In vitro cytogenetic assessment and comparison of vildagliptin and sitagliptin

Abstract

Vildagliptin and sitagliptin are commonly used antidiabetic drugs. Chromosomal aberration (CA), sister chromatid exchange (SCE) and cytokinesis-block micronucleus (CBMN) assays were employed to assess and compare cytotoxic and genotoxic effects of these drugs. Peripheral lymphocytes were exposed to 125 μg/ml, 250 μg/ml and 500 μg/ml of vildagliptin and 250 μg/ml, 500 μg/ml and 1000 μg/ml of sitagliptin for 24 h and 48 h with and without exogenous metabolic activation. At the end of the study, it was determined that these drugs and their metabolites had no genotoxic effects on CA, SCE and CBMN. On the other hand, parallel to the increase in dose, vildagliptin showed weak cytotoxicity on the mitotic index, and depending on its increase in dose; sitagliptin caused potential cytotoxicity and cytostatic effect on the mitotic index, nuclear division index and proliferation index. Due to their cytotoxic and cytostatic potential, these drugs inhibit cell proliferation.

Introduction

One of the most serious global health problems of the twenty-first century is “Diabetes Mellitus” (DM) which is a chronic, progressive and serious disease. DM disturbs the organ systems and occurs when the pancreas cannot secrete enough insulin or the body can’t use the available insulin efficiently. The prevalence of diabetic patients has recently increased almost four times since the 1980s. The International Diabetes Federation’s 2015 data has revealed that there are 415 million diabetic patients worldwide and 5 million deaths due to diabetes. Besides, the federation reported that 318 million adult patients with impaired glucose tolerance would be diabetic in the future. In 2015, one in every 11 adults had diabetes and one in 10 will have diabetes in 2040. In the same report’s Turkey’s data, one in eight adults had diabetes and totally 6339 million people had the disease. Also, it was estimated that 2179 million adults had non-diagnosed diabetes. The prevalence of DM in our country was 12.5% in 2015, and it was estimated that the prevalence of DM in 2040 would be 16%.

The possibility of having cancer and DM at the same time is greater than expectations (Giovannucci et al. 2010). The existence of a relationship between cancer and diabetes was first recognised in the 1930s (Marble 1934). Moreover, epidemiological studies have shown that patients with diabetes who has received chemotherapy or underwent surgery have had a worse prognosis and a higher mortality rate (Sen et al. 2014).

Genotoxicity refers to the ability of chemical substances to damage the cellular components such as spindle fibres, topoisomerases, DNA repair system, DNA polymerases that regulate the DNA or the genome and their side effects on genetic information. Also, the cytotoxicity term is a potential for cell death (Eastmond et al. 2009; Atlı Şekeroğlu and Şekeroğlu 2011; Tokur and Aksoy 2017).

In order to reveal the effects of the substances to be examined from the point of genotoxic and mutagenic features, the most commonly used in vivo and in vitro tests are the Ames Test, the Comet Assay, the Chromosome Aberration Test, the Micronucleus Test and the Sister Chromatid Exchange Test (Tucker and Preston 1996; Jena et al. 2002; Mateuca et al. 2006, Eastmond et al. 2009; Atlı Şekeroğlu and Şekeroğlu 2011, Kocaman et al. 2014).

Vildagliptin and sitagliptin, which exist in the group of incretin mimetic drugs, function as Dipeptidyl Peptidase-4 (DPP-4) inhibitors (TEMD 2014). Oral DPP-4 inhibitors are widely used in combination therapies because of their effects on lowering haemoglobin A1C, without weight gain and hypoglycemia risk (Levetan 2007). Most diabetic patients are treated with medications for the rest of their lives. Therefore, it is important to have information about the long-term efficiency and safety of these drugs to identify management strategies and reduce the economic burden of T2DM (Caballero 2017). Sitagliptin is the first approved DPP-4 inhibitor by the Food and Drug Administration (FDA) in 2006 (Matteucci and Giampietro 2011; Plosker 2014). It is still being used alone or in combination treatments in 130 countries around the world (Scott 2017). Vildagliptin was released to market in 2007 and is still being used in therapies (Ma et al. 2017).

European Medicines Agency (EMEA) reported that, in 2-year mouse carcinogenicity study, 50, 125, 250, 500 mg/kg/day sitagliptin doses were tested and treatment-related tumour incidence did not increase in any organ at all of these doses. However, and treatment-related non-neoplastic changes in both sexes at 500 mg/kg/day centrilobular hepatocellular hypertrophy were seen and at ≥ 250 mg/kg/day hydronephrosis. Also, at 500 mg/kg/day, due to an increased incidence of hydronephrosis, there was a slight decrease in survival. In 2-year rat carcinogenicity study, EMA informed that at systemic exposure levels; that is 58-times higher than average human exposure levels, there was a treatment-related increase in hepatic tumours. On the contrary, there were no other treatment-related or statistically significant increases in tumour incidence in any other organ. In addition, the researchers thought that this increased incidence of hepatic tumours in rats was a secondary effect on chronic hepatic toxicity because of this high dose (EMA 2007). EMA also published a scientific discussion on vildagliptin (EMA 2012). According to this report, no evidence for a carcinogenic potential was observed in the rat, studies, however, an increased incidence of hemangiosarcomas were observed at highest dose. Similarly, the increased incidence of hemangiosarcoma in mice occurred. Unfortunately, no detailed information on the applied tests was obtained in this report. According to EMA’s report, sitagliptin showed no genotoxic effects in in vitro or in vivo assays on mutagenicity (Ames test), direct DNA damage (in vitro test in primary rat hepatocytes), or clastogenicity (in vitro CA test in CHO cells, in vivo mouse MN). For vildagliptin, it is reported that this drug does not indicate a genotoxic potential in several standard genotoxicity tests.

An in vitro study about genotoxicity of sitagliptin was completed by Yuzbasioglu et al. (2018) In the study, the investigators used 31.25, 62.50, 125.00, 250.00, 500.00, and 1000.00 μg/ml concentrations of sitagliptin and applied chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronucleus (MN) and comet assays for 24 h and 48 h. At the end of the study, the researchers concluded that a higher concentration of sitagliptin had genotoxic effects on the human lymphocytes in vitro. They observed significantly increased frequency of CAs and SCEs at 24 h treatment at the highest concentration and at 48 h treatment at all concentrations (except 250 μg/ml for CA, except 31.25 and 62.50 μg/ml for SCE). Also, it was reported that this compound increased the MN at only the highest concentration. In accordance with this study, replication (RI) and nuclear division (NDI) indices were not affected by the drug at all treatments; however, mitotic index (MI) significantly decreased at the three highest concentrations of sitagliptin at 48 h treatment. Along with these results, Comet assay results indicated that sitagliptin significantly increased mean comet tail intensity (at 62.50 and 1000 μg/ml concentrations) and tail moment (1000 μg/ml concentration), and tail length at all concentrations (except 125 and 500 μg/ml concentrations) (Yuzbasioglu et al. 2018).

An in vivo genotoxicity (SCE, CA, and MN) and cytotoxicity (MI, RI and NDI) research was performed with type-2 diabetes patients treated with different oral anti-diabetic agents for 6 months. In this study, sitagliptin (100 mg/day), pioglitazone (30 mg/day) and rosiglitazone (4 mg/day) were evaluated. According to the results, the researchers stated that sitagliptin and thiazolidinediones may cause genotoxic and cytotoxic effects in patients with T2DM (Oz Gul et al. 2013).

A literature search has shown that there is limited data about genotoxicity and cytotoxicity of vildagliptin and sitagliptin. Yuzbasioglu et al. (2018) investigated in vitro genotoxic effects of just sitagliptin, but vildagliptin did not investigated in this study. Furthermore, there is no research has been carried out comparing genotoxic and cytotoxic effects of vildagliptin and sitagliptin. The primary aim of the study is to compare to genotoxic and cytotoxic potentials of these drugs. In the study CA, MN and SCE assays were applied on human lymphocytes with 125 μg/ml, 250 μg/ml and 500 μg/ml of vildagliptin and 250 μg/ml, 500 μg/ml and 1000 μg/ml of sitagliptin for 24 h and 48 h, with and without exogenous metabolic activation in vitro. The effects of drug applications were compared according to exposure time, exposure dosage and metabolic activation status.

Materials and methods

Chemicals

Dimethyl sulfoxide (DMSO) (Cas no. 67-68-5) (Merck), Mitomycin C (MMC) (Cas no. 50-07-7) (Serva) and Cyclophosphamide (CP) (Cas no. 6055-19-2) (Acros Organics), Cytochalasin B (Cyto-B) (Cas no. 14930-96-2), Bromodeoxyuridine (BrdU) (Cas no. 59-14-3) (Sigma-Aldrich), RPMI 1640 (Cas no. 01-106-1), PSA (Cas no. 03-033-1B), phytohemagglutinin (Cas no. 12-009-1H), and l-glutamine (Cas no. 03-020-1A) purchased from Biological Industries. Human liver microsomes, cytosol, and S9 (Human Liver S9 Pooled Donors Cas no. 452961, NADPH Regenerating System Solution A Cas no. 451220, NADPH Regenerating System Solution B Cas no 451200) were purchased from Corning. Other chemicals used for fixation and staining were obtained from Sigma-Aldrich.

Sitagliptin

Sitagliptin (Cas no. 654671-78-0) was purchased from Cayman. Itschemical name is 7-[(3R)-3amino-1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5,6,7,8-tetrahydro-[3-(trifluoromethyl)-1,2,4triazolo[4,3-a]pyrazine phosphate (1:1) monohydrate according to the IUPAC nomenclature. Some properties of sitagliptin are given below.

Molecular formula: C₁₆H₁₅F₆N₅O·H₃PO₄

Formula weight: 407.32 g/mol

Purity: ≥ 98%

Vildagliptin

Vildagliptin (Cas no. 274901-16-5) was purchased from Cayman. Its chemical name is (2S)-1-[2-[(3-hydroxy-1-adamantyl)amino]acetyl]pyrrolidine-2-carbonitrile according to the IUPAC nomenclature. Some properties of vildagliptin are given below.

Molecular formula: C₁₇H₂₅N₃O₂

Formula weight: 303.406 g/mol

Purity: ≥ 98%

Donors and collection of blood samples

Peripheral lymphocytes were obtained from two male and two female healthy donors. Healthy volunteers without any serious illness aged between 20 and 25 years-old who were not exposed to any genotoxic agent, any drugs and who did not smoke were included. Signed informed consent was obtained from all participants before the study.. Ethical permission was obtained from the Ethics Committee of Clinical Investigations of Ondokuz Mayıs University, Samsun with the number 183 on 09.05.2015. Approximately 5 ml of blood was collected from each donor in tubes containing heparin (20 U/ml) and the cell cultures were started on the same day.

Whole blood culture, culture groups and test concentration

Culture medium was prepared with RPMI 1640 medium, supplemented with 20% fetal bovine serum, 1% PSA,1.2% phytohemagglutinin and 2% l-glutamine. Solvent control cultures were treated with DMSO at a final concentration of 10 μl/ml. Positive control cultures were treated with MMC at a final concentration of 0.16 μg/ml and CP at a final concentration of 45 μg/ml. Test concentration of the drugs were determined according to Organisation for Economic Co-operation and Development (OECD) (2016a, b). Several doses were applied to the cell cultures for 48 h and their mitotic activities were analyzed. The concentration causing 55 ± 5% decrease in MI was chosen as the highest concentration. For vildagliptin, 500 μg/ml concentration was determined as the highest concentration; 250 μg/ml concentration as intermediate dose and 125 μg/ml concentration as minimum dose were selected. For sitagliptin 1000 μg/ml concentration was determined as the highest concentration; 500 μg/ml concentration as intermediate dose and 250 μg/ml concentration as minimum dose were chosen. Each group was exposed to the relevant chemicals for 24 h and 48 h. The culture tubes were incubated at 37 °C for 72 h. To start the culture, 300 μl of whole blood was added to 2500 μl medium in sterile conditions.

Chromosomal aberration assay (Without S9 Mix)

The CA test was performed using the methods of Moorehead and Evans’ (Moorhead et al. 1960; Evans and O’Riordan 1975) with minor modifications. At the 24th and 48th hours of the culture, the test concentrations were added to the concerned tubes. 2 h before harvesting, the cells were exposed to 0.06 μg/ml colchicine. At the end of the 72 h, all culture tubes centrifuged at 1200 rpm for 15 min. Supernatant was removed and  % 0.4 KCL was added by trickling and then, the tubes were incubated at 37 °C for 20 min. Culture tubes centrifuged at 1200 rpm for 15 min again. Supernatant was replaced with cold Carnoy’s fixative (methanol: glacial acetic acid, 3:1 v/v) and incubated at room temperature for 20 min. Centrifugation and fixative stages were carried out two times more. After the last centrifugation, supernatant was removed and the pellet was suspended. Three drops of the suspension were dropped on clean and cold slides. The slides were incubated at room temperature for a night. The slides were stained with 5% Giemsa stain solution (diluted with Sorensen buffer, pH 6.8) for 4–5 min and closed with entellan after 24 h.

Chromosomal aberration assay (with S9 mix)

The cultures were started as mentioned above and at the 48th hours of the culture, %2–4 S9 mix (93 µl S9 fraction; 3326 µl Tris buffer (pH = 7.6); 74.4 µl NADPH B; 186 µl NADPH A) and the test concentrations were added into the tubes. The tubes were incubated at 37 °C for 3 h. Then, the tubes were centrifuged at 2000 rpm for 4 min and the supernatant was replaced with fresh medium. The tubes were incubated at 37 °C till the end of 72 h. 2 h before harvesting, the cells were exposed to 0.06 μg/ml colchicine. At the end of the 72 h, the protocol as mentioned above was applied.

Analysis of chromosomal aberration

After the completion of all laboratory procedures, 100 metaphases were evaluated for the detection of numerical and structural CAs in each tubes. Also, 2000 cells were evaluated; the cells in the metaphase stage were detected and the percentage of metaphase cells was assessed as mitotic index (MI).

Sister chromatid exchange assay (without S9 mix)

The SCE test was performed according to Speit method (Speit 1984) with slight changes. 10 μg/ml Bromodeoxyuridine (BrdU) was added to all culture tubes before the incubation and then the tubes were protected from the light (Gonzalez-Gil and Navarrete 1982; Speit 1984; Kontaş and Atlı Şekeroğlu 2015). The same CA protocols detailed above were performed to apply drugs, to provide the maintenance and to harvest of culture. The slides were kept at room temperature and dark ambience for a night, then exposed to 256 nm UV light for 30 min, within 10% Sorensen buffer. After this process, they were incubated at 60 °C within 1 × SSC solution for an hour. Last of all, the slides were stained with 5% Giemsa stain solution (diluted with Sorensen buffer, pH 6.8) for 20 min and covered with entellan after 24 h.

Sister chromatid exchange assay (with S9 mix)

The cultures were started as mentioned above (expect BrdU) and at the 48th hour of the culture, %2–4 S9 mix (93 µl S9 fraction; 3326 µl Tris buffer (pH = 7.6); 74.4 µl NADPH B; 186 µl NADPH A) and the test concentrations were added into the tubes. The tubes were incubated at 37 °C for 3 h. Then, centrifuged at 2000 rpm for 4 min and the supernatant was replaced with fresh medium and 10 μg/ml BrdU added. The tubes were incubated at 37 °C till the end of 72 h. 2 h before harvesting, the cells were exposed to 0.06 μg/ml colchicine to arrest the cells in metaphase. At the end of 72 h, our protocol, as mentioned above, was applied.

Scoring of sister chromatid exchanges

To analyse the number of SCE, well dispersed 25 cells at the stage of the second mitosis were counted in each slide. Also, 100 cells were evaluated and the cells in the first, second and third mitosis stage (according to the differential staining of the sister chromatids) were detected and the proliferation-or replication index (PI) was calculated with this formula PI = [(MI) + 2(M2) + 3(M3)]/100.

Cytokinesis-block micronucleus assay (without S9 Mix)

The MN test was performed according to Fenech’s method with minor alterations (Fenech 2000). At the 20th and 44th hours of the culture, the test concentrations were added to the concerned tubes. 24 h before harvesting, the cells were exposed to 8 μg/ml Cytochalasin-B. At the end of the 68 h, all culture tubes centrifuged at 1200 rpm for 15 min. Supernatant removed and % 0.4 KCl was added by trickling than tubes were incubated at 37 °C for 10 min. Culture tubes were centrifuged at 1200 rpm for 15 min again. Supernatant was replaced with the first fixative (methanol: glacial acetic acid:  %0.9 NaCl 1:5:6 v/v) and incubated at room temperature for 20 min. The tubes centrifuged at 1200 rpm for 15 min and supernatant was replaced with the second fixative (methanol: glacial acetic acid 1:5 v/v) and incubated at room temperature for 20 min again. The centrifugation and second fixative stages were carried out two more times. After the last centrifugation, supernatant was removed and the pellet was suspended. Three drops of the suspension were dropped on clean and cold slides. The slides were incubated at room temperature for a night. The slides were stained with 5% Giemsa stain solution (diluted with Sorensen buffer, pH 6.8) for 15 min and covered with entellan after 24 h.

Cytokinesis-block micronucleus assay (with S9 mix)

The cultures were started as mentioned above and at the 48th hour of the culture, %2–4 S9 mix (93 µl S9 fraction; 3326 µl Tris buffer (pH = 7.6); 74.4 µl NADPH B; 186 µl NADPH A) and the test concentrations were added into the tubes. The tubes were incubated at 37 °C for 3 h. Then, the tubes were centrifuged at 2000 rpm for 4 min and the supernatant was replaced with fresh medium and also 8 μg/ml Cytochalasin-B added. The tubes were incubated at 37 °C until the end of 72 h. At the end of the incubation period, the protocol was applied as mentioned above.

Scoring of micronucleus

To detect to the MN number 2000, binucleated cells were examined and MNwere scored. Besides, mononucleated, binucleated, trinucleated and tetranucleated cells were recorded to calculate nuclear division index (NDI) according to this formula NBI = [(MI) + 2(MII) + 3(MIII) + 4(MIV)]/2000.

Statistical analysis

Statistical analysis was performed using the IBM SPSS Statistics 22 package program. Statistical decisions were based on the significance levels of 0.05 and 0.01. To evaluate if there is a significant difference between the applied doses and between the controls ANOVA, Tukey test-Post-Hoc analysis was applied. The Student’s t test was performed to compare the effects of 24-h and 48-h exposure of two drugs; the direct effects of two drugs with the effects of their metabolites; the effects of the two drugs and the two metabolites. In addition, the orthogonal polynomial analysis was carried out to determine the dose–effect relationships.

Results

Means for the CA, AC  %, NDI, MN %, PI, SCE at 24 h and 48 h vildagliptin treatment and vildagliptin’s metabolites treatment are shown in Table 1. Vildagliptin doses had no effect on the CA, AC  %, NDI, MN  %, PI, SCE, but linear, and cubic effect on the MI (p < 0.05) for the 24 h period. The applied vildagliptin doses level had no effect on the  %, NDI, MN %, PI and SCE, but had linear effect on the CA, AC  % and MI for the 48 h period (p > 0.05). The effect of metabolite levels was linear on MI, but other parameters weren’t affected (p > 0.05).

Table 1.

Effects of vildagliptin levels

Analysed parameters	S9 mix	Period	Treatments				SEM	p	Effects
			Solvent control	VİLDAGLİPTİN 125 µg/ml	VİLDAGLİPTİN 250 µg/ml	VİLDAGLİPTİN 500 µg/ml			L	Q	C
Chromosomal aberrations	–	24 h	12.00	11.75	13.25	10.75	± 0.97	NS	NS	NS	NS
Abnormal cell	–	24 h	11.25	12.75	13.25	10.25	± 0.96	NS	NS	NS	NS
Mitotic index	–	24 h	2.71	2.09	2.48	1.69	± 0.12	*	*	NS	*
Micronucleus (%)	–	24 h	0.38	0.35	0.29	0.25	± 0.03	NS	NS	NS	NS
Nuclear division index	–	24 h	1.98	1.98	1.95	1.89	± 0.04	NS	NS	NS	NS
Sister chromosome exchange	–	24 h	174.50	173.50	164.00	153.75	± 5.93	NS	NS	NS	NS
Proliferation index	–	24 h	2.56	2.58	2.67	2.63	± 0.02	NS	NS	NS	NS
Chromosomal aberrations	–	48 h	3.00	8.00	12.25	13.00	± 1.27	*	*	NS	NS
Abnormal cell	–	48 h	7.25	7.75	11.50	10.50	± 0.76	*	*	NS	NS
Mitotic index	–	48 h	2.55	1.84	2.00	1.34	± 0.14	*	*	NS	NS
Micronucleus (%)	–	48 h	0.44	0.28	0.36	0.21	± 0.04	NS	NS	NS	NS
Nuclear division index	–	48 h	1.98	1.91	1.89	1.99	± 0.03	NS	NS	NS	NS
Sister chromosome exchange	–	48 h	174.75	175.75	180.00	188.00	± 10.19	NS	NS	NS	NS
Proliferation index	+	48 h	2.43	2.51	2.51	2.48	± 0.02	NS	NS	NS	NS
Chromosomal aberrations	+	3 h	10.50	15.00	13.25	14.00	± 4.71	NS	NS	NS	NS
Abnormal cell	+	3 h	10.00	14.00	11.50	11.25	± 1.35	NS	NS	NS	NS
Mitotic index	+	3 h	2.84	2.64	2.56	2.14	± 0.11	*	*	NS	NS
Micronucleus (%)	+	3 h	0.40	0.41	0.32	0.19	± 0.06	NS	NS	NS	NS
Nuclear division index	+	3 h	1.97	1.99	1.98	1.96	± 0.03	NS	NS	NS	NS
Sister chromosome exchange	+	3 h	1.97	1.94	1.93	1.94	± 0.01	NS	NS	NS	NS
Proliferation index		3 h	1.97	1.99	1.98	1.96	± 0.03	NS	NS	NS	NS

SEM pooled standard error of the mean, L linear, Q quadratic, C cubic effects, NS not significant

* p < 0.05

Means for the CA, AC  %, MI, MN %, NDI, PI, SCE at 24 h and 48 h sitagliptin treatment and metabolites of sitagliptin treatment are shown in Table 2. sitagliptin doses had no effect on the CA, AC  %, MI, MN  %, PI, SCE for the 24 h. period, but the effect of sitagliptin levels was linear on the NDI (p < 0.05). For the 48 h period, the effect of sitagliptin levels was linear and cubic on MI, and linear on NDI and PI and there is no effect on CA, AC  %, MN %, SCE (p > 0.05). None of the metabolites had an effect on the analyzed parameters (p > 0.05).

Table 2.

Effects of sitagliptin levels

Analysed parameters	S9 mix	Period	Treatments				SEM	Effects
			Solvent control	Sitagliptin 125 µg/ml	Sitagliptin 250 µg/ml	Sitagliptin 500 µg/ml		P	L	Q	C
Chromosomal aberrations	–	24 h	14.25	11.25	10.25	17.50	1.86	NS	NS	NS	NS
Abnormal cell	–	24 h	12.75	12.75	9.50	16.25	1.60	NS	NS	NS	NS
Mitotic index	–	24 h	2.80	2.34	2.34	2.26	0.18	NS	NS	NS	NS
Micronucleus (%)	–	24 h	0.26	0.45	0.39	0.30	0.07	NS	NS	NS	NS
Nuclear division index	–	24 h	2.03	1.97	1.86	0.69	0.05	*	*	NS	NS
Sister chromosome exchange	–	24 h	161.50	157.75	179.25	169.50	14.90	NS	NS	NS	NS
Proliferation index	–	24 h	2.45	2.25	2.31	2.29	0.06	NS	NS	NS	NS
Chromosomal aberrations	–	48 h	11.50	12.75	15.25	17.00	1.43	NS	NS	NS	NS
Abnormal cell	–	48 h	10.25	11.50	13.25	16.75	1.26	NS	NS	NS	NS
Mitotic index	–	48 h	2.49	2.00	1.96	0.94	0.23	*	*	NS	NS
Micronucleus (%)	–	48 h	0.65	0.40	0.49	0.40	0.10	NS	NS	NS	NS
Nuclear division index	–	48 h	0.97	0.81	10.62	10.19	0.08	*	*	*	NS
Sister chromosome exchange	–	48 h	179.00	183.00	157.00	259.00	16.42	NS	NS	NS	NS
Proliferation index	–	48 h	2.36	2.18	2.12	1.62	0.09	*	*	NS	NS
Chromosomal aberrations	+	3 h	12.00	10.75	13.50	17.00	1.61	NS	NS	NS	NS
Abnormal cell	+	3 h	11.25	10.50	13.00	15.50	1.43	NS	NS	NS	NS
Mitotic index	+	3 h	3.08	3.20	2.58	2.26	0.21	NS	NS	NS	NS
Micronucleus (%)	+	3 h	0.26	0.30	0.24	0.33	0.04	NS	NS	NS	NS
Nuclear division index	+	3 h	10.97	10.98	10.99	10.91	0.03	NS	NS	NS	NS
Sister chromosome exchange	+	3 h	132.00	158.00	156.50	157.00	7.33	NS	NS	NS	NS
Proliferation index	+	3 h	1.93	1.95	1.94	1.86	0.02	NS	NS	NS	NS

SEM pooled standard error of the mean, L linear, Q quadratic, C cubic effects, NS not significant

* p < 0.05

Vildagliptin, sitagliptin and the effects of the metabolites on CA and MI were given (Table 3). According to this study, vildagliptin and sitagliptin didn’t induce chromosome aberration compared to solvent control. However, MI decreased significantly at the maximum doses of vildagliptin at 24 h and 48 h treatment (p < 0.05, p < 0.01).

Table 3.

Chromosome aberrations induced by vildagliptin and sitagliptin in cultured human lymphocytes

Test substance	Concentration (µg/ml)	Period	S9 mix	Type of chromosomal aberration													CA ± SE	Abnormal cell ± SE (%)	MI ± SE
				ctb	csb	f	m	dm	scu	cte	r	cd	cf	dic	p	e
DMSO	1	24 h	–	24	11	7	1	0	0	0	0	3	0	0		0	12.00 ± 2.45	11.25 ± 2.32	2.71 ± 0.16
MMC	0.16	24 h	–	134	262	71	18	1	10	14	0	0	0	0	2	0	128.00 ± 8.57	71.25 ± 2.56	1.60 ± 0.21
Vildagliptin	125	24 h	–	25	18	0	0	0	1	0	1	0	0	0	2	0	11.75 ± 2.36	12.75 ± 2.29	2.09 ± 0.016
Vildagliptin	250	24 h	–	21	15	7	1	0	1	0	0	4	0	0	2	2	13.25 ± 2.43	13.25 ± 2.43	2.48 ± 0.12
Vildagliptin	500	24 h	–	15	14	6	0	0	2	0	1	4	0	0	1	0	10.75 ± 0.48	10.25 ± 0.25	1.69 ± 0.12*,**
DMSO	1	48 h	–	9	7	5	1	0	2	0	0	1	0	3	0	0	3.00 ± 1.91	7.25 ± 1.38	2.55 ± 0.30
MMC	0.16	48 h	–	216	329	348	15	2	0	135	1	0	0	0	5	0	262.8 ± 33.5	93.25 ± 1.65	1.15 ± 0.32
Vildagliptin	125	48 h	–	13	9	6	0	0	0	0	0	4	0	0	0	0	8.00 ± 2.04	7.75 ± 1.89	1.84 ± 0.07
Vildagliptin	250	48 h	–	22	19	5	1	0	0	0	0	2	0	0	0	0	12.25 ± 1.44	11.50 ± 1.32	2.00 ± 0.08
Vildagliptin	500	48 h	–	26	16	3	1	1	0	0	0	4	0	0	1	0	13.00 ± 1.15	10.50 ± 0.29	1.34 ± 0.18*,**
DMSO	1	3 h	+	19	12	5	1	0	0	0	0	0	0	1	4	0	10.50 ± 2.53	10.00 ± 2.04	2.84 ± 0.33
CP	45	3 h	+	54	64	15	0	0	2	0	0	0	0	0	2	0	34.25 ± 1.25	28.25 ± 1.11	2.10 ± 0.33
Vildagliptin	125	3 h	+	29	19	8	2	0	1	0	0	1	0	0	0	0	15.00 ± 2.04	14.00 ± 1.08	2.64 ± 0.14
Vildagliptin	250	3 h	+	17	24	10	0	0	1	0	0	1	0	0	0	0	13.25 ± 4.96	15.33 ± 2.85	2.56 ± 0.09
Vildagliptin	500	3 h	+	26	14	14	1	0	1	0	0	0	0	0	0	0	14.00 ± 4.71	11.25 ± 3.07	2.14 ± 0.17
DMSO	1	24 h	–	33	11	9	0	0	2	0	0	0	0	1	1	0	14.25 ± 4.50	12.75 ± 3.40	2.80 ± 0.31
MMC	0.16	24 h	–	172	291	118	6	0	6	22	6	0	0	2	0	0	155.75 ± 15.72	80.75 ± 3.84	1.16 ± 0.18
Sitagliptin	250	24 h	–	23	10	2	1	0	1	0	0	4	0	0	4	0	11.25 ± 4.40	12.75 ± 4.13	2.34 ± 0.37
Sitagliptin	500	24 h	–	25	10	2	1	0	1	0	0	0	0	0	2	0	10.25 ± 3.33	9.50 ± 2.90	2.34 ± 0.53
Sitagliptin	1000	24 h	–	34	8	10	3	1	2	0	1	6	0	0	5	0	17.50 ± 2.84	16.25 ± 2.59	2.26 ± 0.24
DMSO	1	48 h	–	20	11	8	1	1	5	0	0	0	0	0	0	0	11.50 ± 1.71	10.25 ± 1.60	2.49 ± 0.33
MMC	0.16	48 h	–	264	448	332	16	3	0	133	0	1	0	4	4	0	301.25 ± 60.54	91.25 ± 2.95	1.18 ± 0.26
Sitagliptin	250	48 h	–	19	19	12	0	1	0	0	0	0	0	0	0	0	12.75 ± 2.02	11.50 ± 1.44	2.00 ± 0.44
Sitagliptin	500	48 h	–	36	13	7	2	0	3	0	0	0	0	0	0	0	15.25 ± 4.66	13.25 ± 3.57	1.96 ± 0.55
Sitagliptin	1000	48 h	–	39	13	10	1	0	0	0	0	0	0	0	5	0	17.00 ± 2.48	16.75 ± 2.50	0.94 ± 0.16
DMSO	1	3 h	+	28	10	6	2	0	0	0	0	0	0	0	2	0	12.00 ± 4.38	11.25 ± 3.71	3.08 ± 0.71
CP	45	3 h	+	60	50	12	0	0	2	0	0	0	0	0	3	0	31.75 ± 3.54	26.25 ± 2.06	1.96 ± 0.27
Sitagliptin	250	3 h	+	27	10	5	0	0	0	0	0	1	0	0	0	0	10.75 ± 1.70	10.50 ± 1.71	3.20 ± 0.50
Sitagliptin	500	3 h	+	20	17	13	1	0	0	0	0	0	0	0	3	0	13.50 ± 0.87	13.00 ± 1.00	2.58 ± 0.15
Sitagliptin	1000	3 h	+	37	13	12	0	1	0	0	0	1	0	0	4	0	17.00 ± 4.64	15.50 ± 4.25	2.26 ± 0.21

ctb chromatid break, crb chromosome break, f fragment, m minute, dm double minute, scu sister chromatid union, cte sister chromatid exhange, r ring, cd centromere disassiaion, cf centrik fusion, dic dicentrik chromosome, p polyploidi, e endoreduplication

* Significantly different from the solvent control p < 0.05 (ANOVA)

** Significantly different from the solvent control p < 0.01 (ANOVA)

Vildagliptin, sitagliptin and the effects of the metabolites on micronucleus formation and NBI were given (Table 4). The doses of vildagliptin and its metabolites had no effect on micronucleus formation and NBI (p < 0.05, p < 0.01). sitagliptin and its metabolites didn’t induce micronucleus formation, but sitagliptin decreased NBI at the highest concentration significantly at 24 h and 48 h treatment (p < 0.05).

Table 4.

Micronucleus frequency induced by vildagliptin and sitagliptin in cultured human lymphocytes

Test substance (µg/ml)	Concentration (µg/ml)	Period	S9 mix	BN cells according to MN number					% MN ± SE	Distribution of cells according to nucleus number				NBI ± SE
				0	1	2	3	>3		1	2	3	4
DMSO	1	24 h	–	7970	30	–	–	–	0.38 ± 0.08	731	2916	53	300	1.98 ± 0.07
MMC	0.16	24 h	–	7779	216	5	–	–	2.76 ± 0.44	1917	2077	1	9	1.53 ± 0.13
Vildagliptin	125	24 h	–	7972	25	3	–	–	0.35 ± 0.05	781	2844	60	320	1.98 ± 0.05
Vildagliptin	250	24 h	–	7977	23	–	–	–	0.29 ± 0.06	767	2918	47	266	1.95 ± 0.07
Vildagliptin	500	24 h	–	7980	19	1	–	–	0.25 ± 0.07	1041	2682	71	236	1.89 ± 0.16
DMSO	1	48 h	–	7965	32	2	1	–	0.44 ± 0.13	731	2916	53	300	1.98 ± 0.03
MMC	0.16	48 h	–	6755	1088	143	–	–	15.39 ± 3.00	1917	2077	1	9	1.41 ± 0.06
Vildagliptin	125	48 h	–	7978	22	–	–	–	0.28 ± 0.07	781	2844	60	320	1.91 ± 0.09
Vildagliptin	250	48 h	–	7968	29	–	–	–	0.36 ± 0.07	767	2918	47	266	1.89 ± 0.07
Vildagliptin	500	48 h	–	7983	15	2	–	–	0.21 ± 0.07	1041	2682	71	236	1.99 ± 0.05
DMSO	1	3 h	+	7968	29	3	–	–	0.40 ± 0.11	731	2916	53	300	1.97 ± 0.09
CP	45	3 h	+	7831	161	8	–	–	2.11 ± 0.26	1968	1722	135	175	1.63 ± 0.09
Vildagliptin	125	3 h	+	7967	31	2	–	–	0.41 ± 0.13	781	2844	60	320	1.99 ± 0.05
Vildagliptin	250	3 h	+	7974	23	3	–	–	0.33 ± 0.13	767	2918	47	266	1.98 ± 0.10
Vildagliptin	500	3 h	+	7985	14	1	–	–	0.19 ± 0.09	1041	2682	71	236	1.96 ± 0.05
DMSO	1	24 h	–	7979	20	1	0	0	0.26 ± 0.04	590	3220	59	228	2.03 ± 0.08
MMC	0.16	24 h	–	7735	255	8	1	1	3.31 ± 1.06	1512	2450	8	10	1.62 ± 0.06
Sitagliptin	250	24 h	–	7964	35	1	0	0	0.45 ± 0.24	579	3173	48	200	1.97 ± 0.04
Sitagliptin	500	24 h	–	7969	29	2	0	0	0.39 ± 0.19	812	3035	39	114	1.86 ± 0.08
Sitagliptin	1000	24 h	–	7976	24	0	0	0	0.30 ± 0.07	1289	2664	8	25	1.69 ± 0.08*
DMSO	1	48 h	–	7948	48	4	0	0	0.65 ± 0.34	631	3089	50	230	1.97 ± 0.05
MMC	0.16	48 h	–	6361	1448	163	24	4	20.49 ± 3.83	2130	1798	57	15	1.49 ± 0.08
Sitagliptin	250	48 h	–	7968	31	1	0	0	0.40 ± 0.15	871	3062	17	50	1.81 ± 0.06
Sitagliptin	500	48 h	–	7961	36	3	0	0	0.49 ± 0.20	1536	2446	6	12	1.62 ± 0.10
Sitagliptin	1000	48 h	–	7968	32	0	0	0	0.40 ± 0.09	3259	737	4	0	1.19 ± 0.05*
DMSO	1	3 h	+	7979	20	1	0	0	0.26 ± 0.04	590	3110	59	228	1.97 ± 0.08
CP	45	3 h	+	7831	161	8	–	–	2.11 ± 0.26	1968	1722	135	175	1.63 ± 0.09
Sitagliptin	250	3 h	+	7976	24	0	0	0	0.30 ± 0.12	525	3220	50	202	1.98 ± 0.05
Sitagliptin	500	3 h	+	7981	17	2	0	0	0.24 ± 0.09	577	3132	62	233	1.99 ± 0.05
Sitagliptin	1000	3 h	+	7974	26	0	0	0	0.33 ± 0.06	598	3256	22	119	1.91 ± 0.05

* Significantly different from the solvent control p < 0.05 (ANOVA)

Vildagliptin, sitagliptin and the effects of the metabolites on sister chromatid exchange and PI were given (Table 5). When the effects of vildagliptin, sitagliptin and the metabolites on sister chromatid exchange were examined, no changes were specified. For PI, just the highest sitagliptin concentration decreased PI significantly at 48 h treatment (p < 0.05).

Table 5.

Effects of sitagliptin in SCE and PI in cultured human lymphocytes

Test substance (µg/ml)	Concentartion (µg/ml)	Period	S9 mix	M1	M2	M3	Min–max SCE	Total SCE	SCE/cell ± SE	PI ± SE
DMSO	1	24 h	–	20	137	243	159–194	698	174.5 ± 7.31	2.56 ± 0.06
MMC	0.16	24 h	–	28	256	116	975–1429	4735	1183.8 ± 111.69	2.22 ± 0.03
Vildagliptin	125	24 h	–	38	122	250	148–201	694	173.5 ± 12.55	2.58 ± 0.04
Vildagliptin	250	24 h	–	15	100	285	145–208	656	164 ± 14.75	2.68 ± 0.02
Vildagliptin	500	24 h	–	16	118	266	119–215	615	153.75 ± 13.42	2.63 ± 0.04
DMSO	1	48 h	–	42	143	215	119–215	699	174.75 ± 20.27	2.43 ± 0.06
MMC	0.16	48 h	–	108	290	2	2380–4049	11988	2997 ± 380.60	1.74 ± 0.13
Vildagliptin	125	48 h	–	24	149	227	160–199	703	175.75 ± 9.20	2.51 ± 0.06
Vildagliptin	250	48 h	–	18	162	220	141–237	720	180 ± 20.47	2.51 ± 0.01
Vildagliptin	500	48 h	–	27	156	217	93–243	752	188 ± 33.54	2.48 ± 0.06
DMSO	1	S9 mix	+	44	324	32	115–174	556	139 ± 14.16	1.97 ± 0.02
CP	45	S9 mix	+	100	297	3	532–628	2310	577.5 ± 22.14	1.76 ± 0.02
Vildagliptin	125	S9 mix	+	49	328	23	136–188	636	159 ± 12.38	1.94 ± 0.02
Vildagliptin	250	S9 mix	+	51	327	22	126–185	587	146.75 ± 13.09	1.93 ± 0.02
Vildagliptin	500	S9 mix	+	45	333	22	127–181	626	156.5 ± 11.71	1.94 ± 0.02
DMSO	1	24 h	–	35	152	213	83–225	646	161.50 ± 35.72	2.45 ± 0.10
MMC	0.16	24 h	–	105	229	66	857–1252	4264	1073.5086.87	1.90 ± 0.16
Sitagliptin	250	24 h	–	63	176	161	121–224	631	157.75 ± 22.98	2.25 ± 0.19
Sitagliptin	500	24 h	–	62	151	187	94–231	717	179.25 ± 29.73	2.31 ± 0.12
Sitagliptin	1000	24 h	–	59	167	174	76–270	678	169.50 ± 40.75	2.29 ± 0.12
DMSO	1	48 h	–	53	150	197	101–267	716	179.00 ± 39.66	2.36 ± 0.19
MMC	0.16	48 h	–	129	263	8	1749–3751	10570	2642.50 ± 413.59	1.70 ± 0.13
Sitagliptin	250	48 h	–	63	212	128	117–230	732	183.00 ± 26.23	2.18 ± 0.13
Sitagliptin	500	48 h	–	57	240	103	91–207	628	157.00 ± 25.34	2.12 ± 0.03
Sitagliptin	1000	48 h	–	173	206	21	203–299	1036	259.00 ± 22.57	1.62 ± 0.08*
DMSO	1	S9 mix	+	59	310	31	114–174	528	132.00 ± 14.17	1.93 ± 0.07
CP	45	S9 mix	+	72	325	3	500–676	2391	597.75 ± 39.46	1.83 ± 0.05
Sitagliptin	250	S9 mix	+	44	334	22	127–216	632	158.00 ± 20.32	1.95 ± 0.04
Sitagliptin	500	S9 mix	+	39	348	13	138–168	626	156.50 ± 7.23	1.94 ± 0.03
Sitagliptin	1000	S9 mix	+	82	293	25	115–189	628	157.00 ± 15.81	1.86 ± 0.05

* Significantly different from the solvent control p < 0.05 (ANOVA)

The genotoxic and cytotoxic effects of vildagliptin and sitagliptin were compared via Student’s t-test. The metabolites of the drugs didn’t show any differences in all analysed parameters at all doses and treatments. Minimum doses had no differences significantly at all treatments (p < 0.05, p < 0.01). The differences were determined at intermediate doses for PI at 24 h (t; 2.934, p < 0.01) and 48 h (t 10.887; p < 0.01 and t 10.887; p < 0.05) treatments. Besides, we found out differences at maximum doses for PI at 24 h (t; 2.738; p < 0.05) and 48 h (t; 8.243; p < 0.01; t; 8.243; p < 0.05), NDI at 48 h (t; 11.738; p < 0.01 and t; 11,738; p < 0.05) and AC  % at 48 h (t; − 2.488; p < 0.05) treatments.

According to our results, it can be asserted that vildagliptin and sitagliptin didn’t have any genotoxic effects on lymphocytes in vitro. In addition, vildagliptin and sitagliptin showed cytotoxic potential.

Discussion

T2DM is a deteriorating global health problem and has become a major public health concern. T2DM is characterized by dysregulation of carbohydrate, lipid and protein metabolism. As a result, impaired insulin secretion, insulin resistance or a combination of both can appear (DeFronzo et al. 2015). There are 415 million diabetic patients worldwide according to The International Diabetes Federation’s 2015 data. Moreover, the federation reported that 318 million patients with impaired glucose tolerance will develop diabetes in the future. In the same report, one in every 11 adults had diabetes and one in 10 would have diabetes in 2040 (IDF 2015).

The relationship between diabetes and cancer is still unclear. Recently there are some discussions whether diabetes may be a marker of a biological factor underlying cancer (Giovannucci et al. 2010). Researches in this area have shown that the incidence of certain cancers among diabetic patients in different populations is increasing. In diabetic patients, several studies have indicated that there is an increase in the incidence of pancreatic, liver, endometrium, colorectal, breast and gynaecological cancers; in addition kidney and bladder tumours; in contrast decreases in prostate cancer risk (Okutur 2015). In addition, the relationship between diabetes and cancer may be due to common risk factors such as obesity. Non modifiable factors such as age, gender, race and ethnicity, and factors that can be modifiable such as obesity, nutrition, physical activity, smoking and alcohol consumption have been reported to be common risk factors for diabetes and cancer (Giovannucci et al. 2010). Hyperglycemia is a defining characteristic of diabetes. Also, increased use of glucose is evident in malignant cells. Increased glucose metabolism in cancer cells was described by Warburg in 1956 and this situation is now known as the “Warburg effect”. High glucose obtained by hyperglycemia is thought to function as a “fuel pump” for tumour cells (Warburg 1956). Anti-diabetic drugs have been reported to have acute and chronic side effects such as weight gain, hypoglycemia, oedema, gastrointestinal intolerance. In recent years, it has been suggested that antidiabetic drugs can cause cancer (Cordero et al. 2009; Giovannucci et al. 2010; Matteucci and Giampietro 2011). Since the incidence of cancer is higher in diabetic patients compared to the healthy population, these drugs have become subjects of genotoxicity and cytotoxicity researches (Oz Gul et al. 2013; Engüzel 2015; Harishankar et al. 2015).

Genotoxicity is analyzed during the chemical safety assessment together with other toxicological endpoints in general (Hayashi 2016). The analysis of genotoxicity represents an essential component of the safety assessment of all types of substances such as pesticides, biocides, industrial chemicals, food additives, pharmaceuticals (Corvi and Madia 2017).

Genotoxicity tests defined in vitro and in vivo are used to determine the causative agents that induce genetic damage directly or indirectly by various mechanisms. The application of genotoxicity tests is also important for the design, implementation and interpretation of long-cycle carcinogenicity studies. Due to high sensitivity and rapid yields, in vitro methods are preferred for genotoxicity studies (Jena et al. 2002). With respect to cell kinetic assessment, MI, PI and NDI are analyzed. These parameters are used in the evaluation of cell proliferation, cell division, DNA replication and cell death (Rojas et al. 1993; Mark et al. 1994; Fenech 2000; Atlı Şekeroğlu and Şekeroğlu 2011). In addition, they are used in assessing the cytotoxic effects of chemical or physical agents. Thus, it is possible to measure the cell cycle kinetics with these parameters. Decreases in these parameter ratios are thought to be the result of a delay in the cell cycle. The delay in the process of the cell cycle permits the repair systems to be activated before the DNA replication begins in order to repair the existing genotoxic damage (Laffon et al. 2001). At all of the cell cycle phase, there are check points that allow the cell cycle to stop and the repair mechanisms to be stimulated. If the DNA damage cannot be repaired during the recognized period, the cell goes apoptosis (Maddika et al. 2007).

The presence of numerical and structural CA, MN formation and increasing rate of SCE are important parameters to be used in the detection of damages in genetic material and in the evaluation of genotoxicity of drugs and chemicals (Rojas et al. 1993; Mark et al. 1994; Fenech 2000; Atlı Şekeroğlu and Şekeroğlu 2011).

After the treatment of cultured lymphocytes with sitagliptin concentrations for 24 h and 48 h, our results showed that maximum concentration of the drug is cytotoxic in the normal healthy cells. When compared with solvent control, only the maximum concentration of sitagliptin significantly decreased the MI and NDI at 24 h and 48 h treatment and the PI at 48 h treatment. The results of our study related to sitagliptin are partially in agreement with the previous studies since it was reported that sitagliptin decreased the MI of cultured human lymphocytes at 250–1000 μg/ml concentrations and 48 h treatment, but have no effect on PI and NDI (Yuzbasioglu et al. 2018). An in vivo evaluation of genotoxic and cytotoxic features of oral anti-diabetic agents in patients using these drugs revealed that sitagliptin, rosiglitazone, and pioglitazone have negative effects on three cell-division indices NDI, PI, and MI compared with those who received medical nutrition therapy (Oz Gul et al. 2013).

Presence of cytotoxic effects of sitagliptin were determined by Femia et al. (2013), similar to our study. In the study, the long-term administration of the sitagliptin effects on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis. In the study, male F344 rats were fed on a high-fat diet promoting colon carcinogenesis and insulin resistance and they were induced with DMH (100 mg/kg × 2 times). And then, 1 week later, the animals were allocated into two groups: one continuing with high fat diet and one receiving sitagliptin mixed diet (260 ppm). At the end of the study, 15 weeks after DMH induction, it was identified that the number of the precancerous lesions mucin-depleted foci was significantly lower in rats treated with sitagliptin.

In another study aiming to investigate the direct effect of DPP4 in immune response, it was found that 50 μg/ml and higher concentrations of sitagliptin can inhibit cell proliferation in human lymphocyte cultures (Pinheiro et al. 2017). With having parallel results, this concentration is lower than detected concentration in our study.

A study on healthy cells that evaluates direct cytotoxic effects of vildagliptin in the literature does not exist. On the contrary, it is reported that vildagliptin may increase the cytotoxicity of parthenolide which has anti-leukemic features via selective toxicity against leukaemia cells. The combination of parthenolide and vildagliptin reduced the viability and clonogenic growth of cells from acute myeloid leukaemia patients and had limited effects on the viability of normal human blood stem cells (Spagnuolo 2013). According to our results, vildagliptin significantly decreases the MI alone in the maximum concentration compared with solvent control at 24 h and 48 h treatment.

After analyzing the genotoxic endpoints, we conclude that both vildagliptin and sitagliptin have no genotoxic effects on lymphocytes. These findings are similar to EMEA’s reports about sitagliptin and vildagliptin (EMA 2007, 2012). However, some of the studies in the literature contradict this situation. Yuzbasioglu et al. (2018) observed a significantly increased frequency of CAs and SCEs at 24 h treatment at the highest concentration (1000 μg/ml) and at 48 h treatment of all concentrations of sitagliptin (except 250 μg/ml for CA, except 31.25 and 62.50 μg/ml for SCE). Also, it is reported that this compound increased the MN at only the highest concentration. Another study claim that SITA (100 mg/day) may have genotoxic and cytotoxic effects on patients with T2DM (Oz Gul et al. 2013).

In our study, it was found that there were no differences between the minimum doses of vildagliptin (125 μg/ml) and sitagliptin (250 μg/ml) in terms of genotoxicity and cytotoxicity. In applications of intermediate doses (vildagliptin 250 μg/ml and sitagliptin 500 μg/ml) and maximum doses (vildagliptin 500 μg/ml and sitagliptin 1000 μg/ml), it was determined that sitagliptin was more effective on PI than vildagliptin. It was also found that sitagliptin had higher AC percent and lower NDI in the long-term administration of the maximum dose. On the other hand, it was determined that vildagliptin only affects the MI; therefore, it had a potential to suppress only mitosis. However, sitagliptin affects both MI, NDI and PI and thus suppresses mitosis, nuclear division and replication. At the end of the study, we conclude that sitagliptin was more effective in suppressing cell proliferation.

According to an in vivo mice study sitagliptin and vildagliptin had a mutagenic and toxic effect on the pregnant females and their embryos (Roshdy and Kassem 2013). The researchers evaluated the cytogenetic effects of sitagliptin (0.04 mg/kg/day) alone or with metformin (0.04 mg sitagliptin, 0.2 mg metformin) and vildagliptin (0.04 mg/kg/day) alone or with metformin (0.04 mg vildagliptin, 0.2 mg metformin) in pregnant female mice and their embryos. According to their results, sitagliptin and vildagliptin had a mutagenic and toxic effect on the pregnant females and their embryos. While sitagliptin-metformin combination and vildagliptin-metformin combination had slightly mutagenic and toxic effects on pregnant females and their embryos, these effects decreased significantly in sitagliptin. Besides, vildagliptin treated groups and were similar to the control group.

Amritha et al. (2015) treated HT-29 cell lines with vildagliptin and sitagliptin and used MTT assay to evaluate cytotoxicity. Applied concentrations of sitagliptin and vildagliptin were 7.8, 15.6, 31.2, 62.5, 125, 250, 500, 1000 μg/ml, and the cell viability of sitagliptin was 68.25%, 61.90%, 53.96%, 46.03%, 34.92%, 25.39%, 17.46% and 9.52% and the cell viability of vildagliptin was 74.60%, 69.84%, 65.07%, 57.14%, 49.20%, 39.68%, 28.57% and 15.87%. The researchers reported that the IC 50 value of sitagliptin was 31.2 μg/ml and vildagliptin was 125 μg/ml. Therefore, in this study sitagliptin was reported to be more effective in HT-29 cells. It was reported that both drugs had anticancer effects but the potential of sitagliptin was higher (Amritha et al. 2015). However, in our study we have determined that the IC value is 500 μg/ml for sitagliptin and 250 μg/ml for vildagliptin in healthy peripheral lymphocytes. The determined IC₅₀ concentrations for vildagliptin and sitagliptin in the HT-29 colon cancer cell line were lower when compared to our study with peripheral lymphocytes from healthy volunteers. Besides dose differences, found potantiel effects were different. Amritha et al. (2015) were found more potent sitagliptin than vildagliptin.

The results of CA, SCE and MN assays showed that sitagliptin and vildagliptin were not genotoxic at all the concentrations tested; meanwhile, MI, PI and NDI results showed a dose-dependent decrease and significant differences were found in at least one concentration. Our results demonstrated that these drugs do not represent a significant risk at the genetic level in human lymphocytes cultures. In conclusion, the results of this study suggest that sitagliptin has cytotoxic and cytostatic effects and vildagliptin has cytotoxic effects on human peripheral blood lymphocyte cultures.

This study and previous studies indicate that sitagliptin and vildagliptin may have negative effects on genetic material, cell viability and division (Amritha et al. 2015; Roshdy and Kassem 2013; Yuzbasioglu et al. 2018). Due to this, because of the necessity of long-term use of anti-diabetic drugs, genotoxic and cytotoxic effects of these drugs should be considered when chosing the drug. Especially in some situations such as pregnancy and cancer, the choice of drug should be done carefully owing to potential risks (Andrade et al. 2004; Yang et al. 2012).

Based on our observations about the suppression of cell proliferation by vildagliptin and sitagliptin, these drugs are believed to have a potential to affect the genes and the gene products which are responsible for cell cycle with manage and control cell cycle check point mechanisms. Another issues that need to be clarified are suppression mechanisms of these drugs on cell cycle, without inducing mutations and having genotoxic effects on genes; the possibility of making alterations in expression profiles or epigenetics.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflicts of interest.