PC-SOD (3,000 U/mg) was obtained from the laboratory stock (Igarashi et al., 1992). Oxaliplatin (Code: 4291410A2149) was obtained from NIPRO CORPORATION (Tokyo, Japan). The luminol-based chemiluminescent probe L-012 (Code: 120–04891), RPMI 1640 medium (Code: 189–02025), and isoflurane (Code: 099–06571) were obtained from Fujifilm Wako Pure Chemical Corporation (Tokyo, Japan). Hydrogen peroxide chemiluminescent detection kit (Code: ADI-907-012) was purchased from Enzo Life Sciences (Farmingdale, NY, United States). Fetal bovine serum (Code: 10270106) was purchased from Thermo Fisher Scientific, and EDTA-2K (Code: 340–01511) was purchased from Dojindo Laboratories (Kumamoto, Japan). CellTiter-Glo^® 2.0 (Code: G9242) was purchased from Promega Corporation (Madison, WI, United States), and 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) (Code: D6883) was obtained from Merck KGaA (Darmstadt, Germany). ISOGEN (Code: 311–02501) was obtained from Nippon Gene (Toyama, Japan), ReverTra Ace^® qPCR RT Master Mix (Code: FSQ-201) was purchased from Toyobo (Osaka, Japan), and KAPA SYBR Fast qPCR Kits (Code: KK4602) were obtained from Nippon Genetics (Tokyo, Japan).

Male ICR mice (6–7 weeks old) were purchased from Charles River (Yokohama, Japan) and kept in dedicated cages (maximum 4 mice/cage) in individual ventilation systems (MVCS-140, ITEC Co., Ltd., Tokyo, Japan) under a 12 h light/dark cycle and fed a controlled diet (MF, Oriental Yeast Co., Ltd., Tokyo, Japan). The experiments and procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the National Institutes of Health (Bethesda, MD, United States), and were approved by the Animal Care Committee of Musashino University (approval number 09-A-2024). The animal studies in this manuscript were based on humane endpoints.

Male ICR mice (8–9 weeks old) weighing approximately 35 g were used for the animal experiments. PC-SOD (3000 U/kg = 1.0 mg/kg) was dissolved in 0.45% sterile NaCl solution and administered intravenously once daily from 1 day before oxaliplatin administration (day 0) to day 4. Oxaliplatin (12.5 mg/kg) was dissolved in sterile saline and administered only once intraperitoneally 5 min after day 1 PC-SOD administration. The dose of oxaliplatin that induced leukopenia was determined from previous studies (Karlsson et al., 2012). In addition, the dose of PC-SOD was determined based on previous studies, specifically the dose at which efficacy analyses were conducted for oxaliplatin-induced peripheral neuropathy (Tanaka et al., 2017; Tanaka et al., 2022b; Qiao et al., 2024).

On days 0, 4, 7, and 10, mice were anesthetized using a mixture of 0.75 mg/kg medetomidine, 4.0 mg/kg midazolam, and 5.0 mg/kg butorphanol on days 0, 4, 7, and 10. After anesthesia, whole blood was collected from the tail vein using an EDTA-coated blood collection tube (Code: 2909000; Paul Marienfeld, Lauda-Königshofen, Germany). An automated hematology analyzer (MEK-6450, Nihon Kohden, Tokyo, Japan) was used to measure the white blood cell (WBC), platelet (PLT), and red blood cell (RBC) counts and hemoglobin (HGB) levels in whole blood. Please refer to Scheme 1 for the protocol for grouping and time course of animal experiments.

Mice were euthanized, and the bones of both legs were isolated 9 days after oxaliplatin administration (day 10). Total RNA was extracted from bone marrow cells using ISOGEN, following the manufacturer’s protocol. Pure RNA was purified from crude RNA, and cDNA was synthesized using the reverse transcriptase, ReverTra Ace^® qPCR RT Master Mix. Real-time RT-PCR was performed using the KAPA SYBR Fast qPCR Kit and analyzed using the CFX96™ Real-Time System (Bio-Rad, Hercules, CA, United States) and CFX Manager™ software (version 3.1, Bio-Rad). Primers were designed using the Primer-BLAST website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primer sequences are described in Supplementary Figure S1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal standard. Gene expression data in each figure are shown relative to the control value of 1.00.

ROS production in the bone marrow of mice was measured using the FUSION chemiluminescence imaging system (Vilber Lourmat, Collégien, France), as previously described (Tanaka et al., 2017; Tanaka et al., 2021; Tanaka et al., 2022a; Tanaka et al., 2022b). At 24 h after intraperitoneal administration of oxaliplatin or sterile saline, mice were subcutaneously administered L-012 (75 mg/kg), a ROS-sensing chemiluminescence probe, dissolved in sterile saline. After 20 min, the bones of both legs were removed from the mice, and the muscle and fatty tissues were carefully stripped from the bones. The cleaned bones were placed in plastic Petri dishes, and brightfield and chemiluminescence images (5-min exposure) were obtained using a FUSION chemiluminescence imaging system. The superimposition of bright-field and chemiluminescence images and quantification of chemiluminescence were performed using the FUSION chemiluminescence imaging system software (version 18.02, Vilber Lourmat).

Bone marrow cells were harvested from the bones of both legs of untreated healthy mice. After isolating the bones of both legs for ROS measurement, RPMI1640 medium containing 10% heat-inactivated FBS was injected into the medullary cavity of each bone, and the bone marrow cells were washed out. A cell strainer with a mesh size of 40 µm (Code: VCS-40, AS ONE Corporation, Osaka, Japan) was used to remove tissue fragments and other impurities. RBCs were lysed using RBC Lysis Buffer (Code: 420301, BioLegend, San Diego, CA, United States), and bone marrow cells were seeded in RPMI1640 medium containing 10% heat-inactivated FBS and 50 μmol/L 2-mercaptoethanol (Code: 21438–82, Nacalai Tesque, Kyoto, Japan) in 96-well plates.

The cells were pre-treated with PC-SOD, followed by oxaliplatin treatment. After 24 h, cell viability was measured using CellTiter-Glo^® 2.0, which emits chemiluminescence in reaction with intracellular ATP. Intracellular ROS levels were measured using H₂DCFDA (10 μmol/L). Measurements were obtained using a microplate reader (Tecan, Kawasaki, Japan).

All values are expressed as mean ± S.E.M. The Mann–Whitney U test or Kruskal–Wallis test, followed by Steel’s multiple comparison test, was used to evaluate differences between groups in animal experiments (Figures 1–3). One-way ANOVA followed by Dunnett’s test was used to evaluate the differences between groups in the cellular experiments (Figures 4, 5). Mac Statistical Analysis Ver. 3.0 (ESUMI Co., Ltd., Tokyo, Japan) was used for all the statistical analyses. P < 0.05 indicated statistical significance.

Oxaliplatin was administered once on day 1, and PC-SOD was administered once daily from day 0 to day 4, for a total of five doses. WBC and PLT counts decreased on days 4, 7, and 10 after oxaliplatin treatment, with a slight recovery observed on day 10 (Figure 1). RBC count and HGB levels decreased on days 4, 7, and 10 after oxaliplatin treatment. These two indices showed no recovery trend on day 10. In contrast, a trend toward improvement in oxaliplatin-dependent WBC count reduction was observed on days 4 and 7 with prophylactic administration of PC-SOD, and the reduction was significantly improved on day 10 (P = 0.007). PC-SOD administration significantly improved the oxaliplatin-dependent decrease in PLT count on day 7 (P = 0.006), although the effect was not as pronounced as the recovery in the WBC count. PC-SOD had little effect on the oxaliplatin-dependent reductions in RBC counts and HGB levels. These results suggest that PC-SOD exerts a prophylactic effect against oxaliplatin-induced myelosuppression, especially leukopenia.

Next, we analyzed the gene expression of colony-stimulating factor 2 and 3 [CSF-2 and CSF3, also known as granulocyte-macrophage (GM)-CSF and G-CSF], glycoprotein hormones required for hematopoietic progenitor cell differentiation and proliferation (Ogawa, 1993; Huang et al., 1999), using real-time RT-PCR. Bone marrow cells were harvested from the bones of both legs of mice on day 10, RNA was purified, and cDNA was synthesized and used for analyses. Significant downregulation of CSF-2 and CSF-3 mRNAs was observed in bone marrow cells from oxaliplatin-treated mice compared with bone marrow cells from control mice (Figure 2A). This downregulation was significantly ameliorated in the bone marrow cells from mice pre-treated with PC-SOD (CSF-2: P = 0.001, CSF-3: P = 0.001). We also examined the gene expression of other cytokines associated with hematopoietic progenitor cell differentiation and proliferation (Ogawa, 1993; Huang et al., 1999) and found that the gene expression of interleukin (IL)-3, IL-4, IL-5, IL-6, IL-9, and stem cell factor (SCF) was significantly downregulated by oxaliplatin treatment compared with that in the controls, whereas pre-treatment with PC-SOD significantly reversed this downregulation (IL-3: P = 0.004, IL-4: P = 0.020, IL-5: P = 0.003, IL-6: P = 0.020, IL-9: P = 0.002, SCF: P = 0.006) (Figure 2B). These results indicate that PC-SOD exerts a prophylactic effect against oxaliplatin-induced leukopenia.

Next, we used an in vivo imaging system to analyze ROS production in the leg bones of mice. A chemiluminescent reagent that reacts with ROS was subcutaneously administered to the mice 24 h after oxaliplatin administration, and both leg bones were removed 20 min later. In mice administered oxaliplatin, red-stained areas were detected in the central part of the femur and tibia, indicating areas of increased ROS production (Figure 3A). Pre-administration of PC-SOD markedly suppressed oxaliplatin-induced ROS production in the central part of the femur and tibia. ROS production was increased 2.5 ± 0.3-fold by oxaliplatin and reduced to 1.2 ± 0.3-fold by PC-SOD pre-treatment (P = 0.002) (Figure 3B). We further analyzed ROS production in bone marrow cells from mice in the control, oxaliplatin-treated, and PC-SOD + oxaliplatin groups. As shown in Figure 3C, bone marrow cells from the oxaliplatin-treated group showed marked ROS production, whereas bone marrow cells from mice pre-treated with PC-SOD followed by oxaliplatin treatment showed significantly reduced ROS production (P = 0.025). These results suggest that PC-SOD suppresses oxaliplatin-induced leukopenia by inhibiting ROS production in bone marrow cells.

Finally, we analyzed oxaliplatin-dependent toxicity and ROS production in an experimental cellular system using bone marrow cells from untreated healthy mice. We also analyzed whether PC-SOD directly inhibits oxaliplatin-dependent toxicity and ROS production in vitro. Bone marrow cells cultured for 24 h were treated with oxaliplatin (final concentration: 10–60 μmol/L), and cytotoxicity and ROS production were measured after another 24 h (Figures 4A,B). Oxaliplatin showed toxicity to bone marrow cells, and cell viability decreased in a concentration-dependent manner with oxaliplatin (Figure 4A). We also measured intracellular ROS production using H₂DCFDA and found a significant increase in intracellular ROS production that was dependent on the oxaliplatin concentration (Figure 4B).

Next, we examined the effect of PC-SOD pre-treatment on cytotoxicity and ROS production with 20 μmol/L of oxaliplatin. The oxaliplatin-induced reduction in cell viability was restored by PC-SOD pre-treatment, with significant recovery at 200 U/mL and 300 U/mL PC-SOD pre-treatment (200 U/mL: P = 0.001, 300 U/mL: P < 0.001) (Figure 5A). Oxaliplatin-induced intracellular ROS production was inhibited in a concentration-dependent manner by PC-SOD pre-treatment, with 300 U/mL PC-SOD pre-treatment showing a significant inhibitory effect (P = 0.001) (Figure 5B). These results suggest that PC-SOD exerts a direct inhibitory effect on oxaliplatin-induced cytotoxicity and ROS production in bone marrow cells.