment on the reproductive system. Crit Rev Oncol Hematol 1988; 8: 153–171. (accessed 01 January 1988). DOI: 10.1016/s1040-8428(88)80009-x

21.

Heller

. Kinetics of the germinal epithelium. Recent Prog Horm Res 1964; 20: 545–745.

22.

Meistrich

. Cytotoxic effects of chemotherapeutic drugs on mouse testis cells. Cancer Res 1979; 39: 3575–3582.

23.

Remenár

Számel

Budai

, et al. Increase of hypophyseal hormone levels in male head and neck cancer patients. Pathol Oncol Res 2007; 13: 341–344.

24.

Delessard

Saulnier

Rives

, et al. Exposure to chemotherapy during childhood or adulthood and consequences on spermatogenesis and male fertility. Int J Mol Sci 2020; 21. (accessed 26 February 2020). DOI: 10.3390/ijms21041454

25.

Sieniawski

Reineke

Josting

, et al. Assessment of male fertility in patients with Hodgkin's lymphoma treated in the German Hodgkin Study Group (GHSG) clinical trials. Ann Oncol 2008; 19: 1795–1801.

26.

Takizawa

Horii

. Endocrinological assessment of toxic effects on the male reproductive system in rats treated with 5-fluorouracil for 2 or 4 weeks. J Toxicol Sci 2002; 27: 49–56.

27.

Narayana

Urban

JDS

. 5. po Rouracil (5-FU) induces the formation of giant os and sloughing of seminiferous. Indian J Physiol Pharmacol 2000; 44.

28.

D'souza

Narayana

. Induction of seminiferous tubular atrophy by single dose of 5-fluorouracil (5-FU) in Wistar rats. Indian J Physiol Pharmacol 2001; 45: 87–94.

29.

Skrętkowicz

Sekulska

Danilewicz

, et al. Effect of some anticancer drugs and combined chemotherapy on renal toxicity. Neurosignals 1996; 5: 51–58.

30.

Chemes

. Infancy is not a quiescent period of testicular development. Int J Androl 2001; 24: 2–7.

31.

Franchimont

Millet

Vendrely

, et al. Relationship between spermatogenesis and serum gonadotropin levels in azoospermia and oligospermia. J Clin Endocrinol Metabol 1972; 34: 1003–1008.

32.

Tsatsoulis

Shalet

Morris

, et al. Immunoactive inhibin as a marker of Sertoli cell function following cytotoxic damage to the human testis. Horm Res Paediatr 1990; 34: 254–259.

33.

Wallace

Groome

Riley

, et al. Effects of chemotherapy-induced testicular damage on inhibin, gonadotropin, and testosterone secretion: a prospective longitudinal study. J Clin Endocrinol Metabol 1997; 82: 3111–3115.

34.

Spermon

Ramos

Wetzels

, et al. Sperm integrity pre-and post-chemotherapy in men with testicular germ cell cancer. Hum Reprod 2006; 21: 1781–1786.

35.

Giagulli

Vermeulen

. Leydig cell function in infertile men with idiopathic oligospermic infertility. J Clin Endocrinol Metabol 1988; 66: 62–67.

36.

Booth

Loriaux

. Selective control of follicle-stimulating hormone secretion: new perspectives. New York: Raven Press, 1983.

37.

Rowley

Leach

Warner

, et al. Effect of graded doses of ionizing radiation on the human testis. Radiat Res 1974; 59: 665–678.

38.

Donohue

Thornhill

Foster

, et al. Retroperitoneal lymphadenectomy for clinical stage A testis cancer (1965 to 1989): modifications of technique and impact on ejaculation. J Urol 1993; 149: 237–243.

39.

Peckham

Husband

Barrett

, et al. Orchidectomy alone in testicular stage I non-seminomatous germ-cell tumours. Lancet 1982; 320: 678–680.

40.

Kreuser

Xiros

Hetzel

, et al. Reproductive and endocrine gonadal capacity in patients treated with COPP chemotherapy for Hodgkin's disease. J Cancer Res Clin Oncol 1987; 113: 260–266.

41.

Thomson

Campbell

Irvine

, et al. Semen quality and spermatozoal DNA integrity in survivors of childhood cancer: a case-control study. Lancet 2002; 360: 361–367.

42.

Mohammadnejad

Abedelahi

. Degenerative effect of cisplatin on testicular germinal epithelium. Adv Pharmaceut Bull 2012; 2: 173.

43.

Kotzur

Benavides-Garcia

Mecklenburg

, et al. Granulocyte colony-stimulating factor (G-CSF) promotes spermatogenic regeneration from surviving spermatogonia after high-dose alkylating chemotherapy. Reprod Biol Endocrinol 2017; 15: 1–12.

44.

Delbes

Chan

Pakarinen

, et al. Impact of the chemotherapy cocktail used to treat testicular cancer on the gene expression profile of germ cells from male Brown-Norway rats. Biol Reprod 2009; 80: 320–327.

45.

Velez de la Calle

Soufir

Chodorge

, et al. Reproductive effects of the anti-cancer drug procarbazine in male rats at different ages. Reproduction 1988; 84: 51–61. DOI: 10.1530/jrf.0.0840051

46.

Stumpp

Freymüller

Miraglia

. Sertoli cell function in albino rats treated with etoposide during prepubertal phase. Histochem Cell Biol 2006; 126: 353–361.

47.

Heikens

Behrendt

Adriaanse

, et al. Irreversible gonadal damage in male survivors of pediatric Hodgkin's disease. Cancer: Interdisciplinary International Journal of the American Cancer Society 1996; 78: 2020–2024.

48.

Nurmio

Toppari

Kallio

, et al. Functional in vitro model to examine cancer therapy cytotoxicity in maturing rat testis. Reprod Toxicol 2009; 27: 28–34.

49.

Gerl

Mühlbayer

Hansmann

, et al. The impact of chemotherapy on Leydig cell function in long term survivors of germ cell tumors. Cancer 2001; 91: 1297–1303.

50.

Brämswig

Heimes

Heiermann

, et al. The effects of different cumulative doses of chemotherapy on testicular function. Results in 75 patients treated for Hodgkin's disease during childhood or adolescence. Cancer 1990; 65: 1298–1302.

51.

Smart

Lopes

Rice

, et al. Chemotherapy drugs cyclophosphamide, cisplatin and doxorubicin induce germ cell loss in an in vitro model of the prepubertal testis. Sci Rep 2018; 8: 1–15.

52.

Sklar

. Reproductive physiology and treatment‐related loss of sex hormone production. Med Pediatr Oncol: The Official Journal of SIOP—International Society of Pediatric Oncology (Societé Internationale d'Oncologie Pédiatrique 1999; 33: 2–8.

53.

Tharmalingam

Matilionyte

Wallace

, et al. Cisplatin and carboplatin result in similar gonadotoxicity in immature human testis with implications for fertility preservation in childhood cancer. BMC Med 2020; 18: 1–16.

54.

Chatzidarellis

Makrilia

Giza

, et al. Effects of taxane-based chemotherapy on inhibin B and gonadotropins as biomarkers of spermatogenesis. Fertil Steril 2010; 94: 558–563.

55.

Lynch

Morgan

Panetta

, et al. The ABC transporter MRP4 limits Apoptosome activation 6-mercaptopurine, an important molecular instigator in induced leydig cell death. Drug Metab Rev 2016; 153, Taylor & Francis Ltd.

56.

Waxman

Scher

Hellinger

, et al. Combination cytotoxic-differentiation therapy of mouse erythroleukemia cells with 5-fluorouracil and hexamethylene bisacetamide. Cancer Res 1990; 50: 3878–3887.

57.

Pinedo

Peters

. Fluorouracil: biochemistry and pharmacology. J Clin Oncol 1988; 6: 1653–1664.

58.

O'Dwyer

King

Hoth

, et al. Role of thymidine in biochemical modulation: a review. Cancer Res 1987; 47: 3911–3919.

59.

Schuetz

Wallace

Diasio

. 5-Fluorouracil incorporation into DNA of CF-1 mouse bone marrow cells as a possible mechanism of toxicity. Cancer Res 1984; 44: 1358–1363.

60.

Habas

Anderson

Brinkworth

. Detection of phase specificity of in vivo germ cell mutagens in an in vitro germ cell system. Toxicology 2016; 353: 1–10.

61.

Elstein

Zucker

Shuey

, et al. Utility of the murine erythroleukemic cell (MELC) in assessing mechanisms of action of DNA‐active developmental toxicants: application to 5‐fluorouracil. Teratology 1993; 48: 75–87.

62.

Al-Asmari

Al-Zahrani

Khan

, et al. Taurine ameliorates 5-fluorouracil-induced intestinal mucositis, hepatorenal and reproductive organ damage in Wistar rats. Hum Exp Toxicol 2015; 35: 10–20. DOI: 10.1177/0960327115573597

63.

Miyazaki

. Safety evaluation of oral 5-fluorouracil (acute, subacute, chronic toxicity and teratological studies). Clin Rep 1974; 8: 2603–2640.

64.

Horii

. Toxicity study of doxifluridine (4) Comparative 5 weeks oral toxicity study with FT-207 and 5-FU in rats. J Pharmacol Ther 1985; 13: 89–131.

65.

Russell

. Short‐term morphological response of the rat testis to administration of five chemotherapeutic agents. Am J Anat 1991; 192: 142–168.

66.

D‘souza

. Toxic effects of 5-fluorouracil on sperm count in wistar rats. Malays J Med Sci: MJMS 2003; 10: 43.

67.

Ghafouri-Fard

Shoorei

Abak

, et al. Effects of chemotherapeutic agents on male germ cells and possible ameliorating impact of antioxidants. Biomed Pharmacother 2021; 142: 112040.

68.

Naren

Guo

Bai

, et al. Reproductive and developmental toxicities of 5-fluorouracil in model organisms and humans. Expet Rev Mol Med 2022; 24: e9. 2022/02/01. DOI: 10.1017/erm.2022.3

69.

I Al-Sadi

. Histopathological study of lung, testis, and urinary bladder of mice treated with 5-fluorouracil. Rafidain Journal of Science 2017; 26: 9–16.

70.

Mao

W-W

Mao

L-Y

Jiang

, et al. Early morphological changes in the mouse testis induced by 5-fluorouracil. Zhonghua nan ke xue= National Journal of Andrology 2009; 15: 1064–1067.

71.

Gad El-Hak

Moawad

TIS

Hafez

GA-A

. Effects of 5-fluorouracil on testes histology and sperm morphology assay in mice. Egyptian Academic Journal of Biological Sciences, D Histology & Histochemistry 2017; 9: 1–9.

72.

Fahmy

Abd-Alla

Hassan

, et al. Genotoxicity and sperm defects induced by 5-FU in male mice and the possible protective role of Pentas lanceolata-iridoids. Mutat Res Genet Toxicol Environ Mutagen 2020; 850: 503145.

73.

Choudhury

Misra

Jagdale

, et al. Induction and transmission of cytogenetic toxic effects of 5-fluorouracil in male germline cells of Swiss mice. J Exp Clin Cancer Res: CR 2002; 21: 277–282.

74.

Rao

. 5-Fluorouracil (5-FU) induces the formation of giant cells and sloughin g of seminiferous epithelium in the rat testis. Indian J Physiol Pharmacol 2000; 44: 317–322.

75.

Reddy

Svoboda

. Alterations in rat testes due to an antispermatogenic agent. Light and electron microscopic study. Arch Pathol 1967; 84: 376–392.

76.

El-Sayyad

Hassan

. Effects of adriamycin, cisplatin, and 5-fluorouracil on the testes of albino rats. Br J Med Health Sci 2013; 1: 45–62.

77.

Wang

Liu

Johnson

, et al. Doxorubicin obliterates mouse ovarian reserve through both primordial follicle atresia and overactivation. Toxicol Appl Pharmacol 2019; 381: 114714. (accessed 23 August 2019). DOI: 10.1016/j.taap.2019.114714

78.

Stringer

Swindells

Zerafa

, et al. Multidose 5-fluorouracil is highly toxic to growing ovarian follicles in mice. Toxicol Sci 2018; 166: 97–107.

79.

Ben-Aharon

Bar-Joseph

Tzarfaty

, et al. Doxorubicin-induced ovarian toxicity. Reprod Biol Endocrinol 2010; 8: 20. (accessed 06 March 2010). DOI: 10.1186/1477-7827-8-20

80.

Gui

Jin

Lin

, et al. Rosmarinic acid relieves cisplatin-induced ovary toxicity in female mice via suppression of oxidative stress and inflammation. J Biochem Mol Toxicol 2021; 35: e22839. (accessed 13 July 2021). DOI: 10.1002/jbt.22839

81.

Abdel-Aziz

Mohamed

ASM

Abdelazem

, et al. Cilostazol protects against cyclophosphamide-induced ovarian toxicity in female rats: role of cAMP and HO-1. Toxicol Mech Methods 2020; 30: 526–535. (accessed 28 May 2020). DOI: 10.1080/15376516.2020.1774829

82.

Wang

Tian

Liang

, et al. Mechanistic study on triptorelin action in protecting from 5-FU-induced ovarian damage in rats. Oncology Research 2015; 22: 283.

83.

Lambouras

Liew

Horvay

, et al. Examination of the ovotoxicity of 5-fluorouracil in mice. J Assist Reprod Genet 2018; 35: 1053–1060.

84.

Asaduzzaman

. Role of chemotherapeutics in ovarian failure. Flinders University, College of Medicine and Public Health, 2018.

85.

Almeida

Lima

Vieira

, et al. 5-fluorouracil disrupts ovarian preantral follicles in young C57BL6J mice. Cancer Chemother Pharmacol 2021; 87: 567–578.

86.

Liu

Wang

Niu

, et al. The β6-integrin-ERK/MAP kinase pathway contributes to chemo resistance in colon cancer. Cancer Lett 2013; 328: 325–334.

87.

Aquino Esperanza

Aguirre

Aispuru

, et al. In vivo 5-fluorouracil-induced apoptosis on murine thymocytes: involvement of FAS, Bax and Caspase3. In: Proceedings of the VIIIth conference of the international society for trace element research in humans (ISTERH), the IXth conference of the nordic trace element society (NTES), and the VIth conference of the hellenic trace element society (HTES), 2007, Springer, 2008; 131–142.

88.

K-Y

Lai

Y-H

Yang

Y-C

, et al. 5-Fluorouracil-induced apoptosis changes in cultured corneal epithelial cells. J Ocul Pharmacol Therapeut 2016; 32: 155–162.

89.

Carrillo

Navarro

Ramírez

, et al. 5-Fluorouracil derivatives: a patent review (2012–2014). Expert Opin Ther Pat 2015; 25: 1131–1144.

90.

Wigmore

Mustafa

El-Beltagy

, et al. Effects of 5-FU. Adv Exp Med Biol 2010; 157–164, Springer.

91.

Chu

Callender

Farrell

, et al. Thymidylate synthase inhibitors as anticancer agents: from bench to bedside. Cancer Chemother Pharmacol 2003; 52: 80–89.

92.

Noordhuis

Holwerda

Van der Wilt

, et al. 5-Fluorouracil incorporation into RNA and DNA in relation to thymidylate synthase inhibition of human colorectal cancers. Ann Oncol 2004; 15: 1025–1032.

93.

Conklin

. Chemotherapy-associated oxidative stress: impact on chemotherapeutic effectiveness. Integr Cancer Ther 2004; 3: 294–300.

94.

Chen

Tamaki

Kudo

, et al. Protective effects of resveratrol against 5-fluorouracil-induced oxidative stress and inflammatory responses in human keratinocytes. J Clin Biochem Nutr 2021; 69: 238.

95.

Tavakoli Pirzaman

Ebrahimi

Niknezhad

, et al. Toxic mechanisms of cadmium and exposure as a risk factor for oral and gastrointestinal carcinomas. Hum Exp Toxicol 2023; 42: 9603271231210262. (accessed 23 October 2023). DOI: 10.1177/09603271231210262

96.

Songbo

Lang

Xinyong

, et al. Oxidative stress injury in doxorubicin-induced cardiotoxicity. Toxicol Lett 2019; 307: 41–48.

97.

Han

Kim

Jang

, et al. Cisplatin induces lung cell cilia disruption and lung damage via oxidative stress. Free Radic Biol Med 2021; 177: 270–277.

98.

Baharmi

Kalantari

Kalantar

, et al. Pretreatment with gallic acid mitigates cyclophosphamide induced inflammation and oxidative stress in mice. Curr Mol Pharmacol 2022; 15: 204–212.

99.

Hwang

Chung

Kim

, et al. Drug resistance to 5-FU linked to reactive oxygen species modulator 1. Biochem Biophys Res Commun 2007; 359: 304–310.

100.

Song

Zhang

, et al. ROS and iron homeostasis dependent ferroptosis play a vital role in 5-Fluorouracil induced cardiotoxicity in vitro and in vivo. Toxicology 2022; 468: 153113.