The Mechanism of 5-Fluorouracil-Induced Hyperpigmentation in HRM-2 Hairless Mice: Focus on the Increase of Blood Vessels

Abstract

5-Fluorouracil (5-FU), an effective chemotherapeutic agent for many solid tumors, has long been reported to cause pigmentation in patients treated intravenously, which occurs with increasing frequency of administration and decreases the QOL of the patients. Although melanin accumulation is thought to be the cause, the mechanism of pigmentation induced by 5-FU administration remains unclear, and there is no effective treatment for this problem. In this study, we investigated the mechanism of pigmentation induced by continuous 5-FU administration in 9-week-old male HRM-2 hairless mice for 8 weeks by focusing on the blood vessels for basic verification. In the auricular skin of 5-FU-administered mice, hyperpigmentation caused by melanin accumulation was observed macroscopically and by Fontana–Masson Staining. In addition, the expression of tyrosinase, melanin synthase, and blood vessel markers in the auricular skin was increased by 5-FU-administration in mice auricular skin. Other anticancer agents, cytarabine (Ara-C) and irinotecan (CPT-11), were also administered, and the differences between them and 5-FU were investigated; these changes were not observed in the auricles of these mice. These results suggest that tyrosinase is associated with 5-FU-induced melanin production and that an increase in blood vessels may be involved. Furthermore, pigmentation with melanin accumulation in the basal epidermal layer is a characteristic finding of 5-FU compared with Ara-C and CPT-11. In conclusion, this study indicates that 5-FU causes hyperpigmentation by melanin accumulation in a characteristic manner, including an increase in blood vessels.

INTRODUCTION

Cancer chemotherapy using alkylating agents, platinum agents, and antimetabolites has been reported to cause drug-induced hyperpigmentation as a side effect.^1–6) Anticancer drug therapy causes external changes such as hair loss and darkening of the skin and is a major factor in reducing patients’ QOL.⁴⁾ In a survey of patients undergoing outpatient chemotherapy, Nozawa et al. found that approximately 80% of patients were distressed by changes in appearance. In addition, women felt more distress due to this change than men.⁷⁾ However, changes in appearance caused by cancer chemotherapy are overlooked and underestimated by healthcare providers because they are not life-threatening.

The number of male patients with colorectal cancer (CRC) in Japan has stabilized; however, the number of female patients has increased.⁸⁾ 5-Flurouracil (5-FU), an effective chemotherapeutic agent against CRC, often causes hyperpigmentation. 5-FU is a fluoropyrimidine antimetabolite that was synthesized by Duschinsky et al.¹⁾ and has been evaluated as an anticancer agent in extensive basic and clinical studies led by Heidelberg et al.⁹⁾ This is effective against many solid tumors, including gastric and colorectal cancer, and is a routinely used anticancer drug.^4,7,8) On the other hand, it has been reported that pigmentation occurs in 2–5%³⁾ of patients treated with 900–1750 mg intravenous 5-FU daily. Furthermore, the probability of hyperpigmentation was correlated with the frequency of 5-FU administration, which caused a reduction in the QOL of these patients.^1–3) Accumulation of melanin causes hyperpigmentation.^10,11) However, the mechanism by which 5-FU induces hyperpigmentation remains to be elucidated. There are currently no effective treatments for hyperpigmentation.

Melanin is synthesized by the melanin-forming enzyme tyrosinase.^12,13) Recent studies have reported that melasma pigmentation is associated with blood vessels.¹⁴⁾ Additionally, vascular endothelial cells act on tyrosinase-related signals for melanin production. We previously reported that 5-FU increases melanin synthesis by activating the nuclear factor-kappaB signaling pathway.¹⁵⁾ However, our results suggest the presence of other mechanisms involved in 5-FU-induced hyperpigmentation. This study aimed to elucidate the mechanism of hyperpigmentation associated with continuous 5-FU administration by performing basic studies focusing on tyrosinase and blood vessels.

MATERIALS AND METHODS

Animal Experiments

HRM-2 hairless mice (9 weeks old, male) were purchased from SLC (Hamamatsu, Shizuoka, Japan). Mice were individually maintained in cages in an air-conditioned room at 23 ± 1 °C under specific-pathogen-free conditions with a 12 h light–12 h dark cycle, free access to drinking water, and a pelleted basal diet. All experimental protocols were approved by the Ethics Committee of the Suzuka University of Medical Science. The study was performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of Suzuka University of Medical Science (Approval No. 82) and carried out according to the ARRIVE guidelines. All surgeries of drug administration, measurement of body weight, euthanasia procedure were performed under pentobarbital anesthesia, and efforts were made to minimize animal suffering.

Experimental Design

Mice were randomly divided into two or four groups, and control mice were intraperitoneally administered physiological saline. Mice were used as controls. The other group was intraperitoneally injected with 5-FU (15 mg/kg),¹⁶⁾ cytarabine (Ara-C) (20 mg/kg),¹⁷⁾ or irinotecan (CPT-11) (20 mg/kg).¹⁷⁾ The drugs were administered 6 d a week for a total of 8 weeks. On the final day of examination, skin samples were taken from the auricular and buttock areas, with areas of grossly darkened skin defined as hyperpigmented. The pigmented areas of the buttock skin were photographed after collection and quantified using ImageJ, version 1.47 (NIH, Bethesda, MD, U.S.A.). Auricular skin samples were collected from each mouse. We did not perform a statistical analysis of the area of pigmentation in the auricular due to technical aspects, but determined it by a gross comparison. Therefore, Fontana–Masson (FM) staining was performed to measure pigmentation. The drugs 5-FU, Ara-C, and CPT-11 were obtained from Kyowa Kirin (Tokyo, Japan), Nippon Shinyaku (Kyoto, Japan), and Daiichi-Sankyo (Tokyo, Japan), respectively.

Sections Preparation and Hematoxylin–Eosin (H&E) Staining

Skin samples were fixed with formaldehyde (4%) in phosphate-buffered saline (PBS), embedded in paraffin blocks, and cut into 3 µm sections using Microsystems (Wetzlar, Germany) by routine procedures. The sections were stained with H&E in accordance with established procedures to enable histological analysis of the skin.

FM Staining

After deparaffinization, the 6 µm sections were deparaffinized and incubated in Fontana ammoniacal silver solution (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) overnight in the dark at room temperature, rinsed three times with distilled water, and then incubated in 0.2% gold chloride solution for 30 s. Afterward, the tissues were washed in distilled water and 5% sodium thiosulfate solution for 1 min, counterstained with a nuclear fast red solution for 5 min, washed in distilled water, dehydrated in absolute alcohol, and mounted for observations. The pigmentation area on one side of the auricular skin was compared using ImageJ software.

Immunohistochemical Study

After deparaffinization and three washes in PBS, the 6 µm sections were blocked with 2% skim milk for 20 min at room temperature. The expression levels of tyrosinase, von Willebrand factor (vWF), and CD31 in skin tissue samples were measured using immunofluorescence staining. Tissue samples were stained overnight at 4 °C. with antibodies against tyrosinase (1 : 100; Bioss, Boston, MA, U.S.A.) or vWF (1 : 100; Bioss), CD31 (1 : 100; Santa Cruz Biotechnology, Dallas, TX, U.S.A.). After washing thrice with PBS, goat anti-rabbit immunoglobulin G Alexa Fluor 488 secondary antibody (A32731, Invitrogen, Waltham, MA, U.S.A.) was incubated at a 1 : 200 dilution in PBS for 2 h at room temperature in the dark. Stained tissues were mounted using a mounting medium (Vectashield HardSet Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI); Vector Laboratories, CA, U.S.A.) and observed under a fluorescence microscope (BZ-X800; Keyence, Osaka, Japan). The protein-positive cells expressed were randomly photographed at four locations. This site (per the same area) was analyzed using ImageJ, the average value was calculated, and a comparative study was performed.

Statistical Analysis

The area of melanin expression on the buttocks was measured using ImageJ, and the obtained measurements were statistically analyzed using Microsoft 365 Excel (Microsoft Corp., Redmond, WA, U.S.A.). Each data is expressed as mean ± standard deviation (S.D.). Differences between the two groups were analyzed using the unpaired Student’s t-test. One-way ANOVA followed by the Student–Newman–Keuls test was used to compare more than two groups. p < 0.05 was considered to be statistically significant.

RESULTS

Macroscopic Comparison of Skin and Melanin Accumulation

Eight weeks after 5-FU administration showed a black area in the auricular region and buttocks (Fig. 1A), and H&E staining showed no obvious histological changes in each group (Fig. 1B). FM staining was performed for melanin accumulation in the auricular region and showed increased melanin in the basal epidermal layer in 5-FU administration mice compared to control mice (Figs. 1C, D). These results indicate that melanin is involved in pigmentation induced by 5-FU administration.

Fig. 1. Pigmentation Induced by 5-FU Administration

(A) The photographs show the pigmentation differences between the auricular and buttocks skin in control and 5-FU-administered mice. (B) The auricular skin sections were stained with H&E staining. This staining was compared to histopathological changes in auricular skin in control and 5-FU-administered mice. Bar: 200 µm. (C, D) FM staining of the auricular skin sections. This staining was used to compare melanin expression in the basal epidermal layer in control and 5-FU-administered mice for statistical analysis. Bar: 100 µm. The values are expressed as the mean  ±  S.D. derived from five mice (unpaired Student’s t-test, ** p < 0.01, * p < 0.05 significant difference). 5-FU, 5-fluorouracil; H&E, Hematoxylin–eosin; FM, Fontana–Masson; S.D., standard deviation.

Effect of 5-FU on Tyrosinase and Blood Vessel

To investigate the involvement of tyrosinase in hyperpigmentation of the auricular region, fluorescent immunostaining (IF) showed that tyrosinase expression was significantly increased in the basal epidermal layer of 5-FU administration mice (Figs. 2A, D). To examine the relationship between melanin production and vascular endothelial cells, we used IF to compare the expression of the vascular markers vWF and CD31. The results showed that the expression of the vascular marker vWF was significantly increased in the dermal layer of the 5-FU administration mice compared to that in the control mice (Figs. 2B, E), while the expression of CD31 showed an increasing trend (Figs. 2C, F). These results suggest that tyrosinase may induce melanogenesis by 5-FU administration in vascular cells.

Fig. 2. Comparison by Fluorescent Immunostaining at the Pigmentation in the Auricular of Control and 5-FU Administered Mice

Tyrosinase (A, D), a rate-limiting enzyme for melanogenesis, vWF (B, E), and CD31 (C, F) were analyzed. Protein-positive cells are indicated by white arrows. (A) Bar: 100 µm. (B, C) Bar: 200 µm. Values are expressed as mean  ±  S.D. derived from three to five mice (unpaired Student’s t-test, * p < 0.05, significant difference). 5-FU, 5-fluorouracil; vWF, von Willebrand factor; S.D., standard deviation.

Comparison of Pigmentation in Other Anticancer Agents-Administration Mice

To investigate whether this hyperpigmentation was specific to 5-FU, mice administrated with 5-FU were compared to those administered with Ara-C and CPT-11. The results showed hyperpigmentation at 8 weeks after 5-FU administration. In addition to 5-FU, some individuals in Ara-C and CPT-11-treated mice also had pigmented skin in the auricular and buttock areas. The auricular region was black compared to controls (Fig. 3A). Furthermore, the pigmentation area in the buttocks' skin was significantly greater than that in controls and Ara-C or CPT-11 treated mice (Figs. 3A, B). H&E staining showed no obvious histological changes in any groups (Fig. 3C). Next, we compared the melanin accumulation in the auricular region using FM staining. Melanin accumulation in the basal epidermal layer was higher in the 5-FU group than that in the control, Ara-C, and CPT-11 groups (Figs. 3C, D). These results suggested that hyperpigmentation of the auricular and buttock skin with melanin accumulation is a characteristic finding of 5-FU.

Fig. 3. Comparison of External Pigmentation in the Auricular and Buttocks with 5-FU, Ara-C, and CPT-11 Administered Mice

Visual and histological changes in the auricular area and buttocks of the control, 5-FU, Ara-C, and CPT-11 groups were compared. (A) External color changes of the auricular skin. (B) Comparison of external color changes and areas of pigmentation on the buttocks. Values are expressed as the mean  ±  S.D. derived from four mice. Student–Newman–Keuls test, * p < 0.05, significant difference. (C, D) H&E and FM staining of auricular skin sections. This staining was used to compare histopathological changes and melanin expression in the auricular skin of control, 5-FU, Ara-C, and CPT-11 administered mice. Bar: 200 µm H&E, Bar: 100 µm FM staining. Values are expressed as the mean  ±  S.D. derived from five mice. Student–Newman–Keuls test, ** p < 0.01, * p < 0.05. 5-FU, 5-fluorouracil; H&E, Hematoxylin and eosin; FM, Fontana–Masson; S.D., standard deviation.

Finally, we compared the expression levels of various proteins using immunofluorescence staining. Tyrosinase expression was significantly increased in the basal epidermal layer in the 5-FU group compared to the Ara-C and CPT-11 groups and showed an increasing trend compared to the control group (Figs. 4A, D). The expression of vWF was significantly increased in the dermis of the 5-FU group (Figs. 4B, E). CD31 expression showed an increasing trend in the dermis of the 5-FU group compared with that in the control, Ara-C, and CPT-11 groups (Figs. 4B, F). These results suggest that 5-FU-induced pigmentation-is caused by a distinctive mechanism involving tyrosinase and vascular markers compared to anticancer drugs with different mechanisms.

Fig. 4. Comparison by Fluorescent Immunostaining at the Pigmentation in the Auricular of Control, 5-FU, Ara-C, and CPT-11 Administered Mice

The fluorescence intensities of tyrosinase (A, D), vWF (B, E), and CD31 (C, F) were compared in 5-FU, Ara-C, and CPT-11 administered mice. Tyrosinase was expressed in the basal epidermal layer, whereas vWF and CD31 were expressed in the dermis. Protein-positive cells are indicated by white arrows. (A) Bar, 100 µm (B, C) Bar, 200 µm. Values are expressed as mean  ±  S.D. derived from three to five mice. Student–Newman–Keuls test, ** p < 0.01, * p < 0.05. 5-FU, 5-fluorouracil; vWF, von Willebrand factor; S.D., standard deviation.

DISCUSSION

In this study, the administration of 5-FU to mice induced skin pigmentation in the auricular and gluteal terminal regions. Furthermore, 5-FU-administration mice produced melanin by increasing tyrosinase expression in melanocytes in the basal epidermal layer, suggesting that melanin is produced by melanocytes in the basal layer of the epidermis. In addition, an abnormal increase in blood vessels may be involved in the increased tyrosinase expression.

In one study, a 5-FU ointment caused pigmentation after erosion and ulceration in hairless mice.¹⁸⁾ However, 5-FU systemic administration caused pigmentation, as observed in the present study,^3,6,19) suggesting a cause of pigmentation other than inflammation. In a previous study, human umbilical vein endothelial cells promoted melanogenesis by activating the melanogenic pathway in human melanocyte cells.¹⁴⁾ In addition, patients with colorectal cancer receiving chemotherapy, including 5-FU, experience damage to the vascular endothelium with increased vWF.²⁰⁾ Melanin formation was induced by 5-FU administration and increased vascular marker expression, suggesting an association between increased vascular endothelial cells and 5-FU-induced pigmentation. Furthermore, 5-FU increased the expression of vascular markers compared with Ara-C and CPT-11. This result indicates that pigmentation via abnormal vascular proliferation in the auricular region may be specific to 5-FU.

The mechanism of hyperpigmentation by anticancer drugs is speculated to involve various mechanisms (e.g., direct stimulation of melanocytes, subsequent enhancement of melanin production, oversecretion of corticotropin due to adrenal toxicity, oversecretion of melanin-stimulating hormone, deficiency of tyrosinase inhibitor enzyme, formation of melanin complexes with the causative drug, postinflammatory pigmentation after keratinocyte toxicity (with or without photosensitization), and postinflammatory pigmentation after thrombophlebitis.^10,11,21) However, few studies have elucidated the mechanism of its appearance; cytotoxic drug-induced hyperpigmentation occurs at sites of acute or chronic injury and is caused by increased local blood flow.¹¹⁾ The results of the present study shows a link between pigmentation and local blood flow. It may help to understand the mechanisms of pigmentation-induced 5-FU administration. Further investigation is necessary to determine if 5-FU directly stimulates tyrosinase expression and promotes melanin synthesis.

In addition, Ara-C, which is also an antimetabolite, did not show the same results as 5-FU, suggesting that differences in the drug mechanisms of action and metabolic pathways may cause various types of melanin pigmentation, which requires further investigation. Moreover, since many inflammatory factors are involved in melanin production during drug-induced hyperpigmentation,²²⁾ postinflammatory hyperpigmentation induced by anticancer drugs should also be investigated.

In conclusion, the results of the present study suggest that 5-FU-induced skin pigmentation involves melanogenesis in the basal epidermal layer and is associated with vascular endothelial cells, as evidenced by the accumulation of vascular markers in the dermis. These results are important for elucidating the mechanism of 5-FU-induced hyperpigmentation and identifying target proteins for the treatment and prevention of hyperpigmentation. This study may lead to improved QOL in patients with cancer.

Conflict of Interest

The authors declare no conflict of interest.

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