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Abstract

ALC67 is an N-acylated thiazolidine compound with promising anticancer activity that led to the recent discovery of a series of 3-propionyl thiazolidine-4-carboxylic acid ethyl esters as a family of novel antiproliferative agents. Since the mutagenic and genotoxic properties of marketed anticancer molecules constitute a main issue to be addressed, this study focused on the analysis of the mutagenicity, antimutagenecity, and genotoxicity of this molecule. The mutagenicity and antimutagenicity of ALC67 were evaluated by Ames test performed on Salmonella TA98 and TA100 strains. The genotoxicity of this molecule was investigated in the chromosomal aberration assay on human lymphocytes. All results revealed that the analyzed structure is not mutagenic in the two Salmonella strains tested and was not genotoxic in human lymphocytes in vitro. On the other hand, it showed a weak antimutagenic effect in these two bacterial strains. The above results indicate that after performing some more mutagenicity assays using the other recommended strains, this compound can be safely used for the development of new structures exhibiting anticancer activities.

Keywords

ALC67 novel antiproliferative agent mutagenicity assay antimutagenicity assay genotoxicity

Introduction

Anticancer drug development aims the generation of chemical structures that can kill rapidly dividing cells efficiently. Biochemical pathways in cell division are the main target for anticancer drug development strategies. Indeed, molecules that can directly damage DNA by blocking the synthesis of nucleotides or that can disrupt the mitotic spindle assembly are considered as clinically approved antiproliferative compounds.^1–4

As designed for inducing considerable damage to genetic material in cancer cells, antineoplastic agents have to be investigated for their genotoxic effects on normal somatic cells in order to predict their relative potencies to promote the growth of secondary tumors. In addition to somatic cells, germ cells must be studied for genotoxicity since transmissible damage might cause birth defects^5–9 or increase the offspring’s risk for developing genetic diseases or cancer.^10–13

Many studies focused on the evaluation of the genotoxicity of widely used anticancer agents such as topoisomerase inhibitors,^14–18 cisplatin,^19–24 or paclitaxel^20,25,26 on somatic and/or germ cells and many of them were actually shown to present genotoxic effects. Thus, the development of methods for modulating this toxicity also constitutes a topic of significant interest.^19,27,28

ALC67 is an acetylenic thiazolidine molecule that was demonstrated to exhibit promising cytotoxic activity on several cancer cell lines in vitro. Although its direct mechanism of action has not been elucidated yet, it was shown that this heterocyclic compound is inducing apoptosis-activating caspase 9-dependent apoptotic pathways.²⁹ In this study, we aimed to determine the mutagenic and genotoxic properties of ALC67 using the Ames Salmonella assay on TA98 and TA100 strains and human lymphocytes in vitro since optimization studies of ALC67 led to the discovery of a new family of thiazolidine compounds with promising anticancer properties.

Materials and methods

Chemicals

Sodium azide (SA), 4-nitro-o-phenylenediamine (NPD), biotin, histidine, nicotinamide adenine dinucleotide phosphate, glucose-6-phosphate, crystal violet, ampicillin trihydrate, tetracycline, benzo(a)pyrene (B(a)P), mitomycin C (MMC), and colchicine were supplied from Sigma Chemical Company (St Louis, Missouri, USA); agar and nutrient broth were supplied from HiMedia Laboratories Ltd (Mumbai, Maharashtra, India). Peripheral blood karyotyping medium was purchased from the Biological Industries Israel Beit-Haemek Ltd, Kibbutz Beit-Haemek, Israel.

Synthesis of ALC67

ALC67 was synthesized according to the procedure described by Onen-Bayram et al. from l-cysteine ethyl ester (Figure 1).²⁹

Figure 1.

Structure of ALC67 and its antiproliferative derivatives.

Preparation of (2RS,4R)-2-phenylthiazolidine-4-carboxylic acid ethyl ester

The described procedure was followed for l-cysteine ethyl ester hydrochlorate (1 g, 5.39 mmol), and the resulting compound was directly involved to the following step because of stability problem.

Preparation of ALC67 or (2RS,4R)-2-phenyl-3-propionyl-4-thiazolidine-carboxylic acid ethyl ester

The described procedure was meticulously followed, and the expected product was obtained with a yield of 69%.

1H NMR (CDCl₃) δ 1.20–1.27 (m, 3 H), 2.97 (s, 0.6 H), 3.16 (s, 0.4 H), 3.19 (dd, J = 7.0 Hz, J = 12.0 Hz, 0.4 H), 3.29 (dd, J = 7.0 Hz, J = 12.0 Hz, 0.4 H), 3.35 (d, J = 5.60 Hz, 1.2 H), 4.15–4.25 (m, 2 H), 4.91 (t, J = 5.60 Hz, 0.6 H), 5.18 (t, J = 7.0 Hz, 0.4 H), 6.28 (s, 0.4 H), 6.40 (s, 0.6 H), 7.19–7.62 (m, 5 H). 13C NMR (CDCl₃) δ 14.3, 32.8, 33.9, 62.4, 62.7, 64.1, 65.6, 66.6, 67.9, 75.9, 76.4, 79.6, 81.8, 127.1, 127.3, 128.4, 128.6, 140.5, 152.4, 169.3. LC-MS: ELSD 98%, R_t = 9.85 min, m/z 290 (M + H)⁺

Bacterial strains

For mutagenicity and antimutagenicity assays, Salmonella strains TA98 and TA100 were used. The TA98 strain was provided by the Toxicology Department of the Faculty of Pharmacy of Gazi University, Ankara, Turkey, and the TA100 strain from the Marmara Research Center, (Marmara Araştırma Merkezi, MAM) of the Scientific and Technological Research Council of Turkey (Türkiye Bilimsel ve Teknolojik Araştırma Kurumu, TÜBİTAK), Gebze, Turkey. TA98 detects frameshift mutagens and TA100 detects base pair mutagens. These strains were primarily recommended by Maron and Ames³⁰ for routine mutagenicity assays.

Preparation of S9 fraction

Sprague–Dawley male rats weighing 150–200 g were provided from the Yeditepe University Medical School Experimental Research Center (YUDETAM) and were kept as two animals per cage. The experimental protocol was approved by the ethical committee of Yeditepe University. Animals were fed with standard rodent pellet diet and water ad libitum with 12-h light/12-h dark cycle. Room temperature and relative humidity conditions were 28 ± 2°C and 60 ± 5%, respectively.

The rat liver homogenate was prepared according to Ames et al.,³¹ Garner et al.,³² and Halder et al.³³ Sprague–Dawley male rats weighing 175–200 g were fed with 0.1% phenobarbital in their drinking water for 7 days. On day 6, no food was provided to rats. Two rats were used for S9 preparation. The following day, the animals were killed, and the rat liver homogenate (S9) was prepared by centrifugation at 9000g following the method of Maron and Ames.³⁰ Approximately 2 mL of S9 fractions were distributed in different small sterile cryo vials and quickly frozen and stored at −80°C.

Mutagenecity and antimutagenecity assays

Standard mutagenicity assays in plate incorporation tests were carried out by following the method of Maron and Ames.³⁰ Both mutagenecity and antimutagenicity assays were performed with Salmonella tester TA98 and TA100 strains. ALC67 was dissolved in dimethyl sulfoxide (DMSO) and different concentrations (0, 10, 100, 1000, 5000, and 10,000 µg/plate) in 50 µL of DMSO were used for both mutagenicity and antimutagenicity assay against known mutagens and carcinogen. For testing mutagenicity, plates were co-incubated with the bacterial strains and the different concentrations of chemical, inverted and placed at 37°C for 48 h in dark and revertant colonies were counted after incubation. To evaluate the impact of ALC67 metabolites, similar experiments were also carried out by incubating bacteria and chemical with liver S9. Four plates were used for each concentration tested with and without S9 experiments.

Antimutagenicity assays were carried out similarly using the same bacterial strains and the same ALC67 concentration range with or without S9 homogenate. NPD was used as a positive mutagen for TA98 strain without S9 and SA was used as a positive mutagen for TA100 strain. B(a)P was used as a positive mutagen for both TA98 and TA100 strains with S9 experiment. After incubation of inverted plates at 37°C for 48 h in dark, revertant colonies were counted. Four plates were used for each concentration tested for both with and without S9 experiments.

In vitro CAs assay in human lymphocyte culture

For chromosomal aberration (CA) analysis, blood samples from one male and one female volunteers (aged between 25 and 35 years) with no confounding factors such as medication, X-ray treatment, smoking habit, or any other history of disease were collected in the heparinized vials, and 0.5 mL of blood was added to 5 mL of Karyomax medium and incubated at 37°C. After 24 h, four different concentrations of ALC67 (0.75, 1.5, 3.0, and 6.0 µg/mL of culture) were added to the culture medium. The stock solution was prepared by dissolving ALC67 in DMSO first, this solution is subsequently diluted with Karymax medium to be added to the culture media in the concentrations mentioned above. Two cultures were treated with each concentration of chemical (one from male and one from female blood samples) as recommended by the Organization for Economic Cooperation and Development’s (OECD) guideline.³⁴ Two sets of cultures were also treated with only DMSO and MMC (0.50 µg/mL) and were used as negative and positive controls. After 70 h of the culture, colchicine was added to the culture media to arrest the metaphase cells. At 72 h, cultures were harvested and slides were prepared for metaphase chromosomes following the method of De Chaudhuri et al.³⁵ and also OECD test guideline.³⁴ All slides were coded, and 75 well-spread metaphase cells (46 ± 2 chromosomes) per culture were scored for CA. A total of 150 metaphase cells were scored for each concentration of chemical and for both negative and positive controls. For the mitotic index (MI) analysis, 1000 cells/culture were scored, and the MI was expressed in percentages. CA was scored according to the World Health Organization³⁶ and OECD guideline.³⁴ Frequency of aberrations per cell for chromatid type and chromosome types were calculated. Gaps were recorded as indicated in the OECD guideline but were not included neither as percentage of aberrant cells nor as frequency of aberrations per cell.

Statistical analysis

All the results were expressed as mean ± standard deviation. Dunnett’s multiple comparison test was carried out for both mutagenicity and antimutagenicity data in Salmonella assay and for CA assay in vitro on human lymphocytes data.³⁷ The values of p < 0.05 was considered statistically significant.

Results

Tables 1 and 2 show the results of the mutagenicity and antimutagenicity assays induced by ALC67 in the TA98 and TA100 Salmonella strains with and without metabolic activation. First, the possible mutagenic effects of this anticancer drug candidate were investigated. As expected, the positive control showed very high frequencies of revertant colonies when compared with both negative control and different concentration of treated compound. Neither the TA98 nor the TA100 strains treated with ALC67 concentrations of 10–1000 µg/plate exhibited significant revertant colonies when compared with the negative control, and for both strains, toxicity was observed to appear for an ALC67 concentration of 5000 µg/plate of culture. For both of the strains, 10,000 µg/plate was found to be a totally toxic concentration (Table 1).

Table 1.

Results of mutagenicity assay induced by acetylenic thiazolidine ALC67 in Salmonella strains TA98 and TA100 in both with and without S9.^a

	ALC67 (µg/plate)	(Mean ± SD)–S9 (n = 4)	(Mean ± SD) + S9 (n = 4)
TA98	Positive control	562.50 ± 83.10	113.00 ± 16.43
	Negative control (DMSO)	29.50 ± 4.43	34.00 ± 7.70
	10	26.75 ± 2.36	37.00 ± 5.35
	100	28.25 ± 6.65	32.75 ± 5.25
	1000	27.50 ± 9.26	30.25 ± 3.30
	5000	17.75 ± 4.65^b	16.25 ± 5.44^b
	10,000	5.25 ± 2.32^c	8.75 ± 2.75^c
TA100	Positive control	701.00 ± 46.45	663.00 ± 81.51
	Negative control (water)	175.75 ± 18.73	181.50 ± 17.75
	10	187.00 ± 11.97	173.00 ± 12.91
	100	185.00 ± 13.04	176.70 ± 11.32
	1000	165.00 ± 21.59	159.75 ± 27.21
	5000	47.00 ± 21.60^c	79.75 ± 26.66^c
	10,000	6.00 ± 4.32^c	8.25 ± 4.98^c

NPD: 4-nitro-o-phenylenediamine; DMSO: dimethyl sulfoxide; SA: sodium azide; B(a)P: benzo(a)pyrene.

^aDunnett’s multiple comparison test was carried out for statistical analysis. NPD was used as positive mutagen for TA98 strain without S9 and SA was used as positive mutagen for TA100 strain. B(a)P was used as positive mutagen for both TA98 and TA100 strains with S9 experiment.

^b p < 0.05: control versus ALC67-treated groups.

^c p < 0.01: control versus ALC67-treated groups.

Table 2.

Results of antimutagenicity assay induced by acetylenic thiazolidine ALC67 in Salmonella strains TA98 and TA100 in both with and without S9.^a

ALC67 (µg/plate)		Mean ± SD (N = 4)	ALC67 (µg/plate)		Mean ± SD (N = 4)
TA98–S9	NPD (20 µg/plate)	587.75 ± 70.21	TA98 + S9	B(a)P (1.0 µg plate)	107.75 ± 16.80
	NPD + 10	586.75 ± 82.95		B(a)P + 10	99.50 ± 19.05
	NPD + 100	569.50 ± 84.52		B(a)P + 100	94.50 ± 73.25
	NPD + 1000	366.00 ± 32.79^b		B(a)P + 1000	73.25 ± 28.69
	NPD + 5000	218.25 ± 24.19^b		B(a)P + 5000	31.00 ± 9.31^b
	NPD + 10,000	99.50 ± 19.50^b		B(a)P + 10,000	14.00 ± 5.35^b
TA100 – S9	SA (1.0 µL/plate)	746.25 ± 53.08	TA100 + S9	B(a)P (1.0 µg/plate)	679.75 ± 66.78
	SA + 10	749.50 ± 14.39		B(a)P + 10	660.75 ± 50.55
	SA + 100	695.25 ± 21.19		B(a)P + 100	618.25 ± 30.67
	SA + 1000	411.50 ± 67.58^b		B(a)P + 1000	572.50 ± 82.31^c
	SA + 5000	176.75 ± 47.97^b		B(a)P + 5000	201.50 ± 36.34^b
	SA + 10,000	88.75 ± 24.57^b		B(a)P + 10,000	96.75 ± 23.67^b

NPD: 4-nitro-o-phenylenediamine; SA: sodium azide; B(a)P: benzo(a)pyrene.

^aDunnett’s multiple comparison test was carried out for statistical analysis.

^b p < 0.01: positive mutagen versus positive mutagen plus ALC67-treated groups.

^c p < 0.01: positive mutagen versus positive mutagen plus ALC67-treated groups.

The antimutagenicity assays were carried out against the positive mutagen NPD for the TA98 strain and SA for the TA100 strain for cultures without S9 activation. Also, since ALC67 was described for some anticarcinogenic activity, B(a)P was used as the positive mutagen/carcinogen for both strain cultures activated with S9. Results of the antimutagenic assays indicated that ALC67 was slightly antimutagenic at the 1000 µg/plate concentration in both the nonactivated strains and TA100 when cultured in the presence of S9. The significant decrease in the number of bacterial revertant colonies observed in both TA98 and TA100 strain cultures activated by S9 or not for the 5000 and 10000 µg/plate concentrations is definitely due to the toxic effects in these doses (Table 2).

Genotoxicity results of thiazolidine compound measured by the CA assay are shown in Table 3. Knowing that ALC67 exhibited half maximal inhibitory concentration (IC₅₀) value of around 5 µM (1.4 µg/mL) when evaluated on various cancer cells, the CA assays were chosen to be carried out at four concentrations centered on this value. Thus, the experiments were conducted at ALC67 concentrations of 0.75, 1.5, 3, and 6 µg/mL. No significant CA (percentage of aberrant cells) were observed in the two lower doses (i.e. 0.75 and 1.5 µg/mL of cultures) when compared to DMSO control culture. A weak, but significantly higher incidence of CA were determined in the two higher doses, that is, 3 and 6 µg/mL of culture medium when compared to DMSO control. This increase was statistically significant only at the highest tested dose. Also, the results of the MI showed a slightly reduced MI for the highest dose even if not statistically significant (Table 3). The positive mutagen MMC showed very high frequencies of CA, which indicated that lymphocytes were cultured under proper conditions.

Table 3.

Chromosomal aberrations induced by acetylenic thiazolidine ALC67 in human lymphocyte in vitro.

Treatment (µg/mL of culture)	Metaphase cells scored	Gaps^a	Aberrations/cells^b		Aberrant cells % (Mean ± SD)^c	Mitotic indices (Mean ± SD)^c
Treatment (µg/mL of culture)	Metaphase cells scored	Gaps^a	Chromatid chromosome type		Aberrant cells % (Mean ± SD)^c	Mitotic indices (Mean ± SD)^c
DMSO	150	7	0.033	0.013	4.66 ± 0.94	2.50 ± 0.73
0.75	150	11	0.047	0.020	6.66 ± 1.89	2.84 ± 0.45
1.5	150	8	0.033	0.027	6.00 ± 2.83	2.32 ± 0.87
3.0	150	13	0.073	0.040	10.66 ± 1.89	2.34 ± 0.23
6.0	150	18	0.093	0.047	12.67 ± 2.83^d	1.59 ± 0.14
MMC (0.50)	150	40	0.273	0.133	34.67 ± 1.89	2.39 ± 0.53

MMC: mitomycin C; DMSO: dimethyl sulfoxide.

^aTotal chromatid and chromosome gaps at each dose were recorded but not included as aberrations/cell.

^bTotal number of aberrations (chromatid and chromosome type)/total number of cells scored per dose. Results are of two cultures (75 metaphase cells/culture).

^cResults at each dose were compared to those of DMSO control using Dunnett’s multiple comparison test.

^d p < 0.05: DMSO control versus ALC67-treated groups using Dunnett’s multiple comparisons.

Discussion

Thiazolidine compounds present a five-membered ring with sulfur and a nitrogen atom in their structure. There are some studies that report the cytotoxic activity of molecules presenting this heterocycle.^38
–40 We also presented the anticarcinogenic property of an acetylenic thiazolidine, namely, the ALC67 molecule in a previous study,²⁹ and showed recently that 3-propionyl-thiazolidine-4-carboxylic acid ethyl esters can be classified as a family of antiproliferative compounds.⁴¹ Given the potency of this core structure, we wanted to investigate the mutagenicity and the genotoxicity of these acetylenic thiazolidine, choosing ALC67 molecule as the reference molecule.

While the mutagenicity and antimutagenicity assays were carried out on Salmonella strains, the in vitro genotoxicity was evaluated on cultured human lymphocytes. Results of the mutagenicity assays clearly indicated that ALC67 was not mutagenic on TA98 and TA100 Salmonella strains whether or not incubated with S9. Also, up to 1000 µg/plate the chemical showed no toxic effects and exhibited weak antimutagenic effects in both strains without S9 activation and also in TA100 when treated with S9.

As recommended by the OECD guideline,³⁴ the genotoxicity of ALC67 was also analyzed by measuring the in vitro CA assay on human cells. The CA assay being a short-term test, the genotoxic effects were evaluated at different doses including two and four times higher than the average IC₅₀ value previously determined on cancer cell lines. Results of the CA assay in human lymphocytes clearly indicated that ALC67 was not genotoxic even at the IC₅₀ value.

Many of the marketed anticancer drugs are described to be mutagenic or genotoxic since they are designed to damage the DNA structure or to inhibit its synthesis. Since this toxicity can result in the emergence of secondary cancers, studying the possible benefits of the use of antioxidant molecules such as vitamin B₁₂ or tempol to prevent the toxicity associated with anticancer treatment constitutes a hot research topic.^27,42
–44 Another approach to avoid secondary cancer development is the generation of structures that do not exhibit any genotoxicity or mutagenicity. Our results showed that the acetylenic thiazolidine derivative ALC67 is neither mutagenic nor genotoxic, a result that is in agreement with the only report that examines the genotoxicity of l-thioproline, a simple thiazolidine compound.⁴⁵ In a previous study, we demonstrated that ALC67-induced apoptosis via caspase 9. The lack of genotoxicity or mutagenicity suggests that the molecular mechanism of action of this compound does not lead to neither DNA damage nor the generation of reactive oxygen species. Thus, the use of ALC67 analogs seems to be suitable for the development of non-mutagenic anticancer agents.

Conclusions

ALC67 is an acetylenic thiazolidine molecule with antiproliferative properties that we previously optimized for the generation of a new class of antiproliferative thiazolidine compounds. Given the promising anticancer activity of all the acetylenic structures that we developed, we aimed to determine in this study the safety of this scaffold analyzing its possible mutagenicity and genotoxicity. In fact, as many anticancer drugs are shown to exhibit some toxicity on somatic and/or germ cells, the generation of anticancer molecules with moderate to low potencies to develop secondary tumors or genetic diseases in long term is of great interest. The overall results of this study indicated that the compound ALC67 is not mutagenic in bacterial strains TA98 and TA100 and also not genotoxic in human lymphocytes in vitro. Although some more work on mutagenicity assays using other bacterial stains and also in vivo studies are required before coming to a final conclusion, given account of the present mutagenicity and genotoxicity data, this structure can be further optimized without causing any damage due to genotoxic effects and the thiazolidine scaffold may be used safely for the development of new structures exhibiting anticancer activities.

Footnotes

Acknowledgments

Authors are grateful to the TÜBİTAK, Istanbul, Turkey, for providing Dr Ashok K Giri, the Senior Visiting Scientist position to work at the Department of Toxicology, Faculty of Pharmacy, Yeditepe University, Istanbul, Turkey.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Evaluation of the mutagenic and genotoxic effects of the ALC67 thiazolidine compound in Salmonella strains and human lymphocytes in vitro

Abstract

Keywords

Introduction

Materials and methods

Chemicals

Synthesis of ALC67

Preparation of (2RS,4R)-2-phenylthiazolidine-4-carboxylic acid ethyl ester

Preparation of ALC67 or (2RS,4R)-2-phenyl-3-propionyl-4-thiazolidine-carboxylic acid ethyl ester

Bacterial strains

Preparation of S9 fraction

Mutagenecity and antimutagenecity assays

In vitro CAs assay in human lymphocyte culture

Statistical analysis

Results

Discussion

Conclusions

Footnotes

Acknowledgments

Declaration of Conflicting Interests

Funding

References