Iron Storage Rather Than Serum Iron Levels Is an Important Contributor to Iron-Induced Oxidative Stress Levels in Mice
Authors: Noguchi-Sasaki Mariko , Sasaki Yusuke , Matsuo-Tezuka Yukari , Kurasawa Mitsue , Yorozu Keigo , Shimonaka Yasushi 
Published: October 2019

Abstract
Introduction:
Iron, an essential element for various biological processes, can induce oxidative stress. In iron overload diseases, cardiovascular events are associated with increased oxidative stress accompanied by elevated iron storage and serum iron. However, it has not been investigated whether it is iron storage or serum iron that is the most important contributor to oxidative stress levels, and the relationship between iron metabolism and oxidative stress is not clear. Moreover, no biomarker that can sensitively detect iron-induced oxidative stress has yet been reported. Therefore, we first investigated the sensitivities of several biomarkers to detect oxidative stress induced in mice by altering the amount of total body iron; then using the most sensitive marker, we investigated mechanisms underlying iron metabolism and oxidative stress by exploring the contributions of iron storage and serum iron levels to oxidative stress levels by modulating body iron status in mice.
Methods:
This study used 8-week-old male BALB/c mice. In the first part of the study, we investigated several oxidative stress markers in iron-loaded mice. Mice were intravenously administered iron-dextran (to load iron) or vehicle for 5 days. Nine days after the first injection, we measured serum oxidative stress markers (derivatives of reactive oxygen metabolites [d-ROMs] analyzed by measuring the total amount of hydroperoxides via the Fenton reaction, malondialdehyde [MDA], and hydroxynonenal [HNE]), serum hepcidin level, and hepatic iron content.
In the second part of the study, iron dynamics was modulated by 2 interventions: (1) Mice were intravenously administered 10 μg/kg of epoetin beta pegol (C.E.R.A.), a long-acting erythropoiesis-stimulating agent, or vehicle. Five days later, we determined hemoglobin, serum hepcidin level, serum iron level, hepatic iron content, and d-ROMs. (2) Mice were fed a ferric citrate diet containing 5000 ppm iron or a control diet containing 100 ppm iron for 28 days; we then evaluated serum iron level, hepatic iron content, and d-ROMs.
In the third part of the study, we also evaluated oxidative stress marker d-ROMs by modulating serum iron levels and iron storage independently by 3 interventions: (1) Mice intraperitoneally administered 200 μg/head of anti-erythropoietin antibody or control IgG every other day were analyzed 96 hours later. (2) Mice injected with 100 μg/head of synthetic hepcidin or vehicle were analyzed 4 hours later. (3) Mice intravenously administered iron-dextran (48 mg/kg) or vehicle once a day for 5 days were analyzed.
Results:
In the first part of the study, compared with the control group, iron-loaded mice exhibited dose-dependent increases in serum hepcidin level, hepatic iron content, and serum d-ROMs, whereas no change was observed in MDA or HNE.
In the second part of the study, hemoglobin level was significantly higher in C.E.R.A.-treated mice than in vehicle-treated mice. Serum hepcidin level, serum iron level, hepatic iron content, and d-ROMs were all significantly lower in C.E.R.A.-treated mice than in vehicle-treated mice. In mice fed the ferric citrate diet, serum iron level, hepatic iron content, and d-ROMs were all higher than in mice fed the control diet.
In the third part of the study, in mice given anti-erythropoietin antibody, serum iron level was elevated, but hepatic iron content was not changed resulted from iron overflow with inhibition of erythropoiesis, and d-ROMs was not changed. Mice given synthetic hepcidin showed decreased serum iron level, but no significant changes were detected in hepatic iron content or d-ROMs. In mice given iron-dextran, no significant change in serum iron level was observed; however, hepatic iron content and d-ROMs levels increased compared to control mice.
Conclusions:
We demonstrated that d-ROMs was a sensitive marker of iron-induced oxidative stress. Modulating body iron status by several interventions, we demonstrated that iron storage, rather than serum iron levels, contributed to the level of oxidative stress marker. The results of our C.E.R.A. treatment study suggest a new rationale for treatment with C.E.R.A.: that enhancement of iron metabolism by C.E.R.A., leading to a decrease in oxidative stress by reducing iron storage, contributes to tissue protective properties. C.E.R.A. may have beneficial implications for improving prognosis by correcting oxidative stress-related disorders.

Disclosures
Noguchi-Sasaki: Chugai Pharmaceutical Co., Ltd.: Employment. Sasaki:Chugai Pharmaceutical Co., Ltd.: Employment. Matsuo-Tezuka:Chugai Pharmaceutical Co., Ltd.: Employment. Kurasawa:Chugai Pharmaceutical Co., Ltd.: Employment. Yorozu:Chugai Pharmaceutical Co., Ltd.: Employment. Shimonaka:Chugai Pharmaceutical Co., Ltd.: Employment.