Phenytoin: Is it genotoxic in isolated cultured human lymphocytes without metabolic activation by S9?

There are many conflicting reports around the phenytoin (PHT)-induced genotoxic effect especially in the in-vitro studies. PHT was claimed to cause genotoxic effect by the oxidative stress of its metabolic intermediates. However, by reviewing the distribution and activity of the enzymes responsible for PHT metabolism, we found that PHT is rarely metabolized by human lymphocytes. So that, we will use isolated cultured human lymphocytes to determine which is genotoxic, PHT itself or its metabolites? PHT 60 μg/ml were added to lymphocytes before and after metabolic activation by S9. Also, this study will investigate the possible antioxidant genoprotective effects of Thymoquinone (TQ) 1 μM and Curcumin (CMN) 15 μM on the chromosomal injury induced by PHT or its metabolites. After the end of culture period, the effects of PHT on the lymphocytes were investigated by measuring levels of chromosomal aberrations (CAs); mitotic index (MI); reduced glutathione (GSH); malondialdehyde (MDA); and 8-hydroxydeoxyguanosine (8-OH-dG). Only PHT after metabolic activation caused oxidative genotoxic effects which were significantly ameliorated by TQ more than CMN. Hence, the present study is the first to record that PHT without metabolic activation in isolated human lymphocytes from non epileptic donors cause dose dependant direct toxic effect rather than genotoxic effect.


INTRODUCTION
Genotoxins are compounds causing chemical or physical alterations in DNA structure leading to inaccurate replication of that region of the genome (Bajpayee et al., 2005).Approximately 30% of all marketed drugs, exhibit genotoxic effect when tested by the standard genetic toxicology tests (Snyder, 2009).Phenytoin (PHT), the well known antiepileptic drug has been suspected for teratogenic and mutagenic effects during pregnancy (Kaul et al., 2001).However, there are many conflicting reports observed around its genotoxic effect especially in the in-vitro studies (NTP, 1993;IARC, 1996;Snyder and Greenb, 2001;Snyder, 2009).PHT was *Corresponding author.E-mail: ahmedmhes@mans.edu.egFax: +20 (50) 2223613-124.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License claimed to cause genotoxic effect by the oxidative stress of its metabolic intermediates.However, by reviewing the distribution and activity of the enzymes responsible for PHT metabolism, we found that PHT is rarely metabolized by human lymphocytes (Basta-Kaim et al., 2008).
For testing indirect chemical mutagens, human lymphocyte was exposed directly to an Ames-type microsomal (S9) activation system (Sbrana et al., 1984).
The aim of the present study is to use isolated cultured human lymphocytes to determine which genotoxic, PHT itself is or its metabolites after metabolic activation by S9? Also, this study will investigate the possible antioxidant genoprotective effects of Thymoquinone and Curcumin on the chromosomal injury induced by either PHT or its metabolites.

Human blood samples
10 ml fresh venous blood samples were taken from 30 adult donors after consent.All donors were of either sexes between the ages of 20-45 years, apparently healthy, non-smoking, non-alcoholic and they did not take any medications recently.The donors were obtained from the blood-banking Center of Mansoura University Hospital, Mansoura Faculty of Medicine, Egypt.All blood samples were taken on heparin to prevent clotting.

Chemicals
All chemicals and reagents used in this study were of the highest analytical grade from Sigma-Aldrich (St. Louis, MO, USA).Phenytoin was purchased as sodium salt ≥ 99%, 25 g soluble in water.

Isolation and culture of human lymphocytes
Lymphocytes were isolated from whole blood samples and cultured as described by Durante et al. (1998) with minor modification.All blood samples were collected in an isolation tube for blood cells.The sample was centrifuged at 1600 g (2900 rpm) for 20 min, and the layer of mononuclear cells and platelets was collected by a pipette and transferred to 10 ml centrifuge tube.PRMI 1640 medium was added up to 10 ml. and the sample was centrifuged at 390 g (1500 rpm) for 10 min.After the removal of the supernatant, the cell pellet was re-suspended in 10 ml RPMI 1640 medium at a density of 1.0 X 10 6 cells/ml.Isolated lymphocytes from each blood sample were cultured in 10 ml RPMI 1640 culture medium for a total period of 72 h at 37°C in the dark in a 5% CO2 humidified atmosphere (Watson, 1992).

Plan of the study and grouping of isolated lymphocytes
Isolated lymphocytes from each blood samples were divided randomly into 5 groups, each of 6 samples: the 1 st group was none treated; the 2 nd treated with PHT 60 ug/ml (Ponnala et al., 2009); the 3 rd treated with PHT 60 ug/ml plus S9 (Sbrana et al., 1984, Ponnala et al., 2009); the 4 th treated with PHT 60 ug/ml (Ponnala et al., 2009) + S9 + TQ (Khader et al., 2009); the 5th treated with PHT 60 ug/ml + S9 + CMN (Siddique et al, 2010).The potential genotoxic drugs were added twice, at 24 and 48 h from the start of culture period and after stimulation of mitotic division with phytohaemagglutinin that was added at the start of culture period to induce mitosis within 24 h according to standard protocol of Poddar et al. (2004).The genoprotective drugs were added as a prophylactic therapy 2 h prior to addition of the genotoxic drug.
In a pilot study we tried to increase PHT dose above 60 ug/ml (90 & 120), but these doses were very toxic and fatal to isolated lymphocytes as indicated by MI, so that we did not include these fatal doses in this study.

Evaluation of the drug effects
To investigate the chromosomal effect induced by PHT before and after metabolic activation cells were harvested at the end of the culture period (72 h) for screening CAs following the standard protocol (Carrano and Natarajan 1988) in order to avoid heterogenecity of cycle stage of the treated cells and to score only the first division mitotic cells.Colcemid 0.1 ml was added to stop mitosis and prevent spindle formation and was left 1.5 h.The isolated lymphocytes after recovery from the incubator were investigated for chromosomal aberrations (CAs), mitotic index (MI), 8-hydroxy-2'-deoxyguanosine (8-OH-dG), reduced glutathione (GSH) and malondialdehyde (MDA).

Assay of chromosomal aberrations (Karyotype) in isolated lymphocytes using Gimsa stain
It was done according to the protocol of Poddar et al. (2004).Cells were stained using 10% Giemsa for 12 minutes immersed in distilled water for washing and air-dried.Analysis of cytogenetic data was performed using light microscopy.Slides were scored blind and individual aberrations were recorded.Fifty metaphases were examined for each sample in the different groups (300 metaphase for each group), searching for any chromosomal anomalies either structurally or numerically.

Determination of mitotic index (MI) as a measure of cytotoxicity
The mitotic index (MI) was used as indicators of adequate cell proliferation.Its inhibition could be considered as cellular death, or delay in the cell proliferation kinetics (Eroğlu, 2011).The mitotic index evaluates the cytotoxicity of chemical agents (Calderón-Ezquerro et al., 2007).MI, is easily assessed when CAs are performed.The number of lymphocytes in metaphase was counted in 2000 lymphocytes per sample to determine the mitotic index (Kannan et al., 2006).

Measurement of intracellular reduced glutathione (GSH)
Intracellular reduced GSH in the isolated lymphocytes was Figure 1.In vitro effects of PHT 60 µg/ml alone; PHT + s9 combined with either TQ 1 µM or CNM 15 µM on mitotic index (MI) in isolated human lymphocytes after 72 h of incubation (one way ANOVA, mean ± SD).Each group consists of 6 samples, 50 metaphases were examined for each sample = 300 metaphase for each group, c SD = standard deviation, d MI was obtained for each sample by counting metaphases in 2000 cells.P = significant difference when compared with control normal group, P1 = significant difference when compared with PHT + S9 group, P2 = significant when compared with PHT 60 + S9 + CMN extracted according to the method described by Anderson, (1985), and then reduced GSH was measured according to the method described by Beulter et al. (1963) employing colorimetric method using spectrophotometer determination method (JENWAY 6405,spectrophotometer).

Measurement of malondialdehyde level (MDA)
Lipid peroxidation products (MDA) were released from isolated lymphocytes by sonication according to the method described by Stacey and Klaassen (1981).Then MDA was measured by thiobarbituric acid (TBA) test according to the method described by Draper and Hadley (1990), employing colorimetric method using spectrophotometer determination method (JENWAY 6405, spectrophotometer).

Statistical analysis of the data
All statistical calculations of the data were performed with SPSS version 21.Multiple comparisons of the data for each biochemical parameter were performed using one-way analysis of variance (ANOVA) followed by post-Hoc test for comparing the different groups with each other.P-value of ≤ 0.05 was considered significant.

RESULTS
The assessment of the types of chromosomal aberrations (CAs) in the isolated cultured human lymphocytes in all groups of this study caused only structural CAs and no numerical CAs were found.
In vitro effects of Phenytoin (PHT) 60 μg/ ml plus S9: They caused significant increase in the structural CAs when compared to control normal group (Figure 2).They caused also cytotoxicity and decrease in lymphocyte proliferation indicated by significant decrease in the MI In vitro effects of PHT 60 µg/ml alone; PHT + s9 and PHT + s9 combined with either TQ 1 µM or CMN 15 µM on structural chromosomal aberrations (CAs) in isolated human lymphocytes after 72 h of incubation (one way ANOVA, mean ± SD).P = significant difference when compared with control normal group, P1 = significant difference when compared with PHT + S9 group, P2 = significant when compared with PHT 60 + S9 + CMN when compared to control normal group (Figure 1).In addition, there was significant increase in the MDA level, 8-OH-dG level and significant decrease in the GSH level when compared to control group (Figures 1, 2, 3, 4, 5).

The effects of combined use of PHT +S9 with Thymoquinone (TQ) or Curcumin (CMN):
The combined use of PHT +S9 with TQ 1 μM, or CMN 15 μM caused significant decrease in the structural CAs when compared to PHT +S9 treated group (Figure 2).It caused also improvement in lymphocyte proliferation indicated by significant increase in the MI when compared to PHT + S9 group (Figure 1).In addition, there was significant decrease in the MDA and 8-OH-dG levels together with significant increase in the GSH level when compared to PHT + S9 group alone (Figures 3, 4, 5).However, the protective effects of PHT + S9 with TQ treated group on CAs, MI, MDA, GSH, and 8-OH-dG were more significant than the protective effects of PHT + S9 with CMN (Figures 1,2,3,4,5).This means that PHT + S9induced toxic changes were significantly ameliorated by TQ more than CMN.

In vitro effects of phenytoin (PHT) and PHT + S9 on the isolated cultured human lymphocytes
To the best of our knowledge, this study was the first to record that PHT is only genotoxic in isolated human lymphocytes after its metabolic activation by S9, and without this activation genotoxicity doesn't occur.This result was collaborated by other studies that reported PHT genotoxic effect after metabolic activation in the presence of an exogenous metabolic activation system (S9) in bacteria (Sezzano et al., 1982) and Chinese hamster ovary cells (Galloway et al., 1987).In addition this result was consistent with some other studies that hold PHT without metabolic activation is not genotoxic.Witczak et al. (2008) assessed the potential genotoxic effect of PHT therapy in pregnancy on DNA of umbilical cord blood lymphocytes using Micronucleus (MN) assay.They did not show any significant differences between the MN rates of PHT-treated patients and controls, indicating a lack of genotoxicity of the PHT.In addition, Schaumann et al. (1990), tested the potential genotoxic effect of PHT using sister chromatid exchange (SCE) assay in isolated cultured lymphocytes from adult epileptic patients treated with PHT.He did not show any significant differences between the SCE rates of PHTtreated patients and controls, indicating a lack of mutagenicity of the PHT.Also, the negative tests for PHT genotoxicity were observed in germ cells of male Drosophila melunogaster (Woodruff et al., 1985), many strains of Salmonella typhimurium (Leonard et al., 1984), and cultured Chinese hamster ovary cells (Kindig et al., 1992).Large body of evidence supports this notation.
This debate may be solved if we can understand the mechanism of PHT induced-genotoxic effects.Most of the genotoxic effects of PHT were due to PHT metabolic intermediates not the PHT itself, mainly the para hydroxyphenyl phenyl hydantion (p-HPPH) metabolite (Kaul et al., 2001).These metabolic intermediates induce production of ROS leading to exhaustion of the cellular antioxidant systems (Jacobsen et al., 2008) with subsequent oxidation of DNA, proteins, and lipids (Zegura et al., 2004).This toxic effect will lead to oxidative DNA base modification with DNA strand breaks, lipid peroxidation and decreased GSH-mediated cytoprotection (Al-Jassabi and Azirun, 2010).Our study supports this explanation because when we used PHT without metabolic activation by S9, its genotoxic effect was insignificant.However, when S9 was added to PHT, its genotoxic effect was significant indicating that PHT genotoxic effect could be achieved only after metabolic activation to reactive metabolic intermediates.This conclusion may be true if we were able to prove that the isolated lymphocytes do not contain any of the enzymes responsible for PHT metabolism.
There are three main metabolic pathways for the conversion of PHT to reactive metabolic intermediates.
The first include the bioactivation of about 80% of PHT to para hydroxyphenyl phenyl hydantion (p-HPPH) (Soga et al., 2004), a process catalyzed mainly by the CYP2C9 and to much lesser by CYP2C18 and CYP2C19 (Al-Jassabi and Azirun, 2010).The second pathway includes the hydroperoxidase component of Prostaglandin endoperoxide synthetase pathway (Parman et al., 1998).
Last pathway included the bioactivation of PHT to reactive free radical intermediates through the myeloperoxidase enzyme commonly present in leukocytes (Mays et al., 1995).However, by reviewing the distribution and activity of the enzymes responsible for PHT metabolism, we found that the CYP450s present in lymphocytes are mainly CYP1A1, CYP1B1, CYP 2E1 and CYP3A4 (Anzenbacher and Anzenbacherová, 2001) while those included in PHT metabolism are CYP2C9, CYP2C18 and CYP2C19 (Al-Jassabi and Azirun, 2010).The prostaglandin endoperoxide synthetase was found in relatively high concentrations in lymphocytes (Dailey and Imming, 1999), but this enzyme can not metabolize PHT without metabolic activation through addition of high amounts of arachidonic acid (Kubow and Wells, 1989).The last enzyme myeloperoxidase was found to be distributed unequally between leucocytes where it is excess in neutrophils and very little in lymphocytes (Tay et al., 1998).These facts about the metabolism of PHT in isolated human lymphocytes support the results of our study and previous studies with similar results and give an alert for contrary studies to reconsider their results specially when used PHT in isolated non liver cells without metabolic activation.Poojan et al. (2015) hold that curcumin combined with selenite in utero may prevent the disruption of Skin Stem Cell through different mechanisms including de novo GSH biosynthesis.In the same direction, Sankar et al. (2014) support protective effects of curcumin on genotoxic effects exerted by Arsenic in bone marrow cells through attenuation of its drawbacks on chromosomal aberrations, micronuclei formation and DNA damage.Tawfik et al. (2013) studied protective effects of curcumin on irradiated mice and found that it had significant radioprotective and radio-recovery activities.Malhotra et al. (2012) reported that combined treatment with curcumin and resveratrol stimulate apoptosis and hence, they modulates mitotic injury in benzo(a)pyrenetreated mice.Curcumin normalize mRNA expression levels and ameliorate progression of diabetic nephropathy (Ibrahim et al., 2016).Nicotinamide phosphoribosyl transferase and sirtuin proteins play crucial roles in threshold of cell death modulation and curcumin can increase their levels, so it can be potentially used to reduce chemotherapy-induced nephrotoxicity (Ugur et al., 2015).Badary et al. (2007) studied daily intake of TQ to rats after and before benzo(a)pyrene exposure, that significantly reduced the frequencies of CAs and damaged cells.Aboul-Ela (2002) also found that TQ can protect against chromosomal aberrations in mouse cells infected with schistosomiasis.El-Sheikh et al. (2016) found that TQ can reverse intestinal microscopic changes induced by methotrexate and improve oxidative/ nitrosative stress, inflammatory and apoptotic markers in intestine.Gökce et al. (2016) stated that TQ improved decreased levels of oxidative products Like MDA and proinflammatory cytokines, and reduce motor neuron apoptosis.Hepatic level of MDA in mice exposed to Aflatoxin B1, was reduced by TQ pre-treatment (Daba et al., 1998).
In conclusion, TQ more than CMN can significantly ameliorate oxidative genotoxic effects exerted by PHT.PHT without metabolic activation in isolated human lymphocytes from non-epileptic donors cause dose dependant direct toxic effect rather than genotoxic effect.
Figure2.In vitro effects of PHT 60 µg/ml alone; PHT + s9 and PHT + s9 combined with either TQ 1 µM or CMN 15 µM on structural chromosomal aberrations (CAs) in isolated human lymphocytes after 72 h of incubation (one way ANOVA, mean ± SD).P = significant difference when compared with control normal group, P1 = significant difference when compared with PHT + S9 group, P2 = significant when compared with PHT 60 + S9 + CMN