Title:Role of Oxidative Stress and Reactive Metabolites in Cytotoxicity & Mitotoxicity of Clozapine, Diclofenac and Nifedipine in CHO-K1 Cells In Vitro
Volume: 23
Issue: 14
Author(s): Ali Ergüç, Fuat Karakuş, Ege Arzuk, Neliye Mutlu and Hilmi Orhan*
Affiliation:
- Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Ege University, İzmir, Bornova, 35040, Türkiye
- Izmir Biomedicine and Genome Center (İBG-İzmir), İzmir, Balcova, 35340, Türkiye
Keywords:
Cytotoxicity, in vitro, drugs, reactive metabolite, oxidative stress, mitochondria.
Abstract:
Background: CHO-K1 cells were used as in vitro model to explore mechanisms of cytotoxicity of the test drugs.
Aim: To provide in vitro data on toxicity mechanisms of clozapine, diclofenac and nifedipine.
Objective: Cytotoxic mechanisms of clozapine (CLZ), diclofenac (DIC) and nifedipine (NIF) were
studied in CHO-K1 cells in vitro. All three drugs induce adverse reactions in some patients with partially
unknown mechanisms.
Methods: Following the determination of time- and dose-dependency of cytotoxicity by the MTT test,
cytoplasmic membrane integrity was explored by the LDH leakage test. Both end-points were further
examined in the presence of soft and hard nucleophilic agents, glutathione (GSH) and potassium cyanide
(KCN), respectively, and either individual or general cytochrome P450 (CYP) inhibitors, whether CYPcatalysed formation of electrophilic metabolites play a role in the observed cytotoxicity and membrane
damage. The generation of reactive metabolites during the incubations was also explored. Formation of
malondialdehyde (MDA) and oxidation of dihydrofluorescein (DCFH) were monitored whether
peroxidative membrane damage and oxidative stress take place in cytotoxicity. Incubations were also
conducted in the presence of chelating agents of EDTA or DTPA to explore any possible role of metals
in cytotoxicity by facilitating electron transfer in redox reactions. Finally, mitochondrial membrane
oxidative degradation and permeability transition pore (mPTP) induction by the drugs were tested as
markers of mitochondrial damage.
Results: The presence of an individual or combined nucleophilic agents significantly diminished CLZand NIF-induced cytotoxicities, while the presence of both agents paradoxically increased DIC-induced
cytotoxicity by a factor of three with the reason remaining unknown. The presence of GSH significantly
increased DIC-induced membrane damage too. Prevention of membrane damage by the hard nucleophile
KCN suggests the generation of a hard electrophile upon DIC and GSH interaction. The presence of
CYP2C9 inhibitor sulfaphenazole significantly diminished DIC-induced cytotoxicity, probably by
preventing the formation of 4-hydroxylated metabolite of DIC, which further converts to an electrophilic
reactive intermediate. Among the chelating agents, EDTA caused a marginal decrease in CLZ-induced
cytotoxicity, while DIC-induced cytotoxicity was amplified by a factor of five. Both reactive and stable
metabolites of CLZ could be detected in the incubation medium of CLZ with CHO-K1 cells, which are
known to have low metabolic capacity. All three drugs caused a significant increase in cytoplasmic
oxidative stress by means of DCFH oxidation, which was confirmed by increased MDA from cytoplasmic
as well as mitochondrial membranes. The addition of GSH paradoxically and significantly increased DICinduced MDA formation, in parallel with the increase in membrane damage when DIC and GSH
combined.
Conclusion: Our results suggested that the soft electrophilic nitrenium ion of CLZ is not responsible for
the observed in vitro toxicities, and this may originate from a relatively low amount of the metabolite due
to the low metabolic capacity of CHO-K1. A hard electrophilic intermediate may contribute to cellular
membrane damage incubated with DIC, while a soft electrophilic intermediate seems to exacerbate cell
death by a mechanism other than membrane damage. A significant decrease in cytotoxicity of NIF by
GSH and KCN suggested that both soft and hard electrophiles contribute to NIF-induced cytotoxicity.
All three drugs induced peroxidative cytoplasmic membrane damage, while only DIC and NIF induced
peroxidative mitochondrial membrane damage, which suggested mitochondrial processes may contribute
to adverse effects of these drugs in vivo.
