Effect Of Hemin Research Articles

Heme oxygenase-1 alleviates advanced glycation end product-induced oxidative stress, inflammatory response and biological behavioral disorders in rat dermal fibroblasts.

Advanced glycation end products (AGEs) are involved in delaying the wound healing of diabetic foot ulcers. The present study investigated the effects of heme oxygenase-1 (HO-1) on oxidative stress, inflammatory insult and biological behaviors in rat dermal fibroblasts in the presence of AGEs. Rat dermal fibroblasts were cultured in the presence of AGEs (100 µg/ml), glucose (1.0 g/l or 4.5 g/l), hemin (5 µM) and chromium mesoporphyrin (CrMP; 20 µM). A bilirubin kit, reverse transcription-quantitative PCR and western blotting were used to measure the activity and mRNA and protein levels of HO-1, respectively. ELISA kits were used to measure the levels of reactive oxygen species (ROS), malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), TNF-α, IL-6, IL-1β and the viability and collagen (hydroxyproline) secretion of fibroblasts. Cell proliferation and apoptosis were measured via flow cytometry. The scratch test was performed to evaluate cell migration. The results revealed that AGEs resulted in oxidative stress, inflammatory response and biological behavioral disorders in fibroblasts, while worsened functional disorders were caused by the combination of AGEs and high-glucose treatment. Hemin treatment induced sustained high HO-1 expression, decreased the levels of ROS, MDA, 8-OHdG, TNF-α, IL-6, IL-1β and cell apoptosis, and increased cellular collagen synthesis, viability, proliferation and migration, whereas CrMP abolished the effects of hemin. It was observed that high HO-1 expression reversed the AGE-induced oxidative stress, inflammatory response and biological behavioral disorders in fibroblasts, but fibroblast function did not return to that observed under normal glucose levels. In conclusion, it was demonstrated that hemin treatment induced high HO-1 expression. HO-1 reduced the AGE-induced functional disorders in fibroblasts and may accelerate the healing of diabetic wounds by improving fibroblast biological behaviors and reducing the oxidative stress and inflammatory response.

The Interaction of Hemin, a Porphyrin Derivative, with the Purified Rat Brain 2-Oxoglutarate Carrier.

The mitochondrial 2-oxoglutarate carrier (OGC), isolated and purified from rat brain mitochondria, was reconstituted into proteoliposomes to study the interaction with hemin, a porphyrin derivative, which may result from the breakdown of heme-containing proteins and plays a key role in several metabolic pathways. By kinetic approaches, on the basis of the single binding centre gated pore mechanism, we analyzed the effect of hemin on the transport rate of OGC in uptake and efflux experiments in proteoliposomes reconstituted in the presence of the substrate 2-oxoglutarate. Overall, our experimental data fit the hypothesis that hemin operates a competitive inhibition in the 0.5–10 µM concentration range. As a consequence of the OGC inhibition, the malate/aspartate shuttle might be impaired, causing an alteration of mitochondrial function. Hence, considering that the metabolism of porphyrins implies both cytoplasmic and mitochondrial processes, OGC may participate in the regulation of porphyrin derivatives availability and the related metabolic pathways that depend on them (such as oxidative phosphorylation and apoptosis). For the sake of clarity, a simplified model based on induced-fit molecular docking supported the in vitro transport assays findings that hemin was as good as 2-oxoglutarate to bind the carrier by engaging specific ionic hydrogen bond interactions with a number of key residues known for participating in the similarly located mitochondrial carrier substrate binding site.

Photoenhanced Dual-Functional Nanomedicine for Promoting Wound Healing: Shifting Focus from Bacteria Eradication to Host Microenvironment Modulation.

Pathogenic bacterial infection has become a serious medical threat to global public health. Once the skin has serious defects, bacterial invasion and the following chain reactions will be a thorny clinical conundrum, which takes a long time to heal. Although various strategies have been used to eradicate bacteria, the treatment which can simultaneously disinfect and regulate the infection-related host responses is rarely reported. Herein, inspired by the host microenvironment, a photoenhanced dual-functional nanomedicine is constructed (Hemin@Phmg-TA-MSN) for localized bacterial ablation and host microenvironment modulation. The "NIR-triggered local microthermal therapy" and positively charged surface endow the nanomedicine with excellent bacterial capture and killing activities. Meanwhile, the nanomedicine exhibits broad-spectrum reactive oxygen species (ROS) scavenging activity via the synergistic effect of hemin and tannic acid with photoenhanced electron and hydrogen transfers. Furthermore, the in vivo experiments demonstrate that the dual-functional nanomedicine not only presents robust bacterial eradication capability, but also triggers the oxidative stress and inflammatory microenvironment regulation. The work not only shows a facile and effective way for infected wound management but also provides a new horizon for designing novel and efficient anti-infection therapy shifting focus from bacteria treatment to host microenvironment modulation.

Hemin-induced increase in saponin content contributes to the alleviation of osmotic and cold stress damage to Conyza blinii in a heme oxygenase 1-dependent manner.

Hemin can improve the stress resistance of plants through the heme oxygenase system. Additionally, substances contained in plants, such as secondary metabolites, can improve stress resistance. However, few studies have explored the effects of hemin on secondary metabolite content. Therefore, the effects of hemin on saponin synthesis and the mechanism of plant injury relief by hemin in Conyza blinii were investigated in this study. Hemin treatment promoted plant growth and increased the antioxidant enzyme activity and saponin content of C. blinii under osmotic stress and cold stress. Further study showed that hemin could provide sufficient precursors for saponin synthesis by improving the photosynthetic capacity of C. blinii and increasing the gene expression of key enzymes in the saponin synthesis pathway, thus increasing the saponin content. Moreover, the promotion effect of hemin on saponin synthesis is dependent on heme oxygenase-1 and can be reversed by the inhibitor Zn-protoporphyrin-IX (ZnPPIX). This study revealed that hemin can increase the saponin content of C. blinii and alleviate the damage caused by abiotic stress, and it also broadened the understanding of the relationship between hemin and secondary metabolites in plant abiotic stress relief.

Role of Heme in the Regulation of Transferrin Receptor Expression in Erythroid Cells

Physiologically, iron is transported between sites of absorption, storage, and use by the plasma glycoprotein transferrin (Tf). Most cells acquire iron via the binding of Tf to the Tf receptor (TfR) on the cell membrane. In non-erythroid cells, pivotal players in the regulation of TfR are the iron regulatory proteins, IRP1 and IRP2, which “sense” iron levels in the labile iron pool. IRPs control the synthesis of TfR by binding to iron-responsive elements (IREs) in the 3'UTR region of TfR mRNA. When cellular iron is scarce, IRPs bind to the IREs stabilizing the TfR mRNA, while excess iron prevents the binding of IRPs to IREs resulting in the rapid degradation of TfR mRNA.Developing red blood cells are the largest consumers of iron, which is delivered to them exclusively by Tf via TfR. Erythroid cells have by far the greatest need for iron required for the rapid and efficient synthesis of hemoglobin. Thus, it is not surprising that transcription plays a dominant role over the regulation via the IRE/IRP system in the control of TfR expression (Lok and Ponka (2000) J. Biol. Chem . 275: 24185-24190). The exceptionally high capacity of immature erythroid cells to synthesize heme is primarily a result of their unique control of iron metabolism as well as their distinct heme biosynthetic enzyme, erythroid-specific aminolevulinic acid synthase (ALA-S2). Here we provide evidence that TfR expression and the cellular uptake of iron from Tf is stimulated by enhanced heme synthesis. Incubation of murine eryhroleukemia cells with 5-aminolevulinic acid (ALA) increased TfR expression as well as iron incorporation into heme. This effect of ALA can be completely prevented by the inhibitors of heme biosynthesis (succinylacetone (blocks ALA dehydratase) or N-methylprotoporphyrin (blocks ferrochelatase)), indicating that the effect of ALA requires its metabolism to heme. The induction of TfR expression by ALA is mainly the result of increased mRNA synthesis, since the effect of ALA can be abolished by actinomycin D. Importantly, the TfR promoter is activated in vitro by addition of ALA and hemin to cultured murine erythroleukemia cells differentiated with DMSO. Direct-site specific mutagenesis of the erythroid active element1 in TfR promoter abolished the effects of ALA and hemin. These results suggest that heme may directly or indirectly interact with TfR promoter, consequently activating gene expression. Hence, our results indicate that in erythroid cells heme serves as a positive feedback regulator that maintains high TfR levels, thus ensuring appropriate iron availability for hemoglobin synthesis. DisclosuresNo relevant conflicts of interest to declare.

Topical application of ‘Hemin’ promotes wound healing in Streptozotocin-induced diabetic rats

Hemin may be of potential therapeutic value in wound healing management in diabetics. It is an inducer of heme oxygenase-1, an enzyme which degrades heme and participates in cellular protection against oxidative stress, inflammation and apoptosis. Thus, in the present study, hemin (0.5%) was applied topically over excision wounds, and its therapeutic effect in wound healing evaluated in diabetic rats. Topical hemin application significantly increased the percentage of wound contraction on day 2 in diabetic rats, however, povidone-iodine did the same on day 7 compared to the diabetic control. A significant increase in hydroxyproline and glucosamine content was found on day 14 in the hemin treated wounds of diabetic rats vs. the diabetic control. The histology of the hemin treated rats was in agreement with the cellular proliferation and collagen synthesis in granulation tissue. Hemin significantly increases cytokine IL-10 and decreases TNF-α in the granulation tissue of the healed wounds of diabetic rats. The finding showing the pro-healing effects of hemin was endorsed by inhibition of mRNA expression of pro-inflammatory cytokine TNF-α and adhesion molecule ICAM-1, and up-regulation of anti-inflammatory cytokine IL-10 mRNA. Hence, topical hemin application (i) helps in early and fast wound contraction (ii) enhances the hydroxyproline and glucosamine content of wounds and (iii) modulates pro-healing mRNA expression of cytokines.

Heme oxygenase-1 induction mitigates burn-associated early acute kidney injury via the TLR4 signaling pathway

ObjectivesEarly acute kidney injury (AKI) after burn contributes to disastrous prognoses for severely burned patients. Burn-induced renal oxidative stress and secondary proinflammatory mediator release contribute to early AKI development, and Toll-like receptor (TLR) 4 regulates inflammation. Heme oxygenase-1 (HO-1) is a stress-responsive enzyme that plays a vital role in protecting against ischemia-induced organ injury via its antioxidant properties and regulation of inflammation. We investigated the potential effect of HO-1 induction in preventing burn-induced early AKI and its related mechanism. MethodsA classic major-burn rat model was established using a 100 °C water bath, and hemin was injected intraperitoneally immediately after the injury to induce HO-1. Histological staining and blood tests were used to assess AKI progression based on structural changes and function. Renal levels of HO-1, oxidative stress, proinflammatory mediators and TLR4-related signals were detected using ELISA, immunostaining, qRT-PCR, and western blotting. The selective TLR4 inhibitor TAK242 and TLR4 inducer LPS were introduced to determine the roles of HO-1 in burn-related renal inflammation and the TLR4 pathway. ResultsHemin improved burn-induced renal histological damage and dysfunction, and this beneficial effect was related to reduced renal oxidative stress and the release of proinflammatory mediators, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6 and intracellular adhesion molecule-1 (ICAM-1). Hemin downregulated the expression of TLR4 and the subsequent phosphorylation of IKKα/β, IκBα, and NF-κB p65;. TAK242 exerted an effect similar to but weaker than hemin; and LPS reversed the antiinflammatory effect of hemin and the regulation of TLR4 signals. These results suggested that the TLR4 signaling pathway mediated the HO-1-facilitated regulation of renal inflammation after burn. ConclusionThe present study demonstrated that HO-1 induction prevented burn-induced early AKI by targeting renal inflammation, which was mediated via regulation of the TLR4/NF-κB signaling pathway.

Exogenous Hemin Confers Cadmium Tolerance by Decreasing Cadmium Accumulation and Modulating Water Status and Matter Accumulation in Maize Seedlings

Cadmium (Cd) contamination harms plant growth and human health. The application of hemin (ferroprotoporphyrin IX) can effectively relieve abiotic stresses in plants. This work investigates the effects of hemin on alleviating Cd toxicity and enhancing Cd tolerance in maize seedlings. In this study, maize seedlings were cultivated in nutrient solutions, with a combination of CdCl2 (464 μmol L−1) and hemin (100 μmol L−1). We measured plant growth status, water status, Cd concentration, and Cd distribution in maize seedlings. The results indicated that Cd stress increased Cd accumulation in plants and inhibited plant growth. However, hemin alleviated the growth inhibition and improved water balance, root morphology, and root vitality under Cd stress. Additionally, hemin increased 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), total phenolic content, and phenylalanine ammonia lyase (PAL) activity to enhance tolerance to Cd stress. Hemin reduced Cd concentration, the translocation factor (TF), and the bioconcentration factor (BCF) in maize seedlings under Cd stress. Furthermore, hemin increased Cd concentrations in the cell wall and the soluble fraction of seedling roots, which helped reduce Cd transport from root to shoot. In summary, exogenous hemin could be used for alleviating adverse impacts on maize seedling induced by Cd stress.

Heme effects of hemin on growth of peridontopathogens

Heme effects of hemin on growth of peridontopathogens

ROS-Dependent Induction of Antioxidant System and Heat Resistance of Wheat Seedlings by Hemin

Effect of 24-h-long treatment with hemin (a donor of carbon monoxide, CO, produced with the participation of heme oxygenase) on heat resistance and the processes of ROS formation and neutralization was investigated in wheat (Triticum aestivum L.) seedlings. It was found that treatment of seedlings with hemin in the concentration range from 0.5 to 10 µM induced an elevation of their resistance to damaging heating (45°C, 10 min). The greatest effect was produced by 5 µM hemin. Hemin treatment was associated with a transient elevation in the roots of extracellular peroxidase activity and a rise in the content of hydrogen peroxide. Elevation of H2O2 content was not observed in the presence of its scavenger dimethyl thiourea (DMTU) and upon treatment of seedlings’ roots with CO absorber hemoglobin and inhibitor of extracellular peroxidase sodium azide; however, this pattern was not followed in the presence of NADPH oxidase inhibitor imidazole. Since decomposition of hemin is associated with the production of redox active Fe2+ ions, the specificity of its action as a CO donor was tested by comparing the effects of hemin and 5 µM FeSO4. At the examined concentration, ferrous sulfate did not affect the generation of hydrogen peroxide in the roots and activity of extracellular peroxidase. In 24 h after the beginning of hemin treatment, activities of superoxide dismutase, catalase, and intracellular peroxidase in the roots rose. Elevation in the activity of antioxidant enzymes induced by hemin was not observed in the presence of DMTU, and treatment of seedling roots with FeSO4 did not modify their activity. Exposure to the CO donor after damaging heating also reduced outflux from the seedling roots of compounds absorbed in the ultraviolet spectral region. Positive influence of hemin on heat resistance of the seedlings was eliminated by CO scavenger hemoglobin and hydrogen peroxide absorber DMTU. Treatment of seedlings with FeSO4 did not alter their survival after heating. Hydrogen peroxide is assumed to participate as a signal mediator in realization of the influence of CO donor on the antioxidant system of wheat seedlings and their heat resistance.

Early Growth Response Protein 1 Knockdown Alleviates the Cerebral Injury in Rats with Intracerebral Hemorrhage via STAT3/NF-κB Pathway by Reducing RXRα Acetylation Level

Brain EGR1 (early growth response protein 1) overexpression aggravates focal ischemic brain injury, but its role in intracerebral hemorrhage (ICH) induced cerebral injury remains obscure. In this study, a rat ICH model was established by injecting type VII collagenase into the brain, and EGR1 knockdown reversed the increase of hematoma area, neurological function score, brain water content, blood–brain barrier (BBB) permeability, inflammation, p300 and retinoid a X receptor-α (RXRα) protein levels, as well as RXRα acetylation level induced by ICH. EGR1 expression was up-regulated in primary brain microvascular endothelial cells (BMECs), neurons, and astrocytes after ICH induction, and the up-regulation was most significant in BMECs. We also found that EGR1 promoted RXRα acetylation level by regulating p300 in BMECs. Silencing EGR1 rescued the upregulation of cell inflammation and the reduction of cell viability and TEER (transendothelial electric resistance) caused by OGD (oxygen glucose deprivation) plus hemin via p300-mediated RXRα acetylation. Furthermore, the STAT3/NF-κB pathway was activated after treatment with OGD plus hemin, which was suppressed by silencing EGR1. Treatment with Stattic (an inhibitor of STAT3) restrained the effect of OGD plus hemin on NF-κB pathway activity, inflammation, cell viability and TEER. In conclusion, EGR1 increased RXRα acetylation level by regulating p300, thereby aggravating brain damage in ICH rat model and dysfunction in BMECs, Through the STAT3/NF-κB pathway.

Heme oxygenase-1 inducer hemin does not inhibit SARS-CoV-2 virus infection

Antiviral agents with different mechanisms of action could induce synergistic effects against SARS-CoV-2 infection. Some reports suggest the therapeutic potential of the heme oxygenase-1 (HO-1) enzyme against virus infection. Given that hemin is a natural inducer of the HO-1 gene, the aim of this study was to develop an in vitro assay to analyze the antiviral potency of hemin against SARS-CoV-2 infection. A SARS-CoV-2 infectivity assay was conducted in Vero-E6 and Calu-3 epithelial cell lines. The antiviral effect of hemin, and chloroquine as a control, against SARS-CoV-2 virus infection was quantified by RT-qPCR using specific oligonucleotides for the N gene. Chloroquine induced a marked reduction of viral genome copies in kidney epithelial Vero-E6 cells but not in lung cancer Calu-3 cells. Hemin administration to the culture medium induced a high induction in the expression of the HO-1 gene that was stronger in Vero-E6 macaque-derived cells than in the human Calu-3 cell line. However, hemin treatment did not modify SARS-CoV-2 replication, as measured by viral genome quantification 48 h post-infection for Vero-E6 and 72 h post-infection for the Calu-3 lineages. In conclusion, although exposure to hemin induced strong HO-1 up-regulation, this effect was unable to inhibit or delay the progression of SARS-CoV-2 infection in two epithelial cell lines susceptible to infection.

Exogenous hemin improves Cd2+ tolerance and remediation potential in\xa0Vigna radiata\xa0by intensifying the HO-1 mediated antioxidant defence system

The present study evaluated the effects of exogenous hemin on cadmium toxicity in terms of metal accretion and stress resilience in Vigna radiata L. (Wilczek). One-week-old seedlings were treated with CdCl2 (50 μM) alone and in combination with hemin (0.5 mM) in half-strength Hoagland medium for 96 h. The optimum concentrations of Cd and hemin were determined on the basis of haem oxygenase-1 activity. The results demonstrated that under Cd stress, plants accumulated a considerable amount of metal in their tissues, and the accumulation was higher in roots than in leaves, which significantly reduced the plant biomass and chlorophyll content by increasing the oxidative stress (MDA and H2O2 content). However, hemin supplementation under Cd,-stress improved plant growth by enhancing the harvestable biomass and photosynthetic pigments, increasing antioxidant activities (SOD, APX, POD, HO-1 and proline), lowering oxidative damage and increasing Cd tolerance in plants. Furthermore, the application of hemin enhances the removal efficiency of Cd in V. radiata by increasing the uptake of Cd via roots and its translocation from roots to foliar tissues. Thus, the study suggests that hemin has the potential to improve the stress tolerance and phytoremediation ability of heavy metal-tolerant plants so that they can be used instead of hyperaccumulators for remediation of Cd-contaminated environments.

Effect of ascorbic acid and hemin on experimental colon carcinogenesis in mice

Colon cancer is known as the third most widespread cancer in the world. The interaction of heme-iron and ascorbic acid (AA) in colon carcinogenesis is not evident. Hemin (ferric chloride heme) is an iron-containing porphyrin with chlorine that can be formed from a heme group. The purpose of this study was to investigate the protective effect of AA on the formation of pre-neoplastic lesions induced by azoxymethane (AOM)/dextran sodium sulfate (DSS) plus hemin in mice. Forty-five ICR male mice were divided into three experimental groups; AOM/ DSS treatment (control group), hemin (2 g hemin/kg of b.w.), hemin + AA (1.0% in drinking water). The mice had three s.c. injections (0–2nd weeks of the experiment) of AOM (10 mg/kg b.w.) weekly and 2% DSS as drinking water for the next one week and the animals fed on AIN-76A purified rodent diet for 6 weeks. The numbers of aberrant crypt foci (ACF) and aberrant crypts (ACs) in colonic mucosa were counted after methylene blue staining. Lipid peroxidation in feces was measured by the thiobarbituric acid-reactive substances (TBARS) assay. The numbers of ACF and ACs per colon significantly increased in Hemin group compared to the control group. However, the numbers of ACF and ACs per colon notably decreased in hemin + AA group compared to the control group or hemin group (p<0.05). In feces, the TBARS value of hemin group was higher than the control group (p<0.01). The TBARS value of hemin + AA group was slightly decreased compared to Hemin group. These results indicate that hemin can promote the experimental colon carcinogenesis in ICR mice. On the other hand, additional supplement of AA via drinking water has a protective effect against the colon carcinogenesis. The related mechanisms need to be illustrated by further studies in future.

Hemin Prevents Increased Glycolysis in Macrophages upon Activation: Protection by Microbiota-Derived Metabolites of Polyphenols.

Meat consumption plays a critical role in the development of several types of cancer. Hemin, a metabolite of myoglobin produced after meat intake, has been demonstrated to be involved in the cancer initiation phase. Macrophages are key components of the innate immunity, which, upon activation, can prevent cancer development by eliminating neoplastic cells. Metabolic reprogramming, characterized by high glycolysis and low oxidative phosphorylation, is critical for macrophage activation. 3,4-dihydroxyphenylacetic acid (3,4DHPAA) and 4-hydroxyphenylacetic acid (4HPAA), both microbiota-derived metabolites of flavonoids, have not been extensively studied although they exert antioxidant properties. The aim of this study was to determine the effect of hemin on the anticancer properties of macrophages and the role of 3,4DHPAA and 4HPAA in metabolic reprogramming and activation of macrophages leading to the elimination of cancer cells. The results showed that hemin inhibited glycolysis, glycolytic, and pentose phosphate pathway (PPP) enzyme activities and hypoxia-inducible factor-1 alpha (HIF-1α) stabilization, which interferes with macrophage activation (evidenced by decreased interferon-γ-inducible protein 10 (IP-10) release) and their ability to eliminate cancer cells (via cytotoxic mediators and phagocytosis). Hemin also reduced the mitochondrial membrane potential (MMP) and mitochondrial mass in macrophages. 3,4DHPAA and 4HPAA, by stimulating glycolysis and PPP, prevented the impairment of the macrophage anticancer activity induced by hemin. In conclusion, 3,4HPAA and 4HPAA administration could represent a promising strategy for preventing the reduction of macrophage activation induced by hemin.

Effect of myoglobin, hemin, and ferric iron on quality of chicken breast meat.

ObjectiveThe objective was to evaluate the impact of different forms of iron including myoglobin, hemin, and ferric chloride on the quality of chicken breast meat.MethodsChicken breast muscles were subjected to 1, 2, 3 mg/mL of FeCl3, myoglobin and hemin treatment respectively, and the production of reactive oxygen species (ROS) and malondialdehyde, meat color, tenderness, water holding capacity and morphology of meat was evaluated.ResultsHemin was found to produce more ROS and induce greater extent of lipid oxidation than myoglobin and ferric chloride. However, it showed that hemin could significantly increase the redness and decrease the lightness of the muscle. Hemin was also shown to be prominent in improving water holding capacity of meat, maintaining a relatively higher level of the immobilized water from low-field nuclear magnetic resonance measurements. Morphology observation by hematoxylin-eosin staining further confirmed the results that hemin preserved the integrity of the muscle.ConclusionThe results indicated that hemin may have economic benefit for the industry based on its advantage in improving water holding capacity and quality of meat.

The Microbiota-Derived Metabolite of Quercetin, 3,4-Dihydroxyphenylacetic Acid Prevents Malignant Transformation and Mitochondrial Dysfunction Induced by Hemin in Colon Cancer and Normal Colon Epithelia Cell Lines.

Meat diet plays a pivotal role in colorectal cancer (CRC). Hemin, a metabolite of myoglobin, produced after meat intake, has been involved in CRC initiation. The compound, 3,4-dihydroxyphenylacetic acid (3,4HPAA) is a scarcely studied microbiota-derived metabolite of the flavonoid quercetin (QUE), which exert antioxidant properties. The aim of this study was to determine the protective effect of 3,4HPAA against malignant transformation (increased cell proliferation, decreased apoptosis, DNA oxidative damage and augmented reactive oxidative species (ROS) levels) and mitochondrial dysfunction induced by hemin in normal colon epithelial cells and colon cancer cells. The effect of 3,4HPAA was assessed in comparison to its precursor, QUE and to a known CRC protective agent, sulforaphane (SFN). The results showed that both, tumor and normal cells, exposed to hemin, presented increased cell proliferation, decreased caspase 3 activity and cytochrome c release, as well as augmented production of intracellular and mitochondrial ROS. In addition, hemin decreased the mitochondrial membrane potential (MMP) and the activity of complexes I and II of the electron transport chain. These effects of hemin were prevented by the action of 3,4HPAA. The metabolite showed to be more active than QUE and slightly less active than SFN. In conclusion, 3,4HPAA administration could represent a promising strategy for preventing malignant transformation and mitochondrial dysfunction in colon epithelia induced by hemin.

Heme oxygenase 1 protects human colonocytes against ROS formation, oxidative DNA damage and cytotoxicity induced by heme iron, but not inorganic iron

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.

Reactive Metamizole Metabolites Enhance the Toxicity of Hemin on the ATP Pool in HL60 Cells by Inhibition of Glycolysis.

Metamizole is an analgesic, whose pharmacological and toxicological properties are attributed to N-methyl-aminoantipyrine (MAA), its major metabolite. In the presence of heme iron, MAA forms reactive metabolites, which are toxic for granulocyte precursors. Since decreased cellular ATP is characteristic for MAA-associated toxicity, we studied the effect of MAA with and without hemin on energy metabolism of HL60 cells, a granulocyte precursor cell line. The combination MAA/hemin depleted the cellular ATP stronger than hemin alone, whereas MAA alone was not toxic. This decrease in cellular ATP was observed before plasma membrane integrity impairment. MAA/hemin and hemin did not affect the proton leak but increased the maximal oxygen consumption by HL60 cells. This effect was reversed by addition of the radical scavenger N-acetylcysteine. The mitochondrial copy number was not affected by MAA/hemin or hemin. Hemin increased mitochondrial superoxide generation, which was not accentuated by MAA. MAA decreased cellular ROS accumulation in the presence of hemin. In cells cultured in galactose (favoring mitochondrial ATP generation), MAA/hemin had less effect on the cellular ATP and plasma membrane integrity than in glucose. MAA/hemin impaired glycolysis more than hemin or MAA alone, and N-acetylcysteine blunted this effect of MAA/hemin. MAA/hemin decreased protein expression of pyruvate kinase more than hemin or MAA alone. In conclusion, cellular ATP depletion appears to be an important mechanism of MAA/hemin toxicity on HL60 cells. MAA itself is not toxic on HL60 cells up to 100 µM but boosts the inhibitory effect of hemin on glycolysis through the formation of reactive metabolites.

Protective Effect of Hemin Against Experimental Chronic Fatigue Syndrome in Mice: Possible Role of Neurotransmitters.

Chronic fatigue syndrome (CFS) is a disorder characterized by persistent and relapsing fatigue along with long-lasting and debilitating fatigue, myalgia, cognitive impairment, and many other common symptoms. The present study was conducted to explore the protective effect of hemin on CFS in experimental mice. Male albino mice were subjected to stress-induced CFS in a forced swimming test apparatus for 21days. After animals had been subjected to the forced swimming test, hemin (5 and 10mg/kg; i.p.) and hemin (10mg/kg) + tin(IV) protoporphyrin (SnPP), a hemeoxygenase-1 (HO-1) enzyme inhibitor, were administered daily for 21days. Various behavioral tests (immobility period, locomotor activity, grip strength, and anxiety) and estimations of biochemical parameters (lipid peroxidation, nitrite, and GSH), mitochondrial complex dysfunctions (complexes I and II), and neurotransmitters (dopamine, serotonin, and norepinephrine and their metabolites) were subsequently assessed. Animals exposed to 10min of forced swimming session for 21days showed a fatigue-like behavior (as increase in immobility period, decreased grip strength, and anxiety) and biochemical alteration observed by increased oxidative stress, mitochondrial dysfunction, and neurotransmitter level alteration. Treatment with hemin (5 and 10mg/kg) for 21days significantly improved the decreased immobility period, increased locomotor activity, and improved anxiety-like behavior, oxidative defense, mitochondrial complex dysfunction, and neurotransmitter level in the brain. Further, these observations were reversed by SnPP, suggesting that the antifatigue effect of hemin is HO-1 dependent. The present study highlights the protective role of hemin against experimental CFS-induced behavioral, biochemical, and neurotransmitter alterations.