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. 2021 Jan 5;10(1):11–17. doi: 10.1093/toxres/tfaa091

4-Hydroxyhalcone effects on cisplatin-induced genotoxicity model

Aref Nazari 1, Mina Mirian 2, Mahmoud Aghaei 3, Mehdi Aliomrani 4,✉
PMCID: PMC7885192  PMID: 33613968

Abstract

Background

The genotoxicity of cisplatin (CP) as a platinum-based antineoplastic agent due to its oxidative stress induction was well known. In this research, we examined 4-hydroxychalcone (4-HCH) as a natural food that presents flavonoid effects on reactive oxygen species (ROS) production and CP-induced in vivo genotoxicity.

Method and materials

Cytotoxicity of CP and 4-HCH was measured on human embryonic kidney 293 cells with MTT assay. Then, intracellular ROS content at IC50 concentration of CP was measured with 2′,7′-dichlorofluorescein diacetate (DCFDA) dye. Finally, 4-HCH was administered intraperitoneally at 10 and 40 mg/kg/BW doses as a pre and post-treatment schedule in a mice model of CP genotoxicity (7 mg/kg). Acridine-orange-stained bone marrow cells were quantified for micronucleus presence examination.

Results

The calculated IC50 of CP and 4-HCH were reported around 19.4 and 133.6 μM, respectively, on HEK293 cells. Also, it was observed that 4-HCH at 0.2, 2 and 10 μM concentrations did not show obvious cytotoxicity. The fluorimetry confirmed that pre-treatment with 10 μM and co-treatment with 2 μM of 4-HCH could attenuate the CP-induced ROS production (P < 0.05 and P < 0.01, respectively). Also, the lowest micronucleated cells were seen in 10 mg/kg 4-HCH-treated group after CP exposure (39 ± 7.9, P < 0.0001).

Discussion

Our results demonstrated the antigenotoxic action of 4-HCH in CP-treated mice bone marrow cells for the first time in both concentrations of 10 and 40 mg/kg especially in the form of co-treatment. Further studies required clinical application of this compound in a combination of CP to attenuate the normal cells’ genotoxicity side effects.

Keywords: 4-hydroxychalcone, cisplatin, micronucleus test, oxidative stress, DCFDA

Introduction

cis-Diamminedichloroplatinum(II) (CP), the first platinum compound approved by FDA in 1978, is one of the most potent agents available for cancer chemotherapy [1]. It is used in various types of cancer treatment regimen including head and neck, cervical, breast, lung, ovarian, gastric and bladder with around 90% cure rate [2]. However, using the platinum-based agents is often associated with several toxic side effects, including, hepatotoxicity [3], cardiotoxicity [4]and more importantly nephrotoxicity [5].

The molecular mechanism of CP (CP) toxicity is associated with its biological activation by the sequential thermal exchange of two chloride groups with hydroxyl ligands [6]. After activation, CP that acts as a potent nucleophilic agent, which attacks DNA targets leading to the formation of inter/intra cross-links [7], protein adducts and strand breaks, [8] resulted in inhibition of replication, transcription and cell death [9,10].

On the other hand, oxidative stress is also involved in CP-induced genotoxicity. It is capable of generating reactive oxygen species (ROS) such as superoxide and hydroxyl radicals [11] and suppressed antioxidant defensive system including enzymes, non-enzymatic molecules and reduced glutathione [12,13]. Several normal tissue malignancies have been reported based on the broad mechanism of action and genotoxic activity of this antitumor drug.

Chalcones are naturally presented aromatic ketones with an α,β-unsaturated carbonyl group between aryl rings [14]. This class of open-chain flavonoids is present in grains, roots, fruits, tea, vegetables, flowers and wines, which are routinely used [15]. These compounds have been suggested to have a wide range of pharmacological effects, such as antiosteoporosis, anticonvulsant, anti-inflammatory, antimalarial, antimicrobial, larvicidal, antimutagenic and anticancer with a tyrosinase inhibitory activity [16–21]. In addition, the potent antioxidant activity due to the presence of the reactive keto-vinylene group in their structure has also been shown in [22].

Hydroxychalcones are compounds with two benzene or phenol aromatic rings linking to an unsaturated side chain. In the past decades, a large number of reports have been published on the beneficial effects of hydroxy chalcones especially due to their antioxidant and antitumor activities [23,24]. 4-HCH is an alpha, beta-unsaturated ketone with the core structure of chalcone and one hydroxyl substituent on the four positions of the A ring. Also, it can inhibit the angiogenesis by affecting vascular endothelial growth factor and basic fibroblast growth factor intracellular signaling pathways [25]. These agents can act as chemopreventive and chemotherapeutic drugs with several different mechanisms [23,24,26].

The aim of the present study was to investigate the administration effects of 4-hydroxy chalcone on CP-induced genotoxicity. For this purpose, we performed the micronucleus assay in mice treated with CP and 4-hydroxy chalcone bone marrow cells.

Material and Methods

Chemicals

4-Hydroxy chalcone was purchased from Indofine (Hillsborough, NJ, USA). CM-H2DCFDA was supplied from Invitrogen (Waltham, Massachusetts, USA), CP and all the other commercially available reagents were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

Cell culture

Human embryonic kidney 293 cells or HEK293 cells were obtained from the National Cell Bank of Iran (Pasteur Institute, Tehran, Iran) and were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and antibiotics (100 μg/ml streptomycin, 100 U/ml penicillin G) at 37°C in a humidity-controlled incubator with 5% CO2. The cells were subcultured at 50–80% confluences using 0.05% trypsin-0.02% EDTA [27].

Cell survival

This assay is based on a direct relationship between the viable cells and absorbance provided by the enzymatic reduction phenomenon of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye. The effect of CP and 4-hydroxy chalcone on cell viability was evaluated by MTT assay as performed earlier [28]. Selective doses viz. 0.2, 2, 10, 20, 30, 50, 100 and 200 μM of CP and 4-hydroxy chalcone were prepared in the final volume of 100 μL media. After 48 h of exposure, 10 μL of MTT solution (5 mg/ml stock solution) was added in each well and re-incubated for 3 h at 37°C until formazan blue crystal developed. Media were discarded from each well, and 100 μL of DMSO was added to dissolve formazan crystals for 10 min at 37°C. The absorbance was recorded at 540 nm by ELISA microplate reader and relative percentage cell viability was calculated.

ROS measurement

Cytosolic reactive oxygen species (ROS) was evaluated using the 2′,7′-dichlorofluorescein diacetate (DCFDA) dye. The cells in positive-control wells (only CP treatment), pre-treatment (exposed to the safe dose of 4-HCH 18 h, before CP exposure) and co-treatment (co-exposure to CP and 4-HCH) were washed after 60 min, incubated with DCFDA for 30 minute and processed as shown in Fig. 1. The fluorescent intensity was analyzed using the Synergy HT Multi-Mode Microplate Reader (BioTek Instruments, USA) at 485 nm excitation and 528 nm emission. Finally, the relative florescence unit (CFI) was calculated based on the unstained cells and positive-control florescent emission [29,30].

Figure 1.

Figure 1

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Graphical abstract representing the cells preparation for fluorescent detection of intracellular reactive oxygen species using the 2′,7′-dichlorofluorescein diacetate (DCFDA) dye. CP (CP) and hydrogen peroxide were chosen as a positive control and HEK293 cells were pre- and post-treated with 0.2, 2 and 10 μM of 4-HCH.

Animal exposure

Healthy, young male adult (8–10 weeks) Swiss mice, weighing 18–22 g, obtained from the animal facilities of the Isfahan University of Medical Science, Faculty of Pharmacy and Pharmaceutical Sciences, were brought to the laboratory five days before the experiments and housed in plastic cages (40 × 30 × 16 cm), lined with wood shavings, kept at 25 ± 3°C and 55 ± 10% humidity, with a 12-h light–dark cycle. Standard food pellets and water were provided ad libitum. All animal studies were carried out according to the approved guidelines of the Iranian Institutional Animal Ethics Committee (IR.MUI.RESEARCH.REC.1397.158).

As shown in Fig. 2 Thirty-five mice were randomly divided into seven groups. The animals in Group 1 received phosphate buffer saline as vehicle intraperitoneally (IP) for five consecutive days, while Group 2 received 7 mg/kg/BW CP in a single IP administration (negative control). Group 3 received 40 mg/kg/BW 4-HCH for five consecutive days. The animals in Groups 4 and 5 received 10 and 40 mg/kg BW 4-hydroxy chalcone, respectively, for five consecutive days (pretreatment). On the last day, the animals in both groups received a single dose of 7 mg/kg BW CP IP. The animals in Groups 6 and 7 were treated with a single dose of 7 mg/kg BW CP IP. After 6 and 12 h, the animals in Group 6 received 10 mg/kg BW 4-hydoxychalcone, whereas the animals in Group 7 received 40 mg/kg BW 4-hydroxy chalcone (post-treatment). All the animals were euthanized by cervical dislocation after ketamine–xylazine injection (100–10 mg/kg).

Figure 2.

Figure 2

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Animal experiment was done with seven groups consisting five male mice in each group. CP (CP) as a genotoxic drug (7 mg/kg/bw) administrated intraperitoneally at day 5 except in Groups 1 and 3. Group 1 received vehicle for five consecutive days. Groups 3–7 received 4-HCH (10 and 40 mg/kg/bw) intraperitoneally as a pre- and post-treatment schedule. All of subjects were euthanized at the end of day 6 and both femoral bone marrows were used for micronucleus assay.

Bone marrow micronucleus test

The micronucleus test was done according to a previously published protocol [31]. Briefly, 5 μL of the femoral of bone marrow cells was flushed out with fetal bovine serum and smeared on an acridine orange-coated glass slides and stored at room temperature for 30 min. Five slides per each animal were coded for blind count and analysis. Two thousand polychromatic erythrocytes per animal were observed using an Olympus fluorescent microscope (Olympus, Tokyo, Japan) with blue excitation filter. The frequencies of the micronucleated polychromatic erythrocytes (MNPCE) and the normochromatic erythrocytes were counted based on the OECD 474 guideline in vivo MNT assay protocol.

Statistical analysis

The statistical significance of the data was tested by using one-way analysis of variance test (ANOVA) with Tukey’s multiple comparison post hoc test using Graph Pad Prism 8 (Graph Pad Software, Inc., CA, USA). Values of P ≤ 0.05 were considered as statistically significant.

Results

Cytotoxicity assay

The effect of different concentration of CP on cell survival in HEK293 cells are shown in Fig 3a. The percent cell survival data indicate that at 0.2 and 2 μM concentrations. CP did not have any cytotoxic effects on HEK293 cells as compared to the control group. As CP concentration was increased (30, 50, 100 and 200 μM), further dropped in the percent of cell survival was observed as compared to the control group (P < 0.01). The half-maximal inhibitory concentration (IC50) of CP in our experiment was obtained at 19.4 μM concentration for further studies.

Figure 3.

Figure 3

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MTT assay of (A) CP and (B) 4-HCH in HEK293 cells. Values are expressed as means ± SD from three independent experiments. *Significant difference compared to the control group (P < 0.05), **(P < 0.01) and ***(P < 0.001). IC50 value for each item was calculated with GraphPad prism software.

It was observed that 4-HCH at 0.2, 2 and 10 μM concentration did not have any cytotoxic effect on HEK293 cells, so we selected these three optimum doses for further studies. However, significant cell reduction was observed at 100 μM (P < 0.05) and 200 μM (P < 0.001) as compared to the control group; with this regards, the IC50 of 4-HCH was obtained around 133.6 μM (Fig. 3b).

Intracellular ROS

The measured intracellular ROS was presented as CFI values in Fig. 4. As it was shown, CP at IC50 concentrations (19.15 μM) significantly increased free radical production at the first hour of cellular exposure in comparison with the normal control (P < 0.0001). On the whole, adding the 4-HCH to cells showed a dose-dependent reduction in CFI value; however, the statistically significant effects were observed at pre-treatment with 10 μM (P < 0.05) and co-treatment with 2 and 10 μM of 4-HCH (P < 0.01). In general, the stronger protective effect against produced ROS was observed in co-treated groups with 4-HCH.

Figure 4.

Figure 4

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HEK293 intracellular ROS measurement via 2′,7′-dichlorofluorescein diacetate dye. Cells were exposed to the H2O2 as a positive ROS inducer control. CP at an IC50 concentration (19.15 μM) was used in mentioned groups. All values are presented as mean ± SD from three independent experiments. *Significant difference compared to the CP (positive control) group (P < 0.05), **(P < 0.01). # CP group as compared to the normal cells **(P < 0.0001).

Micronucleus test

The ratio of micronucleated polychromatic erythrocytes (MNPCE) in 2000 polychromatic erythrocytes (PCE) was shown in Fig. 5. As expected, the negative-control group (Vehicle) had low MNPCE frequency (15.96 ± 5.32), and the CP-treated control group demonstrated a significant jump in MNPCE frequency as compared to the negative control group (125.6 ± 6.28, P < 0.0001), which is supporting the genotoxicity of the CP with single 7-mg/kg IP dose administration. However, measured MNPCE cells in 4-HCH-treated subjects confirmed the absence of obvious genotoxic effect at 40 mg/kg IP administration (21.85 ± 4.52). As it was shown in pre- and post-treated groups, the frequency of micronucleated cells was significantly decreased in comparison with CP control group (P < 0.0001), in which the lowest MN cells were seen in 10-mg/kg-treated subjects with 4-HCH after CP exposure (39 ± 7.9).

Figure 5.

Figure 5

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Effects of treatments with different doses of the 4-HCH (4HCH) on the number of MNPCE (A) and the frequency of MNPCE per 2000 PCE in bone marrow cells of mice (B) were shown. Orange–red micronuclei were indicated with white arrow in part I with 10X and II with 100X magnification.

CP: received 7 mg/kg BW CP. All drugs were administered intraperitoneally (IP). *Significant difference compared to the vehicle group (P < 0.001) and # significance in comparison with CP positive control group (P < 0.001).

Discussion

CP is an antineoplastic agent, which is a prime choice for the treatment of different cancers such as head and neck, ovarian, cervical and lung through platinum-based chemotherapy. Its exposure is frequently associated with increased free radicals and subsequently different types of damages to a cellular genetic material, which can cause subsequent malignancies in normal cells [32]. It has been proved that CP undergoes aquation inside the cells, and the platinum atom covalently binds to the N7 position of purines nucleotides of DNA leading to mutation, intra-strand and inter-strand cross-linking and strand breaks [33]. Charles et al. suggested that some natural products such as arbutin, chalcones, epicatechin, myricetin and sakuranetin induced non-homologous end-joining and DNA double-strand break repair, which is the major pathway used by higher eukaryotic cells to repair double-strand breaks and genomic instabilities [24].

The MTT assay showed that CP can reduce the HEK293 cells survival in a concentration-dependent manner. About 80% of cells were destroyed after 50 μM CP exposure, which confirmed the high cytotoxic potency of CP in HEK293 cells. Our finding is in collaboration with previous studies on CP cytotoxicity on various cell lines [34,35]. Which previous works suggested that CP cytotoxicity is probably because of increased free radicals such as ROS generation, induction of endoplasmic reticulum (ER) stress, inflammatory response and autophagy activation [36].

Flavonoids such as chalcones and hydroxy chalcones have been showed to be remarkable potential anticancer agents due to their ability to scavenge free radicals and so have the capacity to inactivate carcinogens [37]. 4-Hydroxy chalcone is one of the most potent chalcones in terms of free radicals scavenging based on its chemical structure and also is one of the safest one due to its slight cytotoxic properties [38]. It was known that chalcones with a benzene B-ring and polyphenol A-ring may have a less cytotoxic effect than those with phenols on both A- and B-rings. In our study, the absence of significant cytotoxic effect of 4-hydroxy chalcone in HEK293 cells up to 100 μM concentration was observed and it was suggested that this natural occurring flavonoid is safe in selected optimum doses for further studies.

Berndtsson et al. (2007) found that CP can induce DNA damage, whereas superoxide anion formation reaches its highest level after 3 h of administration, suggesting that the protective agent must be used at the same time in order to neutralize the generated free radicals [39]. It was previously observed that maltol as a natural antioxidant could protect HEK293 cells from cytotoxicity, ROS generation and apoptosis induced by CP through the AMPK-mediated PI3K/Akt and p53 signaling pathways in form of co-treatment [40]. Also, it was reported that CP-induced oxidative stress in HEK293 and cholangiocarcinoma cells can completely be suppressed by N-acetylcysteine as a precursor of glutathione and superoxide scavenger in a form of co-treatment [41].

One of the most important mechanisms of tumor induction is free-radicals-mediated damages to the cells such as DNA mutation, chromosomal aberrations and formation of micronuclei, which can lead to cancerous cells, aging or cell death [42]. It was observed that increased intracellular ROS due to CP exposure was attenuated with 4-HCH at concentrations as low as 2 μM in form of co-treatment, also in both pretreatment and co-treatment of 10 μM concentration. This finding is in accordance with the previous studies, which were reported that lower concentrations of chalcones including phloretin or quercetin can significantly reduce ROS generation due to CP exposure [43]. It was suggested that chemical keto-vinylene reactive group in the backbone structure of chalcones and its derivatives such as 4-hydroxy chalcone can neutralize ROS and their toxic effect to cells [44]. On the other hand, Perjési et al. found that Quercetin and some synthetic chalcones have the most antioxidant activity at the first 10 min of incubation and this activity decrease significantly after 240 min of incubation [45]. These time-basing evidence may justify why in our experiment we observed a stronger protective effect of co-treatment groups than pre-treatment groups of 4-HCH in order to reverse ROS generation induced by CP.

In addition to free radical’s generation, CP-produced platinated tubulin in the cells, which could not assemble into microtubules and subsequently cause strong tubulin aggregation, resulted in micronucleus formation [46]. As it was expected in accordance with the previous discussed findings, CP showed a strong genotoxic effect through a significant increase in the frequency of bone marrow MNPCE. On the other hand, recent findings showed that chalcones, 4-hydroxyderricin, and xanthoangelol and also sulfonamide chalcone blocked the formation of micronucleus induced by genotoxic agents [47]. It was observed that 4-HCH treatment even though in a lower concentration such as 10 mg/kg.BW 24 h after CP exposure could significantly decrease the induced genotoxicity.

Conclusion

Our results demonstrated the antigenotoxic action of 4-hydroxy chalcone in CP-treated mice bone marrow cells for the first time in both concentrations of 10 and 40 mg/kg especially in the form of co-treatment. Further studies required clinical application of this compound in a combination of CP to attenuate the normal cells’ genotoxicity side effects.

Highlights

  • The measured IC50 of CP and 4-HCH were 19.4 μM 4 and 133.6 μM on HEK293 cells.

  • 2 μM of 4-HCH could attenuate the CP-induced ROS production.

  • Treatment with 10 mg/kg of 4-HCH significantly lowered the CP-induced MN cells.

  • Time dependency of antioxidant treatment in CP genotoxicity.

Acknowledgments

We thank our colleagues specially Dr. Mehdi Eftekhari who helped us in this research.

Conflict of interest statement

The authors state that they have no conflict of interests.

Funding

This work was supported by the Isfahan University of Medical Sciences and Pharmaceutical Research Center (grant number 397312).

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