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. 2019 May 22;8(4):544–551. doi: 10.1039/c9tx00070d

Photoactive CO-releasing complexes containing iron – genotoxicity and ability in HO-1 gene induction in HL-60 cells

Daniel Wysokiński a, Patrycja Lewandowska a, Daria Zątak a, Michał Juszczak a, Magdalena Kluska a, Daria Lizińska b, Bogna Rudolf b, Katarzyna Woźniak a,✉
PMCID: PMC6621133  PMID: 31367337

graphic file with name c9tx00070d-ga.jpgThis paper presents the results of research on the biological properties of two photoactive CO-releasing molecules containing iron, i.e. (η5-C5H5)Fe(CO)2(η1-N-maleimidato) (complex A) and (η5-C5H5)Fe(CO)2(η1-N-succinimidato) (complex B).

Abstract

This paper presents the results of research on the biological properties of two photoactive CO-releasing molecules containing iron, i.e. (η5-C5H5)Fe(CO)2(η1-N-maleimidato) (complex A) and (η5-C5H5)Fe(CO)2(η1-N-succinimidato) (complex B). We studied their cytotoxicity, genotoxicity and the ability of inducing the HO-1 gene in HL-60 cells. We also investigated the kinetics of DNA damage repair induced by complexes A and B. We demonstrated that complex B was not toxic to HL-60 cells in high doses (above 100 μM). The ability to induce DNA damage was higher for complex A. Importantly, there was no difference in irradiated and non-irradiated cells for both complexes. DNA damage induced by complex B was repaired efficiently, while the repair of DNA damage induced by complex A was disturbed. Complex B had a minor effect on HO-1 gene expression (less than 2-fold induction), while complex A had induced HO-1 gene expression to a great extent (over 17-fold for 10 μM) – similarly in irradiated and non-irradiated HL-60 cells. The results of our research indicate that the ability of both complexes to damage DNA and to upregulate HO-1 gene expression is not related to the release of CO. Further research is needed to test whether these compounds can be considered as potential CO carriers in humans.

1. Introduction

Carbon monoxide (CO) is a gas commonly present in the environment. Exogenous CO is mostly produced during incomplete combustion of carbon-containing organic materials.1 It is a toxic agent in high doses and CO poisoning is responsible for more than 50% of all fatal poisoning cases.2,3 Its toxicity is displayed primarily through binding to hemoglobin with 230 times stronger affinity than oxygen, thus preventing oxygen transport in the body and causing acute hypoxia.4,5 The brain and heart are the first organs affected by acute CO poisoning.6,7 At the cellular level, high CO doses are cytotoxic, partially due to mitochondrial function disruption. It has been shown that CO blocks cellular respiration, inhibits cytochrome c oxidase and diminishes oxidative phosphorylation in cells.8–10

Besides the toxic nature of exogenous CO in high doses, it is also endogenously produced in organisms. The major endogenous pool of CO is generated by heme oxygenases (HO-1/2/3) in a heme catabolism reaction, generating ferrous iron, CO and biliverdin, which is next reduced to bilirubin.11 It is currently known that CO plays numerous roles in the human body. Like another endogenously produced gas, i.e. nitric oxide (NO), CO acts as a signaling molecule. In the same way as NO, carbon monoxide relaxes smooth muscles and lowers blood pressure.12 Moreover, there is a crosstalk between CO and NO signaling. CO is capable of activating nitric oxide synthase (NOS) to stimulate NO generation, while NO influences HO-1 and HO-2 expression.13 CO has some cytoprotective functions in different tissues. It activates the p38 MAPK kinase pathway14 and regulates nuclear hormone receptors and a number of ion channels.15 It seems that CO ensures cytoprotection through mitochondria. It decreases mitochondrial membrane permeabilization and limits the release of pro-apoptotic factors, as well as regulates the uptake of Ca2+ ions into mitochondria.16 CO may regulate the expression of some genes, e.g. by means of binding to transcription factors; it has been presented that CO binds with the NPAS2 transcription factor – a hemoprotein involved in circadian clock regulation.17 Furthermore, it has been shown that CO may have anti-hypertensive and anti-inflammatory properties18 as well as may inhibit blood platelet activation on the level of the entire organism.17 It may also display a therapeutic potential in some neurological pathologies, such as ischemic stroke or multiple sclerosis.19

According to numerous reports, the CO/HO-1 system seems to be important in cancer research and may be a target in new cancer treatment strategies. Under many conditions, the HO-1 fraction translocates to the nucleus and leads to the upregulation of HO-1 gene expression. Such an effect is observed in a number of different types of malignant cells in, for example, lung and prostate cancers.22 The HO-1/CO system has been shown to influence apoptosis and migration of tumor cells, immunosuppression, angiogenesis and synthesis of growth factors in cancerous cells, reviewed in ref. 25–27. Nowadays, numerous new CO-releasing carriers have been developed to check its potential therapeutic role in different pathological conditions, including cancer treatment, and various promising results have been reported.28–32

One of the most promising methods of triggering CO release from metallocarbonyl complexes is via photoexcitation. Photoinduced release of CO enables the control of location, timing and dosage of CO available to the target tissue. The main method used to monitor CO release in a myoglobin assay is based on the conversion of deoxy-myoglobin (deoxy-Mb) into the myoglobin-CO complex (MbCO) by UV-vis spectroscopic analysis.33 Recent studies have shown that complexes bearing imidato ligands are photoactive CO-releasing molecules.34 The (η5-C5H5)M(CO)n(η1-N-imidato) complexes (M = Fe, Mo, W; n = 2, 3) released CO under illumination with visible light. The mechanism of CO loss upon illumination has been investigated and examined using several methods, including myoglobin assay, LC/QTOF, IR and 1H NMR. Performed analyses reveal that CO release is due to the total decomposition of these complexes.34

This paper provides data from a study of two photoactive CO-releasing molecules containing iron, i.e. (η5-C5H5)Fe(CO)2(η1-N-maleimidato) (complexA) and (η5-C5H5)Fe(CO)2(η1-N-succinimidato) (complexB). We studied the DNA-damaging potential of these complexes in HL-60 cells using single-cell gel electrophoresis (comet assay). The kinetics of DNA damage repair was also measured. Moreover, we examined the ability of complexes A and B to induce the HO-1 gene in HL-60 cells.

2. Materials and methods

2.1. Chemicals

Complexes (η5-C5H5)Fe(CO)2(η1-N-maleimidato) (complexA) and (η5-C5H5)Fe(CO)2(η1-N-succinimidato) (complexB) (Fig. 1) were synthesized as previously described.35,36 Both complexes were dissolved in PBS. IMDM and fetal bovine serum (FBS) were obtained from Cytogen (Germany). Low-melting-point (LMP) and normal-melting-point (NMP) agarose, phosphate buffered saline (PBS), DAPI (4′,6-diamidino-2-phenylindole), dimethyl sulfoxide (DMSO) and hydrogen peroxide (H2O2) were acquired from Sigma-Aldrich (USA). All other chemicals were of the highest commercial grade available.

Fig. 1. Chemical structures of complex A (η5-C5H5)Fe(CO)2(η1-N-maleimidato) and complex B (η5-C5H5)Fe(CO)2(η1-N-succinimidato).

Fig. 1

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2.2. Cell culture

The HL-60 (human promyelocytic leukemia) cell line was obtained from the American Type Culture Collection (ATCC) and cultured in IMDM with 15% fetal bovine serum, 2 mM l-glutamine and 0.5% penicillin–streptomycin. The HL-60 cells were cultured in flasks at 37 °C in 5% CO2 and sub-cultured every 3–4 days to maintain exponential growth.

2.3. Cell treatment

The HL-60 cells were plated in 96 well plates with the seeding density of 1 × 105 cells per well and incubated in Iscove's Modified Dulbecco's Medium (IMDM) with 15% FBS at room temperature (25 °C) with complex A and complex B at the concentrations ranging from 0.5 μM to 500 μM for 67 min and 48 min, respectively. The incubation time was equal to half-lives of the conversion of deoxy-Mb to Mb-CO after addition of complexes A or B and irradiation with visible light.34 The cells were irradiated with light in the wavelength range of 450 to 650 nm by using the Q·Light Pro Unit lamp. The distance from the light source was 20 cm.

The viability of the HL-60 cells was determined by the trypan blue exclusion assay. An equal volume of 0.4% trypan blue reagent was added to the cell suspension and the percentage of viable cells was evaluated with a TC20 cell counter (Bio-Rad).

2.4. DNA damage

Complexes A and B were added to the suspension of the cells with an intention to ensure final concentrations of 0.5, 1, 5, 10, 50 and 100 μM. The cells were incubated and irradiated under the conditions described above. The experiment included a positive control, i.e. a sample of cells incubated with hydrogen peroxide (H2O2) at 20 μM for 15 min on ice. After treatment and irradiation with complexes A and B, the cells were washed and resuspended in IMDM with 15% FBS. A freshly prepared suspension of the cells in LMP agarose dissolved in PBS was spread onto microscope slides.

The comet assay was performed under alkaline conditions according to the procedure worked out by Singh et al. (1988)37 as described previously by Blasiak and Kowalik (2000).38 A freshly prepared suspension of cells in 0.75% LMP agarose dissolved in PBS was layered onto microscope slides (Superior, Germany), which were pre-coated with 0.5% NMP agarose. Then, the cells were lysed for 1 h at 4 °C in a buffer containing 2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, 1% Triton X-100, pH = 10. After cell lysis, the slides were placed in an electrophoresis unit. DNA was allowed to unwind for 20 min in the solution containing 300 mM NaOH and 1 mM EDTA (pH > 13).

Electrophoretic separation was carried out in the solution containing 30 mM NaOH and 1 mM EDTA (pH > 13) at an ambient temperature of 4 °C (the temperature of the running buffer did not exceed 12 °C) for 20 min at an electric field strength of 0.73 V cm–1 (28 mA). Then, the slides were washed in water, drained, stained with 2 μg ml–1 DAPI and covered with cover slips. In order to prevent additional DNA damage, the procedure described above was conducted under limited light or in the dark.

2.5. DNA repair

The cells incubated with complexes A and B at 5 μM were washed and resuspended in fresh IMDM with 15% FBS preheated to 37 °C. Aliquots of the suspension were taken immediately (“time zero”), as well as 30 min, 60 min, 90 min and 120 min later. The samples were placed in an ice bath to stop DNA repair. Next, the preparation of the samples was conducted as described above. DNA repair was assessed by the extent of residual DNA damage detection at each time-point using the comet assay.

2.6. Comet analysis

The comets were observed at 200× magnification in an Eclipse fluorescence microscope (Nikon, Japan) attached to a COHU 4910 video camera (Cohu, Inc., San Diego, CA, USA) equipped with a UV-1 A filter block and connected to a personal computer-based image analysis system Lucia-Comet v. 6.0 (Laboratory Imaging, Prague, Czech Republic). Fifty images (comets) were randomly selected from each sample and the mean value of DNA in the comet tail was taken as an index of DNA damage (expressed in percent).

2.7. HO-1 gene expression analysis

Total RNA was extracted from each sample using the Universal RNA Purification Kit (EurX, Gdansk, Poland). The RNA concentration and purity were measured by BioTek Synergy HT Microplate Reader (BioTek Instruments, Winooski, VT, USA). The isolated RNA was stored at –20 °C until further steps. 90 ng of total RNA from each sample was used for RT-qPCR reaction. Reverse transcription and real-time PCR reaction were performed using the SensiFAST™ Probe No-ROX One-Step Kit (Bioline, London, UK) on the CFX96 C1000 real-time system (Bio-Rad, Hercules, CA, USA). The relative HO-1 expression was evaluated using the TaqMan gene expression assay (ThermoFisher Scientific, Waltham, MA, USA). The analysis was performed in triplicate. The GAPDH gene was utilized as a reference gene. Relative gene expression was calculated as fold-change according to the control sample based on the double delta Ct method.

2.8. Data analysis

The values of the comet assay in this study were expressed as mean ± S.E.M. from three experiments, i.e., data from three separate experiments were pooled and the statistical parameters were calculated. The values of the cell viability experiments were presented as mean ± S.D. from three experiments. The Mann–Whitney U-test was used during data analysis of the comet assay to determine the statistical differences between the samples with the abnormal distribution (Kolmogorov–Smirnov test). The differences between the samples with the normal distribution were evaluated by applying the Student's two-tailed t-test.

HO-1 gene expression was calculated by double delta Ct. Statistics were performed using Student's two-tailed t test. Data were presented as a mean ± SD, relative to control. HO-1 expression was normalized to GAPDH (as a reference gene).

3. Results and discussion

Based on the results obtained, we found that complex A evoked a concentration-dependent decrease in the viability of HL-60 cells – both irradiated and non-irradiated (Fig. 2A). At the maximum concentration applied, i.e. 500 μM, the viability was 39.3% ± 12.6 (p < 0.001) in the irradiated cells and 41% ± 9.6 (p < 0.001) in the non-irradiated cells. In the case of complex B, we did not observe any changes in the viability of both irradiated and non-irradiated HL-60 cells, in comparison to the cells that were not incubated with complex B (Fig. 2B).

Fig. 2. Effect of complex A (A) and complex B (B) on the viability of HL-60 cells measured by the trypan blue exclusion method. The figure shows mean results from three independent experiments. Error bars denote S.D.; *** p < 0.001 compared with non-treated cells.

Fig. 2

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We used single cell gel electrophoresis (comet assay) in the alkaline version for the analysis of the DNA-damaging potential of complexes A and B. In this assay, the cells are immersed in low melting point agarose (LMP), placed on microscopic slides, and then lysed. As a result, released DNA is subject to electrophoresis under alkaline conditions (pH > 13). The comet assay in the alkaline version enables the identification of DNA single strand breaks (SSBs) and DNA double strand breaks (DSBs), as well as alkali labile sites (ALSs). Fig. 3A shows the level of DNA damage, including SSBs and DSBs, as well as ALSs, induced by complex A in irradiated and non-irradiated HL-60 cells. We detected a statistically significant increase in DNA damage in irradiated and non-irradiated cells after incubation with this complex at all concentrations used (0.5, 1, 5, 10, 50 and 100 μM). We did not observe significant differences in the level of DNA damage in irradiated and non-irradiated cells. Fig. 3B shows DNA damage induced by complex B in irradiated and non-irradiated HL-60 cells. We showed an increase in DNA damage, but it was statistically significant (p < 0.001) only in the HL-60 cells incubated with complex B at the two highest concentrations, i.e. 50 and 100 μM. As in the case of complex A, we did not observe any significant changes in the level of DNA damage in irradiated and non-irradiated cells.

Fig. 3. DNA damage, measured as the comet tail DNA of HL-60 cells incubated with complex A (A) and complex B (B), and analyzed by the comet assay. The number of cells scored in each treatment was 50. The figure shows mean results from three independent experiments. Error bars denote S.E.M.; ** p < 0.01, *** p < 0.001 compared with non-treated cells. The concentration axis is not linear and distances between points are chosen arbitrarily.

Fig. 3

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Since no significant differences were observed in the level of DNA damage in irradiated and non-irradiated cells for complexes A and B, we decided to investigate the kinetics of DNA damage repair only in non-irradiated cells. Fig. 4 shows DNA damage in the HL-60 cells incubated with complex A and complex B, respectively, at 5 μM immediately after incubation and after 30, 60 and 120 min of repair incubation. In the case of complex A, we did not observe any effective repair of DNA damage induced by this complex (Fig. 4A). After 120 min of repair incubation, there was a statistically significant difference (p < 0.001) in the level of damage between the cells incubated with complex A compared to the cells non-incubated with this complex. The efficiency of DNA damage repair induced by H2O2 (positive control) was 42%. DNA damage induced by complex B was effectively repaired during 120 min of incubation (Fig. 4B). In this experiment, the efficiency of repair of DNA damage induced by H2O2 (positive control) was 36%.

Fig. 4. Time course of the repair of DNA damage, measured as the comet tail DNA in the comet assay in HL-60 incubated with complex A (A) and complex B (B) at 5 μM. Error bars denote S.E.M.; *** p < 0.001 compared with “time 0”; ### p < 0.001 compared with non-treated cells after 120 min post-incubation. The number of cells scored and number of experiment repeats are the same as in Fig. 3.

Fig. 4

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Viability testing showed that complex B was not toxic to HL-60 cells in a broad concentration range, while complex A decreased cell viability but only in high doses – above 100 μM. However, the testing showed differences in the toxicity of both compounds, as complex A in 500 μM concentration decreased cell viability to about 40% (Fig. 2A). The ability of DNA damage induction by both complexes, evaluated by the comet assay, was similar and dose-dependent for complexes A and B, although the extent of DNA damage was higher for complex A (Fig. 3). Importantly, there was no difference in the irradiated and non-irradiated conditions for both complexes, indicating that the mechanism of toxicity and DNA damage induction was independent of carbon monoxide release. It is probable that these properties are linked with metal-induced ROS generation, especially in the case of complex A. The activity of complex A containing the maleimidato ligand could be also explained by the presence of the maleimide double bond in the structure. Maleimide derivatives are useful Michael acceptors which react easily with different heteronucleophiles like thiols and amino groups present in biomolecules. Similarly, the efficacy of DNA damage repair evaluated by the comet assay differed between both complexes. While DNA damage induced by complex B was efficiently repaired, in the case of complex A the process of repair was disturbed (Fig. 4). Some may speculate that, similarly as in the case of DNA damage generation and cytotoxic properties, the core metal of the decayed complex may generate ROS and interfere with cellular homeostasis, including the DNA repair mechanism.

There are three isoforms of heme oxygenase in humans: HO-1, HO-2 and HO-3. HO-2 and HO-3 are constitutive, while HO-1 is an inducible form and a high expression level of this gene is observed in tissues of, for example, the spleen and liver. Based on numerous reports, HO-1 seems to display a strong cytoprotective and therapeutic potential.20 HO-1 is also inducible in response to a number of factors.21 HO-1 is induced under many conditions. The HO-1 level increases in response to conditions such as hypoxia, oxidative stress, cytokines and exposure to heavy metals.22 Its level in lung epithelial cells is increased when exposed to lipopolysaccharide or cigarette smoke.23 Furthermore, some natural compounds extracted from plants, like curcumin and carnosol, are capable of HO-1 induction.24 It is observed that a fraction of HO-1 is translocated to mitochondria and this action protects cells from mitochondria-dependent cell death.25

HO-1 overexpression is observed in different types of tumors such as pancreatic, prostate and bladder cancers, Kaposi sarcoma, hepatoma and melanoma.39–41 It has been demonstrated that HO-1 expression correlates with cancer growth and is crucial for the success of therapy.42 Lin et al.43 have shown that CORM-3 induces HO-1 expression and promoter activity in RBA-1 cells. In other studies conducted by these researchers, the findings suggest that CORM-2 induces HO-1 expression in various cell types such as RBA-1 cells,44 human tracheal smooth muscle cells,45 and human cardiomyocytes.46

HO-1 gene was slightly yet significantly upregulated in the HL-60 cells incubated with complex B at concentrations of 10 and 50 μM, but only in the non-irradiated cells (1.67- and 1.39-fold as compared to control, respectively) (Fig. 5B). No significant results were observed for complex A in the cells incubated at two of the lowest concentrations (0.5 and 1 μM) (Fig. 5A). However, complex A at 5 μM resulted in almost two-fold induction of HO-1 gene in the non-irradiated cells, while the concentration of 10 μM caused 17.6- and 14-fold induction of the HO-1 gene, respectively, in the irradiated and non-irradiated cells (Fig. 5A). A high level of HO-1 gene expression was also observed in the non-irradiated cells incubated with complex A at 50 μM (5.7-fold induction), while the cells incubated with complex A at 100 μM induced only a 1.6-fold increase in irradiated samples (Fig. 5A).

Fig. 5. Relative expression of HO-1 gene in HL-60 cells incubated with complex A (A) and complex B (B), presented as a fold-change in accordance to control (double delta Ct method). Data were normalized to the GAPDH gene as a reference. Columns represent mean values ± S.D.; * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig. 5

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We have shown that the two investigated complexes induce HO-1 gene expression, although the scale of induction differs strongly among them. HO-1 is highly inducible in response to numerous factors, such as thrombin47 or LPS.48 Complex B, on the other hand, has a minor effect on HO-1 gene expression (less than 2-fold induction). Complex A, to a similar extent in irradiated and non-irradiated cells, greatly induces HO-1 expression – even over 17-fold for 10 μM in non-irradiated cells (Fig. 5). Importantly, the peak of HO-1 gene induction is in the middle of the complex A concentration range.

Further research is needed to establish the mechanism of cyto- and genotoxicity of complex B and, in particular, of complex A, and whether these compounds can be considered as potential CO carriers in humans. Perhaps there is a relationship between the ability of complex A to induce DNA damage, non-effective DNA repair and HO-1 gene expression. It would be also interesting to check the combined effect of complexes A or B together with heme oxygenase inhibitors or with the application of HO-1-depleted cells.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgments

This research was financed from a student research grant of the University of Lodz, given to Daria Zątak. This work was supported financially by the University of Lodz, Poland, the Faculty of Chemistry and the Faculty of Biology and Environmental Protection. We wish to express our gratitude to Professor Maria Bryszewska from the Department of General Biophysics, University of Lodz, for making the Q·Light Pro Unit lamp available.

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