Note
Sex Differences in Genital Area Hyperpigmentation Induced by 5-Fluorouracil Administration in Mice
2025 Volume 48 Issue 8 Pages 1260-1264
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2025 Volume 48 Issue 8 Pages 1260-1264
Pigmentation is one of the most prominent side effects caused by anticancer drugs, especially in female patients, as these changes in appearance can decrease QOL. A typical drug causing such pigmentation is 5-fluorouracil (5-FU); we have previously shown that 5-FU-induced pigmentation is associated with increased adrenocorticotropic hormone (ACTH) and reactive oxygen species (ROS) levels. In male Hos:HRM-2 mice, 5-FU administration resulted in pigmentation appearing in the genital area, accompanied by elevations in neutrophils, ACTH, and ROS. By contrast, female mice showed increases in neutrophils and noradrenaline, but not in ACTH or ROS levels; furthermore, they did not develop pigmentation. In addition, estradiol levels were markedly decreased in female mice, which may have enhanced neutrophil apoptosis and suppressed ROS production. In addition, noradrenaline reflects the stress response and may contribute to the decrease in estradiol, suggesting the hypothalamus–pituitary–adrenal axis and sex hormones may interact in the formation of sex differences. These results suggest that sex differences exist in the development of 5-FU-induced hyperpigmentation and that fluctuations in estradiol and associated changes in neutrophils, ROS, and ACTH may underlie this phenomenon.
Anticancer drug-induced pigmentation has recently garnered attention as a factor that reduces patient QOL. In particular, changes in appearance and cancer-related pain have been reported as highly distressing symptoms among female patients with cancer.1) Among the various anticancer drugs, 5-fluorouracil (5-FU) is known to induce hyperpigmentation. We have previously investigated the mechanisms underlying 5-FU-induced hyperpigmentation, demonstrating that 5-FU administration causes pigmentation by increasing adrenocorticotropic hormone (ACTH), cAMP, and reactive oxygen species (ROS) levels, all of which are involved in melanin production.2,3)
Meanwhile, sex differences in the effects of 5-FU administration have also been previously reported. For instance, female mice exhibit more pronounced weight gain than male mice following 5-FU treatment, which has been attributed to increased ghrelin levels due to decreased estradiol.4) Estradiol is also known to reduce ROS levels and has been implicated in the differences in skin tone between males and females.5) Based on these findings, we hypothesized that, similar to the observed sex differences in weight changes and skin tone, 5-FU-induced hyperpigmentation may also exhibit sex-dependent differences. Therefore, we investigated the sex differences in 5-FU-induced hyperpigmentation and the underlying mechanisms in pigmented hairless mice.
A total of 20 Hos:HRM-2 mice (9 weeks old; 10 males and 10 females) were purchased from SLC (Hamamatsu, Shizuoka, Japan). The mice were individually caged and housed under specific pathogen-free conditions in a room regulated at 23 ± 1°C, with a 12-h/12-h light/dark photoperiod, free access to drinking water, and a basic diet in pellet form. All experimental protocols were approved by the Ethics Committee of the Suzuka University of Medical Sciences. This study was conducted in strict adherence to the recommendations of the Suzuka University of Medical Sciences Laboratory Animal Care and Use Guide (Approval No. 82) and in accordance with the ARRIVE guidelines. All surgical procedures were performed under isoflurane anesthesia and adjusted to minimize animal distress.
Experimental DesignAs in previous experiments, the 5-FU-treated group (five males and five females) received 15 mg/kg 5-FU (adjusted with saline solution).2–4) The control group (five males and five females) received saline solution. The administration schedule was the same as that used in previous experiments, with intraperitoneal administration once daily for 5 d, followed by a 2-d rest period for 4 cycles.6) The required samples were collected under isoflurane anesthesia on the last day of the experiment.
StainingGenital area skin samples were fixed in 4% paraformaldehyde in phosphate-buffered saline, embedded in frozen Tissue-Tek OCT compound (Sakura Finetech, Tokyo, Japan), and sectioned into 5-μm thick slices. 3,4-Dihydroxyphenylalanine (DOPA)-positive melanocytes in the sections were stained using a previously described method.6)
Additionally, other skin sections were used for immunofluorescence staining of tyrosinase, neutrophils, and estrogen receptors. The following primary antibodies were used: rabbit polyclonal anti-tyrosinase (1:100; Cell Signaling Technology, Danvers, MA, U.S.A.), mouse monoclonal anti-lymphocyte antigen 6 complex locus G6D (Ly6G; neutrophil marker) (1:100; BD Biosciences, Franklin Lakes, NJ, U.S.A.), rabbit polyclonal anti-estrogen receptor alpha, and rabbit polyclonal anti-estrogen receptor beta (1:100; Bioss, Woburn, MA, U.S.A.).
The sections were then incubated with appropriate secondary antibodies (1:30 dilution; fluorescein isothiocyanate-conjugated anti-rabbit, anti-mouse, anti-rat, or anti-goat secondary antibodies [Dako Cytomation, Glostrup, Denmark]) for 2 h in the dark. The expression levels of tyrosinase, Ly6G, and estrogen receptors were observed under a fluorescence microscope and quantified using ImageJ software (ver. 1.53; National Institutes of Health, Bethesda, MD, U.S.A.).
Measurement of Noradrenaline, Estradiol, ROS, and ACTHGenital area and plasma samples were collected on the final day of the study. The genital area samples were homogenized in lysis buffer (Kurabo, Osaka, Japan), the tissue extracts were centrifuged at 20400 × g for 10 min, and the supernatants were collected. Commercially available enzyme-linked immunosorbent assay kits were used according to the manufacturers’ instructions: Noradrenaline (Abcam, Cambridge, U.K.), estradiol (Cayman Chemical, Ann Arbor, MI, U.S.A.), ROS (Cell Biolabs Inc., San Diego, CA, U.S.A.), and ACTH (Abcam).
Statistical AnalysisAll data are expressed as mean ± standard deviation. Statistical analyses were performed using Microsoft Excel 2010 and SPSS version 20 (SPSS Inc.). Data were analyzed using one-way ANOVA, and Tukey’s post hoc test. Differences were considered statistically significant at p < 0.05.
Gross observations showed that male mice treated with 5-FU had pigmentation of the genital area, whereas female mice did not (Fig. 1A). In addition, Dopa staining, which stains cells with tyrosinase, the rate-limiting enzyme in melanogenesis, showed significantly more Dopa-positive cells on the genital area of the 5-FU-treated group compared with the control group (Fig. 1B). By contrast, there was no increase in the number of Dopa-positive cells in the female 5-FU-treated group. Furthermore, when tyrosinase was evaluated by immunofluorescence staining, it was significantly activated in male 5-FU-treated rats but not in female 5-FU-treated rats (Fig. 1C).
Gross observation of the buttocks (A), Dopa staining (B), and tyrosinase levels (C). Values are expressed as mean ± standard deviation (S.D.) of 5 animals. * : p < 0.05, ** : p < 0.01, N = 5 cases per group.
Blood noradrenaline levels increased following 5-FU administration in both males and females (Fig. 2A). Estradiol was not changed by 5-FU administration in males but was significantly decreased by 5-FU administration in females (Fig. 2B).
Plasma noradrenaline (A) and estradiol (B) levels. Values are expressed as mean ± S.D. of 5 animals. * : p < 0.05, ** : p < 0.01, N = 5 per group.
Neutrophil count increased after 5-FU administration in both males and females (Fig. 3A). By contrast, estrogen receptor expression was not altered by 5-FU administration in either male or female mice (Figs. 3B, 3C).
The expression levels of neutrophils (A), estrogen receptor alpha (B), and estrogen receptor beta (C) in the genital area. Values are expressed as mean ± S.D. of 5 animals. * : p < 0.05, ** : p < 0.01, N = 5 cases per group.
The ROS levels in the genital area were significantly increased by 5-FU administration in males, but not in females (Fig. 4A). Furthermore, similar to ROS, genital area ACTH levels were also increased in males treated with 5-FU, but was unchanged in females (Fig. 4B).
Genital area ROS (A) and ACTH (B) levels. Values are expressed as mean ± S.D. of 5 animals. * : p < 0.05, ** : p < 0.01, N = 5 per group.
In this study, we showed that 5-FU administration is associated with sex differences in pigmentation development in the mouse genital area. Male mice showed increased neutrophil counts and elevated ACTH and ROS levels, which are involved in melanogenesis, along with the development of pigmentation. By contrast, in female mice, similar increases in neutrophil counts and noradrenaline were observed, but no changes in ACTH or ROS levels were observed, nor did pigmentation develop.
This difference is due to the involvement of sex hormones, particularly estradiol. In female mice, a marked decrease in estradiol levels was observed following 5-FU administration. An increase in noradrenaline levels indicates a common stress response and noradrenaline is known to inhibit estradiol secretion.7) This suggests that the stress caused by 5-FU administration may have caused the decrease in estradiol via noradrenaline.
Estradiol is also known to inhibit neutrophil apoptosis and prolong their lifespan.8) Therefore, a decrease in estradiol levels may promote neutrophil apoptosis, resulting in limited survival time and neutrophil activity. By contrast, the expression levels in neutrophils increased in both male and female mice. Although this may seem contradictory at first glance, it may be due to the common enhancement of neutrophil mobilization signals by the stress response to 5-FU administration. In other words, although neutrophil release from the bone marrow occurred in a sex-neutral manner, it is likely that neutrophils have shortened life spans in female mice due to reduced estradiol and that apoptosis progressed before ROS production and other functions could be expressed. However, the possibility that estradiol deficiency promotes neutrophil apoptosis, thereby triggering compensatory production and mobilization, remains purely speculative. Further verification, such as experiments involving estradiol supplementation, would be necessary to clarify this mechanism. This leaves a challenge to be addressed in future studies. Notably, estradiol-induced neutrophil apoptosis is believed to be estrogen receptor-mediated. Therefore, if neutrophil apoptosis is accelerated in female mice treated with 5-FU, it is likely to be due to decreased estradiol levels and not due to a change in estrogen receptor expression. At present, there are no clear reports on the relationship between neutrophils and pigmentation, and the findings of this study provide initial data suggesting a relationship.
Another important finding of this study is that the increase in ACTH was limited to male mice; ACTH is secreted via the hypothalamus–pituitary–adrenal (HPA) axis and promotes steroid hormone secretion from the adrenal glands as part of the stress response. It is also known to be involved in melanocyte activation in the skin.9,10) However, ACTH was not elevated in female mice. This sex-based difference could be attributed to multiple factors. First, estradiol is known to inhibit the activity of the HPA axis, and a decrease in estradiol levels in female mice may have inhibited HPA axis activation.11) In addition, ACTH secretion is regulated not only by systemic stress signals but also by local inflammation-related factors such as ROS.12) Here, female mice also showed no increase in ROS, suggesting weak stimulation of the HPA axis and that induction of ACTH was unlikely to occur. Thus, the sex difference in ACTH likely occurs because of the combined effects of the endogenous hormone environment and local inflammatory response. Future detailed analysis focusing on the crosstalk between these pathways is required. It should be noted that ACTH is mentioned in this study as one potential factor contributing to pigmentation, and not as the main cause. In our hypothesis, ACTH functions as an auxiliary pathway that may regulate these phenomena. The conclusions of this study are not based on a single factor, but are instead built on the premise that multiple stress responses and hormonal pathways interact in a complex manner.
As described above, administering 5-FU caused an increase in neutrophil counts in male mice, which may have increased ROS and ACTH levels. These factors may be associated with an increase in tyrosinase activity, which may have contributed to pigmentation. However, this study did not directly evaluate neutrophil activation; thus, this point remains to be investigated in future studies. By contrast, in female mice, the promotion of neutrophil apoptosis and decreased production of ROS/ACTH, associated with decreased estradiol levels, may have contributed to the suppression of pigmentation.
However, this study has several limitations. First, as the analysis was based on a mouse model, its potential applicability to humans must be carefully examined. In addition, these findings remain hypothetical as there have been no previous reports on the association between neutrophils and hyperpigmentation. Verifying this causal relationship would require assessing the effects of estradiol on neutrophil function and longevity. Future prospects include intervention experiments using sex hormone supplementation or blockade and analysis of the time course of neutrophil apoptosis. In addition, differences in local skin characteristics, such as stratum corneum thickness, enzyme activity, hormone receptor expression, and skin sensitivity to drugs may also influence pigmentation outcomes. Although these factors were not directly evaluated in this study, they may contribute to sex differences in pigmentation, along with systemic factors; therefore, they should be addressed in future studies. Furthermore, by matching human-derived cells and clinical data, we may gain a better understanding of sex differences in the adverse effects of anticancer drugs, which may help prevent adverse effects and develop personalized treatment strategies. This study is also expected to contribute to preventing adverse effects and establishing personalized treatment strategies.
The authors declare no conflict of interest.
