Abstract
The consumption of red meat is probably carcinogenic to humans and is associated with an increased risk to develop colorectal cancer (CRC). Red meat contains high amounts of heme iron, which is thought to play a causal role in tumor formation. In this study, we investigated the genotoxic and cytotoxic effects of heme iron (i.e., hemin) versus inorganic iron in human colonic epithelial cells (HCEC), human CRC cell lines and murine intestinal organoids. Hemin catalyzed the formation of reactive oxygen species (ROS) and induced oxidative DNA damage as well as DNA strand breaks in both HCEC and CRC cells. In contrast, inorganic iron hardly affected ROS levels and only slightly increased DNA damage. Hemin, but not inorganic iron, caused cell death and reduced cell viability. This occurred preferentially in non-malignant HCEC, which was corroborated in intestinal organoids. Both hemin and inorganic iron were taken up into HCEC and CRC cells, however with differential kinetics and efficiency. Hemin caused stabilization and nuclear translocation of Nrf2, which induced heme oxygenase-1 (HO-1) and ferritin heavy chain (FtH). This was not observed after inorganic iron treatment. Chemical inhibition or genetic knockdown of HO-1 potentiated hemin-triggered ROS generation and oxidative DNA damage preferentially in HCEC. Furthermore, HO-1 abrogation strongly augmented the cytotoxic effects of hemin in HCEC, revealing its pivotal function in colonocytes and highlighting the toxicity of free intracellular heme iron. Taken together, this study demonstrated that hemin, but not inorganic iron, induces ROS and DNA damage, resulting in a preferential cytotoxicity in non-malignant intestinal epithelial cells. Importantly, HO-1 conferred protection against the detrimental effects of hemin.
Highlights
Heme is an important iron source in human nutrition and occurs mainly in food of animal origin such as meat[1]
In human colonic epithelial cells (HCEC), hemin caused a dose-dependent increase in reactive oxygen species (ROS) levels already 30 min after incubation, with 200 μM hemin resulting in a 25-fold induction as compared to control cells (Fig. 1a and Supplementary Fig. S1A)
We demostrated that hemin (≥20 μM) caused substantial ROS formation in a concentration-dependent manner in both HCEC and colorectal cancer (CRC) cells, whereas inorganic iron had only very little or no effects
Summary
Heme is an important iron source in human nutrition and occurs mainly in food of animal origin such as meat[1]. Heme b is the most abundant heme, consisting of a protoporphyrin ring with a central iron (Fe2+). As such, it is the prosthetic group of the oxygen transporter proteins. The dietary uptake of heme iron has attracted great attention due to its involvement in the etiology of colorectal cancer (CRC)[7]. Heme occurs abundantly in red meat[8], whose consumption has been classified as possibly carcinogenic to humans by the International Agency for Research on Cancer[9]. Several lines of evidence indicate that heme iron is the critical constituent of red meat driving colorectal tumorigenesis. The cellular resistance to heme triggered cytotoxicity was mediated by key enzymes responsible for 4-HNE detoxification[19] and by the activation of the transcription factor Nrf[220], which is known to be engaged by oxidative stress and electrophilic compounds[21]
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