Behavioral profile alterations and predation susceptibility of Japanese medaka fish exposed to phenytoin, an antiepileptic drug.

Antiepileptic drugs, such as phenytoin, are often leaked into aquatic systems through sewage facilities due to their low metabolic rate. Fish, such as the Japanese medaka (Oryzias latipes), demonstrate abnormal swimming behavior such as equilibrium abnormalities, rotational behavior, and vertical swimming, when exposed to phenytoin. Therefore, it is hypothesized that predator avoidance may be hindered. This study aimed to investigate the effects of phenytoin exposure-induced behavioral abnormalities in predator avoidance in Japanese medaka. The results showed that individuals with behavioral abnormalities had a reduced ability to avoid danger. Furthermore, the fish demonstrated a delayed recognition reaction to approaching predators. Additionally, predatory fish, such as silver pike characin (Ctenolucius hujeta), were more likely to prey upon abnormal individuals. In conclusion, the fish exposed to phenytoin demonstrated behavioral changes that increased its predation risk. This study is the first to determine the effects of behavioral abnormalities in Japanese medaka which was induced after phenytoin exposure on predator risk avoidance.

Involvement of microRNA-4680-3p against phenytoin-induced cell proliferation inhibition in human palate cells.

Cleft palate (CP) is one of the most common birth defects and is caused by a combination of genetic and/or environmental factors. Environmental factors such as pharmaceutical exposure in pregnant women are known to induce CP. Recently, microRNA (miRNA) was found to be affected by environmental factors. The aim of the present study was to investigate the involvement of miRNA against phenytoin (PHE)-induced inhibition of proliferation in human embryonic palatal mesenchymal (HEPM) cells. We demonstrated that PHE inhibited HEPM cell proliferation in a dose-dependent manner. We found that treatment with PHE downregulated cyclin-D1 and cyclin-E expressions in HEPM cells. Furthermore, PHE increased miR-4680-3p expression and decreased two downstream genes (ERBB2 and JADE1). Importantly, an miR-4680-3p-specific inhibitor restored HEPM cell proliferation and altered expression of ERBB2 and JADE1 in cells treated with PHE. These results suggest that PHE suppresses cell proliferation via modulation of miR-4680-3p expression.

Multi-biomarker approach to evaluate the neurotoxic effects of environmentally relevant concentrations of phenytoin on adult zebrafish Danio rerio.

Several studies have reported the presence of phenytoin (PHE) in wastewater treatment plant effluents, hospital effluents, surface water, and even drinking water. However, published studies on the toxic effects of PHE at environmentally relevant concentrations in aquatic organisms are scarce. The present study aimed to determine the effect of three environmentally relevant concentrations of PHE (25, 282, and 1500 ng L-1) on behavioral parameters using the novel tank test. Moreover, we also aimed to determine whether or not these concentrations of PHE may impair acetylcholinesterase (AChE) activity and oxidative status in the brain of Danio rerio adults. Behavioral responses suggested an anxiolytic effect in PHE-exposed organisms, mainly observed in organisms exposed to 1500 ng L-1, with a significant decrease in fish mobility and a significant increase in activity at the top of the tank. Besides the behavioral impairment, PHE-exposed fish also showed a significant increase in the levels of lipid peroxidation, hydroperoxides, and protein carbonyl content compared to the control group. Moreover, a significant increase in brain AChE levels was observed in fish exposed to 282 and 1500 ng L-1. The results obtained in the present study show that PHE triggers a harmful response in the brain of fish, which in turn generates fish have an anxiety-like behavior.

Acute exposure to environmentally relevant concentrations of phenytoin damages early development and induces oxidative stress in zebrafish embryos.

Phenytoin (PHE) is an antiepileptic drug that has been widely used in clinical practice for about 80 years. It is mainly used in the treatment of tonic-clonic and partial seizures. The widespread consumption of this drug around the world has led to PHE being introduced into water bodies through municipal, hospital, and industrial effluent discharges. Since the toxic effects of this drug on aquatic species has been scarcely explored, the aim of this work was to investigate the influence of low (25-400 ngL-1) and high (500-1500 ngL-1) environmentally relevant concentrations of PHE on the development and oxidative status of zebrafish (Danio rerio) embryos. The toxicity of PHE was evaluated from 12 to 96 h after fertilization in D. rerio at concentrations between 25 and 1500 ngL-1. In both the control group and the 0.05% DMSO system, no malformations were observed, all embryos developed normally after 96 h. The severity and frequency of malformations increased with increasing PHE concentration compared to embryos in the control group. Malformations observed included developmental delay, hypopigmentation, miscellaneous (more than one malformation in the same embryo), modified chorda structure, tail malformation, and yolk deformation. Concerning the biomarkers of oxidative stress, an increase in the degree of lipid peroxidation, protein carbonylation, and hydroperoxide content was observed (p < 0.05) concerning the control. In addition, a significant increase (p < 0.05) in antioxidant enzymes (SOD, CAT, and GPx) was observed at low exposure concentrations (25-400 ngL-1), with a decrease in enzyme activity at high concentrations (500-1500 ngL-1). Our IBR analysis demonstrated that oxidative damage biomarkers got more influence at 500ngL-1 of PHE. The results demonstrated that PHE may affect the embryonic development of zebrafish and that oxidative stress may be involved in the generation of this embryotoxic process.

The effect of phenytoin on embryonic heart rate in Vivo.

Phenytoin is a known human teratogen with unknown etiology. Several mechanisms have been proposed including disturbances in folate metabolism, induction of embryonic hypoxia following phenytoin-induced bradycardia, free radical formation following re-oxygenation and phenytoin-induced maternal hyperglycemia. Using high frequency ultrasound, we demonstrated that phenytoin induced a dramatic decrease in the heart rate of embryos. This coincided with a moderate transient decrease in maternal heart rate and blood glucose levels. Embryonic heart rate had not fully recovered 24 h later in some embryos despite normal maternal physiological parameters. In a separate study, extent of hypoxia was measured using the marker pimonidazole. Phenytoin-exposed embryos did not demonstrate increased hypoxia compared to control embryos at 2, 4, 8 or 24 h dosing. Together our results show that phenytoin induces malformations as a result of a combination of insults: embryonic bradycardia, maternal bradycardia and maternal hyperglycemia. However, this does not appear to result in measurable embryonic hypoxia in our animal model.

Phenytoin Inhibits Cell Proliferation through microRNA-196a-5p in Mouse Lip Mesenchymal Cells.

Cleft lip (CL) is one of the most common birth defects. It is caused by either genetic mutations or environmental factors. Recent studies suggest that environmental factors influence the expression of noncoding RNAs [e.g., microRNA (miRNA)], which can regulate the expression of genes crucial for cellular functions. In this study, we examined which miRNAs are associated with CL. Among 10 candidate miRNAs (miR-98-3p, miR-101a-3p, miR-101b-3p, miR-141-3p, miR-144-3p, miR-181a-5p, miR-196a-5p, miR-196b-5p, miR-200a-3p, and miR-710) identified through our bioinformatic analysis of CL-associated genes, overexpression of miR-181a-5p, miR-196a-5p, miR-196b-5p, and miR-710 inhibited cell proliferation through suppression of genes associated with CL in cultured mouse embryonic lip mesenchymal cells (MELM cells) and O9-1 cells, a mouse cranial neural crest cell line. In addition, we found that phenytoin, an inducer of CL, decreased cell proliferation through miR-196a-5p induction. Notably, treatment with a specific inhibitor for miR-196a-5p restored cell proliferation through normalization of expression of CL-associated genes in the cells treated with phenytoin. Taken together, our results suggest that phenytoin induces CL through miR-196a-5p induction, which suppresses the expression of CL-associated genes.

A model-based analysis of phenytoin and carbamazepine toxicity treatment using binding-competition during hemodialysis.

Hemodialysis (HD) has limited efficacy towards treatment of drug toxicity due to strong drug-protein binding. In this work, we propose to infuse a competitor drug into the extracorporeal circuit that increases the free fraction of a toxic drug and thereby increases its dialytic removal. We used a mechanistic model to assess the removal of phenytoin and carbamazepine during HD with or without binding-competition. We simulated dialytic removal of (1) phenytoin, initial concentration 70 mg/L, using 2000 mg aspirin, (2) carbamazepine, initial concentration 35 mg/L, using 800 mg ibuprofen, in a 70 kg patient. The competitor drug was infused at constant rate. For phenytoin (~ 13% free at t = 0), HD brings the patient to therapeutic concentration in 460 min while aspirin infusion reduces that time to 330 min. For carbamazepine (~ 27% free at t = 0), the ibuprofen infusion reduces the HD time to reach therapeutic concentration from 265 to 220 min. Competitor drugs with longer half-life further reduce the HD time. Binding-competition during HD is a potential treatment for drug toxicities for which current recommendations exclude HD due to strong drug-protein binding. We show clinically meaningful reductions in the treatment time necessary to achieve non-toxic concentrations in patients poisoned with these two prescription drugs.

Phenytoin - An anti-seizure drug: Overview of its chemistry, pharmacology and toxicology.

Phenytoin is a long-standing, anti-seizure drug widely used in clinical practice. It has also been evaluated in the context of many other illnesses in addition to its original epilepsy indication. The narrow therapeutic index of phenytoin and its ubiquitous daily use pose a high risk of poisoning. This review article focuses on the chemistry, pharmacokinetics, and toxicology of phenytoin, with a special focus on its mutagenicity, carcinogenicity, and teratogenicity. The side effects on human health associated with phenytoin use are thoroughly described. In particular, DRESS syndrome and cerebellar atrophy are addressed. This review will help in further understanding the benefits phenytoin use in the treatment of epilepsy.

Intoxicación por fenitoína. Reacción cruzada con barbitúricos / Phenytoin intoxication. Cross-reaction with barbiturates

Las intoxicaciones suponen un motivo frecuente de consulta en Pediatría. El cribado toxicológico en orina es una herramienta de diagnóstico rápido y bajo coste, pero debemos tener precaución en su interpretación, ya que existen reacciones cruzadas con otros fármacos de estructura química similar. Se describe el caso clínico de un paciente de 11 años con afectación neurológica y resultados positivos para barbitúricos en orina. La confirmación de niveles elevados de fenitoína en sangre permite confirmar el diagnóstico y la reactividad cruzada de los resultados en orina

Intoxications are a common reason for consultation in Pediatrics. Urine toxicology screening is a rapid and low-cost tool, but these results have to be interpreted with caution due to cross reactions with other drugs with similar chemical structure. We describe the clinical case of a 11 years old patient with neurological involvement and positive results for barbiturates in urine. Confirmation of high levels of phenytoin in blood guaranteed the diagnosis and cross-reaction results in urine

Dl-3-n-butylphthalide inhibits phenytoin-induced neuronal apoptosis in rat hippocampus and cerebellum.

Rats were divided into six groups: sham/control , Dl-3-n-butylphthalide, P1 (low phenytoin, 100 mg/kg), P2 (high phenytoin, 200 mg/kg), NP1 (Dl-3-n-butylphthalide 80 mg/kg, phenytoin 100 mg/kg), NP2 (Dl-3-n-butylphthalide 80 mg/kg, phenytoin 200 mg/kg). Hematoxylin/eosin and Nissl staining showed that, compared to the sham/control group, the Dl-3-n-butylphthalide group had no obvious hippocampal and cerebellar neuron loss, but there was a significant neuron loss in the P1 and P2 groups (P < 0.05), which was more obvious in the P2 group (P < 0.05). The positive expression of Bax and Bcl-2 proteins in hippocampal and cerebellar neurons was not significantly different between sham and Dl-3-n-butylphthalide groups; however, compared to sham, Bax expression was significantly increased and Bcl-2 was significantly decreased in the hippocampal and cerebellar neurons of rats in both P1 and P2 groups (P < 0.05), being more obvious in the P2 group (P < 0.05). Furthermore, the administration of Dl-3-n-butylphthalide attenuated the deleterious effects of phenytoin (P < 0.05). Our results indicate that phenytoin causes apoptosis of hippocampal and cerebellar neurons in rats in a dose-dependent manner, with the effect of a higher dose being more obvious, whereas, Dl-3-n-butylphthalide inhibits the phenytoin-induced apoptosis of neurons and has a neuroprotective role.

Phenytoin-induced hypothermia.

A 60-year-old man with cerebral palsy and epilepsy was admitted with acute lethargy and deterioration in coordination. He was noted to be hypothermic at 35°C on admission. Routine work-up revealed toxic levels of phenytoin. No cause of hypothermia could be identified but as his phenytoin levels normalised, his body temperature also improved. There are three other reported cases of phenytoin- induced hypothermia in the literature. Could this be a rare cause of hypothermia?

Ameliorative Effects of α-Tocopherol and/or Coenzyme Q10 on Phenytoin-Induced Cognitive Impairment in Rats: Role of VEGF and BDNF-TrkB-CREB Pathway.

Phenytoin is one of the most well-known antiepileptic drugs that cause cognitive impairment which is closely related to cAMP response element-binding protein (CREB) brain-derived neurotrophic factor (BDNF) signaling pathway. Moreover, vascular endothelial growth factor (VEGF), an endothelial growth factor, has a documented role in neurogenesis and neuronal survival and cognitive impairment. Therefore, this study aimed to investigate the influence of powerful antioxidants: α-Toc and CoQ10 alone or combined in the preservation of brain tissues and the maintenance of memory formation in phenytoin-induced cognitive impairment in rats. The following behavioral test novel object recognition and elevated plus maze were assessed after 14 days of treatment. Moreover, VEGF, BDNF, TrkB, and CREB gene expression levels in the hippocampus and prefrontal cortex were estimated using RT-PCR. Both α-Toc and CoQ10 alone or combined with phenytoin showed improvement in behavioral tests compared to phenytoin. Mechanistically, α-Toc and/or CoQ10 decreases the VEGF mRNA expression, while increases BDNF-TrKB-CREB mRNA levels in hippocampus and cortex of phenytoin-treated rats. Collectively, α-Toc and/or CoQ10 alleviated the phenytoin-induced cognitive impairment through suppressing oxidative damage. The underlying molecular mechanism of the treating compounds is related to the VEGF and enhancing BDNF-TrkB-CREB signaling pathway. Our study indicated the usefulness α-Toc or CoQ10 as an adjuvant to antiepileptic drugs with an advantage of preventing cognitive impairment and oxidative stress.

Degradation of 5,5-diphenylhydantoin by chlorination and UV/chlorination: kinetics, transformation by-products, and toxicity assessment.

This study investigated the reaction kinetics and mechanism of the degradation of 5,5-diphenylhydantoin (DPH) during conventional chlorination and UV/chlorination. DPH is one of the antiepileptic drugs, which has frequently been detected in the aquatic environment. For chlorination, the second-order rate constant for the reaction between DPH and free active chlorine (FAC) was determined at pH 5 to 8. At pH 6 to 8, the efficiency of chlorination in the removal of DPH was found to be dominated by the reaction involving hypochlorous acid (HOCl). The result also showed that anionic species of DPH was more reactive toward FAC as compared with neutral DPH. For UV/chlorination, the effect of FAC dosage and pH on the degradation of DPH was evaluated. UV/chlorination is a more effective method for removing DPH as compared with conventional chlorination and UV irradiation. The DPH degradation rate was found to increase with increasing FAC concentration. On the other hand, the degradation of DPH was found to be more favorable under the acidic condition. Based on the identified transformation by-products, DPH was found to be degraded through the reaction at imidazolidine-2,4-dione moiety of DPH for both chlorination and UV/chlorination. Toxicity study on the chlorination and UV/chlorination-treated DPH solutions suggested that UV/chlorination is a more efficient method for reducing the toxicity of DPH.

Phenytoin: neuroprotection or neurotoxicity?

Phenytoin is an 80-year young molecule and new indications are still emerging. The neuroprotective potential of phenytoin has been evaluated for decades. Recently, a positive phase II trial supported its further development in the treatment of optic neuritis in multiple sclerosis. In 1942, however, peripheral neuritis was first reported to be an adverse event of phenytoin, and since then a small but steady stream of publications discussed peripheral polyneuropathy as being a possible adverse event of phenytoin. We have reviewed the literature and concluded there is some supportive evidence for a reversible polyneuropathy after the oral use of phenytoin, though with no evidence for clear neurotoxicity on the level of peripheral nerves. This is probably due to the fact that the pharmacological effects of phenytoin, based on the stabilizing effect of the voltage-gated sodium channels, make impairment of nerve conduction in asymptomatic and symptomatic reversible polyneuropathies plausible. Clear toxically-induced phenytoin-related polyneuropathies, however, are extremely rare and are always related to high dose or high plasma levels of phenytoin, mostly developing during many years of therapy. We could only find one case of a probable reversible chronic phenytoin intoxication resulting in a biopsy proven axonal atrophy with secondary demyelination and signs of remyelination. All case series and case reports published are insufficient in detail to prove a clear causal relation between phenytoin intake and the induction of a peripheral polyneuropathy. Phenytoin does not lead to irreversible toxicity of the peripheral nerves and might, on the other hand, have neuroprotective properties.

Human exposures to pentobarbital-phenytoin combination veterinary drugs.

A combination of pentobarbital and phenytoin is used as a veterinary euthanasia drug. Because of its lethal effect, this study described pentobarbital-phenytoin combination veterinary drug human exposures reported to Texas poison centers during 2000-2015. Of 66 exposures, 73% involved female and 27% male patients. The distribution by patient age was 3% 0-5 years, 5% 6-19 years, 91% 20+ years, and 2% unknown. The most common routes were ocular (41%), ingestion (32%), injection (23%), and dermal (18%). The exposure reasons were unintentional (77%) and intentional (23%). The exposure site was the workplace (52%), patient's own residence (38%), health-care facility (2%), and other/unknown (9%). The management site was managed on site (48%), at/en route to health-care facility (45%), referred to health-care facility (5%), and other (2%). The medical outcomes were no effect (23%), minor effect (30%), moderate effect (8%), major effect (8%), not followed nontoxic (3%), not followed minimal effects (24%), unable to follow potentially toxic (2%), and unrelated (3%). The most common adverse effects were ocular irritation/pain (18%), drowsiness/lethargy (15%), and coma (9%). The most common treatments were dilution/irrigation (70%), intravenous fluids (21%), and oxygen (14%). This study found few pentobarbital-phenytoin combination veterinary drug exposures were reported to Texas poison centers during a 16-year period. Although meant to be administered intravenously, the most common exposure routes were ocular and ingestion. Many of the exposures appeared to be unintentional and occurred at the workplace.

Evaluation of Cytotoxic Effect and Antioxidant Activity of Grape Seed Extract, Crocin, and Phenytoin.

Antioxidant compounds inhibit formation of free radicals, chelate catalytic metals, and scavenge free radicals in biological systems. In addition, antioxidants play a decisive role in prevention of numerous physiological dysfunctions, cancers, and metabolic disorders. This study sought to evaluate the antioxidant capacity and cytotoxic effect of grape seed extract (GSE), crocin (CRO), and phenytoin (PHEN) on a human breast cancer cell line (MCF-7). Methanol extracts of the three mentioned agents were prepared and their antioxidant activity was evaluated by diphenyl-1-picrylhydrazyl method, using Quercetine (QUER) as positive control. The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate the cytotoxic effect of the extracts on Michigan Cancer Foundation-7MCF-7 cell line, using doxorubicin hydrochloride (DOX) as the positive control. Given the results, greater scavenging activity was achieved by using GSE in comparison to CRO and PHEN. Further, a significant correlation was found between the antioxidant activity and cytotoxic effects of these agents, and GSE had the highest antioxidant capacity and cytotoxic effect in comparison to CRO and PHEN.

Phenytoin overdose treated with hemodialysis using a high cut-off dialyzer.

We describe the case of a 52-year-old man who presented after having ingested an unknown quantity of phenytoin. Peak phenytoin concentration was 51.2 mg/L (therapeutic range 10-20 mg/L). Five days after admission, the patient became comatose and was intubated. Because of persistent toxic phenytoin levels and unchanged clinical status for 12 days, hemodialysis (HD) was prescribed to enhance elimination of phenytoin. HD was performed using a Gambro TheraliteTM filter (Baxter International Inc., Deerfield, USA), a high cut-off filter that allows the removal of molecules of up to 45 kDa. Phenytoin concentration readily decreased during the 8-hour HD treatment from 38.9 mg/L to 27.8 mg/L (28.5% decrease); during HD, phenytoin half-life was 18.5h (compared to 1109.8h before HD and 56.3h after HD), phentyoin clearance averaged 80.1 mL/min and a total of 1.1 g of phenytoin was removed. Albumin removal from the Theralite filter was most important at the beginning of HD. The high clearance of phenytoin obtained with this filter was likely due to its high surface area rather than its capacity to remove the albumin-phenytoin complex.

Design, Synthesis, and Evaluation of 2-Amino-6-nitrobenzothiazole-Derived Hydrazones as MAO Inhibitors: Role of the Methylene Spacer Group.

A series of 2-amino-6-nitrobenzothiazole-derived extended hydrazones were designed, synthesized, and investigated for their ability to inhibit monoamine oxidase A and B (MAO-A/MAO-B). The compounds were found to exhibit inhibitory activities in the nanomolar to micromolar range. Some of the compounds showed excellent potency and selectivity against the MAO-B isoform. N'-(5-Chloro-2-oxoindolin-3-ylidene)-2-(6-nitrobenzothiazol-2-ylamino)acetohydrazide (compound 31) showed the highest MAO-B inhibitory activity (IC50 =1.8±0.3 nm, selectivity index [SI]=766.67), whereas compound 6 [N'-(1-(4-bromophenyl)ethylidene)-2-(6-nitrobenzothiazol-2-ylamino)acetohydrazide] was found to be the most active MAO-A inhibitor (IC50 =0.42±0.003 µm). Kinetic studies revealed that compounds 6 and 31 exhibit competitive-type reversible inhibition against both MAO-A and MAO-B, respectively. Structure-activity relationship (SAR) studies disclosed several structural aspects significant for potency and the contribution of the methylene spacer toward MAO-B inhibitory potency, with minimal or no neurotoxicity. Molecular modeling studies yielded a good correlation between experimental and theoretical inhibitory data. Binding pose analysis revealed the significance of cumulative effects of π-π stacking and hydrogen bond interactions for effective stabilization of virtual ligand-protein complexes. Further optimization studies of compound 31, including co-crystallization of inhibitor-MAO-B complexes, are essential to develop these compounds as potential therapeutic agents for MAO-B-associated neurodegenerative diseases.