Comparative Study of the Neurotoxic Effects of Pregabalin Versus Tramadol in Rats.

In Egypt, both pregabalin and tramadol misuse increased in the last decade. Although many studies have confirmed the neurotoxic effects of tramadol, those of pregabalin are understudied. The aim of the study is to evaluate the neurotoxic effects of pregabalin compared with tramadol. Thirty male albino rats were included in this experimental study, and they were randomly allocated into three equal groups: group I (normal saline), group II (tramadol misuse), and group III (pregabalin misuse). All rats received the commenced drugs for 1 month. Open field tests were performed on the day of scarification, and after that, cortical samples were taken for immunohistochemical analysis and quantification of dopamine receptors' gene expression. The drug misuse groups showed a significant decrease in weight gain at the end of the study. Open field testing showed the upper hand of controls regarding all of the tested parameters. Tramadol has a more negative impact on the locomotor parameters compared with pregabalin. Both drugs induced relatively low dopamine-1 receptor (D1Rs) expression to dopamine-2 receptors (D2Rs), mimicking the schizophrenia model. Both tramadol and pregabalin were associated with neurotoxic effects in male albino rats. These effects were less noticed with pregabalin. It is suggested that long-term abuse may end in psychosis.

The teratogenic effect of pregabalin on heart, liver and kidney in rats: a light microscopic, electron microscopic and immunohistochemical study.

BACKGROUND: Pregabalin (PGB) was approved as new anti-epileptic drugs with little information about its teratogenic effect. AIM OF THE WORK: to evaluate the developmental toxicity of PGB. MATERIALS AND METHODS: 60 pregnant albino rats were divided into three groups. PGB (500 mg/kg body weight/day) was given to group II, PGB (1250 mg/kg body weight/day) was given to Group III and no medications were given to group I. The pups were normally delivered. Liver, kidney and heart specimens were prepared for histological, immunohistochemical, and morphometric studies. RESULTS: A dose of 500 mg of PGB had minimal toxic effects in the form of mild collagen deposition and moderate positive caspase-3 immunoexpression. PGB dose of 1250 mg/kg induced gross toxic effects in form of degenerated cardiac myofibres, ruptured blood vessels, vacuolations in the renal cortex, fibrosis and strong positive caspase-3 immunoexpression. CONCLUSION: PGB at dose of 500 mg/kg revealed minimal toxic changes. PGB cause embryotoxicity in a dose-dependent manner, as the higher dose induced more degenerative changes.

Gabapentinoid Pharmacology in the Context of Emerging Misuse Liability.

This article will review the epidemiology and pharmacology of gabapentinoids (gabapentin and pregabalin) relevant to their emerging misuse potential and provide guidance for clinical and regulatory management. Gabapentinoids are γ-aminobutyric acid analogues that produce their therapeutic effects by inhibiting voltage-gated calcium channels and decreasing neurotransmitter release. Recently gabapentinoid prescribing and use have increased tremendously. Although traditionally thought to possess a favorable safety profile, gabapentinoid misuse has also risen significantly. Gabapentinoid misuse generally occurs in combination with other substances, most notably opioids, and may be for purposes of eliciting euphoric effects, enhancing the effects of other substances, or self-treating conditions such as withdrawal, pain, anxiety, or insomnia. Given its faster onset, increased bioavailability and potency, and nonsaturable absorption, pregabalin's pharmacokinetics theoretically enhance its misuse liability versus gabapentin. However, gabapentin can produce similar euphoric effects, and epidemiologic studies have identified higher rates of gabapentin misuse in the United States, likely because of greater availability and less regulated prescribing. Although adverse events of gabapentinoid-only ingestion are relatively benign, a growing body of evidence indicates that gabapentinoids significantly increase opioid-related morbidity and mortality when used concomitantly. In addition, significant withdrawal effects may occur on abrupt discontinuation. As a result of these trends, several US states have begun to further regulate gabapentinoid prescribing, reclassifying it as a controlled substance or mandating reporting to local prescription drug-monitoring programs. Although increased regulation of gabapentin prescribing may be warranted, harm reduction efforts and increased patient and provider education are necessary to mitigate this concerning gabapentinoid misuse trend.

Toxicity assessment of concurrent gabapentin/pregabalin administration with high-dose melphalan in autologous hematopoietic cell transplant recipients.

A theoretical pharmacokinetic interaction mediated through L-amino acid transporter 1 and 2 exists between gabapentin (GP) and pregabalin (PG) with melphalan. Peripheral neuropathy is a common toxicity of various multiple myeloma regimens commonly utilized prior to autologous hematopoietic cell transplant (auto-HCT) with high-dose melphalan (HD-Mel). Therefore, it is likely concurrent administration of either GP or PG will occur in patients receiving HD-Mel conditioning for auto-HCT, which could potentially increase cellular uptake and worsen the mucosal injury. A retrospective chart review of adult patients from January 2012 to July 2016 who received HD-Mel (140-200 mg/m2) at West Virginia University Medicine was performed to assess toxicity and outcomes in these patients. A total of 80 patients were included in the study, with 30 patients receiving GP or PG and 50 control patients. There were no significant differences in grade 2 or higher mucositis, admissions for nausea/vomiting/diarrhea, intravenous opioid requirements, oral topical therapies, antidiarrheal medication use, rescue anti-emetics, days of nausea or vomiting, pain scores, neutrophil or platelet engraftment, treatment-related mortality, progression-free survival, or overall survival. Our data suggest that it is safe to continue GP/PG therapy throughout HD-Mel therapy, with no negative transplant outcomes. Prospective studies or evaluations of larger databases are necessary to better characterize the clinical effect of concomitant therapy.

Pregabalin does not Cause Midline Closure Defect but is not as Innocent as It is Thought.

AIM: To investigate the effects of pregabalin on neural tube closure, and other potential effects on other organ systems in a chick embryo model. MATERIAL AND METHODS: Fertilized chicken eggs were divided into groups, and different doses of pregabalin was administered. All embryos were harvested in the 8th day of incubation, and investigated both macroscopically and microscopically against any developmental malformations caused by Pregabalin. RESULTS: Macroscopically not any malformations were detected but macrosomia was statistically significant in medium and high dose groups. Microscopically, vertebral lamina ossification was delayed in some embryos in high dose group but not interpreted as midline closure defect and also not statistically significant. Decrease in the number of renal glomerulus and increase in the tubular damage was statistically significant in medium and high dose groups. Cardiomegaly was also found in some embryos in middle and high dose groups but not statistically significant. CONCLUSION: The use of pregabalin does not cause neural tube closure defect in the embryo unless not exceed recommended maximum dose. Causing macrosomia instead of developmental retardation by Pregabalin is in conflict with the literature. This study revealed that Pregabalin causes fetal nephrotoxicity and macrosomia. These findings indicate that the use of Pregabalin in pregnancy still needs to be accounted as suspicious.

Neurotoxic effects of pregabalin dependence on the brain frontal cortex in adult male albino rats.

Pregabalin (PGB) is an analog of the inhibitory neurotransmitter gamma-aminobutyric acid. The currently available evidence favors the misuse and abuse potential of PGB. However, its neurotoxicity remains unclear. Therefore, this study assessed the toxic effects of chronic pregabalin dependence as well as withdrawal on the cortical neurons of the frontal lobe. This study included eighty adult male albino rats which were divided into three groups. Group I (Control) included 40 rats and was further subdivided into two equal subgroups (IA and IB) as negative and positive controls. Group II (PGB-dependent) included 20 rats which received PGB starting with the therapeutic dose (300 mg/day), then the doses were gradually increased until they reached the dependent dose (3400 mg/day) by the end of the first month. Further, the dependent dose was given daily for another 2 months. Group III (PGB withdrawal) included 20 rats which received PGB as described in group II. After that, administration of PGB was stopped and the rats were kept for another one month. By the end of the experiment, all animals were sacrificed by cervical decapitation. The specimens were taken from the frontal cortex for histologic and immunohistochemical staining as well as morphometric analysis. Sections of the frontal cortex of group II showed changes in the form of disturbed architectural pattern of cortical layers, apoptotic cells, weak immunoexpression of Bcl-2 and VEGF as well as moderate-strong immunoexpression of iNOS and nestin. These expressions were significantly different from the control groups, but they were non-significant in comparison with group III. These findings indicate that chronic PGB dependence induces neurotoxic effects mainly in the form of neuronal apoptosis, gliosis, and oxidative stress injury of the frontal cortex. The PGB- induced neurotoxic effects persisted after withdrawal. The influence of these neurotoxic effects and their relevance to the cognitive or neurologic disorders in PGB-dependent individuals warrants further research. Furthermore, it is recommended to quantify the behavioral changes related to PGB dependence as well as withdrawal in future studies.

Pregabalin induced reproductive toxicity and body weight changes by affecting caspase3 and leptin expression: Protective role of wheat germ oil.

Pregabalin (PGB) drug abuse is common among the youth. It substituted tramadol before its recent schedule as a controlled drug since April 2019. PGB is an antiepileptic drug acting on the central nervous system. It blocks calcium channels regulating the action of neurotransmitters and causing prolonged depolarization. The present study aimed to investigate the toxic effect of long term pregabalin abuse on the reproductive function and body weight in both male and female albino rats and to evaluate the ameliorative effect of wheat germ oil (WGO). Forty-eight rats were randomly divided into eight groups. The first four groups were males and they were treated as follows: control group (1.5 mL saline), WGO group (1.5 mL L/kg), PGB group (300 mg/kg), and protective group (PGB + WGO). All doses were administrated once per day for 60 days by gastric gavage. The second four groups were females. They were divided and treated the same as the male groups. Pregabalin caused significant weight loss, decreased serum triglyceride level, and increased leptin gene expression in all rats. PGB affected male rats reproduction by decreasing total testosterone serum level and inhibiting spermatogenesis. Reproductive toxicity in females was caused by decreasing pituitary steroids, increasing gonadal hormones, and increasing the number of atretic ovarian follicles. Mechanism of toxicity may be attributed to the PGB oxidative stress effect that induced apoptosis and caused diffuse gonadal atrophy. WGO showed a protective effect on PGB induced toxicity as all measured parameters were relatively improved.

Prenatal Pregabalin Exposure Alters Postnatal Pain Sensitivity and Some Behavioral Responses in Adult Offspring Rats.

BACKGROUND: Prenatal antiepileptic drug exposure could demonstrate both congenital malformations and behavioral impairments in offspring. OBJECTIVE: This study was performed to assess the effects of prenatal exposure to pregabalin (PGB) on pain response, anxiety, motor activity and some behavior of adult offspring rats. METHODS: Pregnant Wistar rats received PGB (7.5, 15 and 30 mg/kg/ip) during embryonic days 9.5- 15.5. The pain response, anxiety-like behaviors, locomotor activity, motor balance and coordination and anhedonia of adult offspring were examined by tail-flick and hot plate test, open field test, elevated plus maze (EPM), beam balance test and sucrose preference test in their 60th day of life, respectively. RESULTS: Prenatal exposure to PGB revealed significant dose-dependent reduction in pain sensitivity (increase in pain latency response) in the hot plate test, especially in females, while anxiety-like behavior assessed in EPM and open field significantly reduced in males. In the open field, locomotor activity reduced significantly after exposure to PGB 30 mg/kg and motor coordination decreased dose-dependently, especially in males. Anhedonia, as an indication of sucrose preference or pleasure response, was not changed. CONCLUSION: These findings suggest that prenatal PGB exposure could be associated with significant changes in pain response, anxiety, locomotor activity and coordination in adult offspring rats.

The neurotoxic effect of long-term use of high-dose Pregabalin and the role of alpha tocopherol in amelioration: implication of MAPK signaling with oxidative stress and apoptosis.

Pregabalin abuse has become an emerging concern; thus, the current study has been designed to study the neurotoxic hazards of prolonged high-dose of pregabalin (akin to that abused by addicts) and to evaluate the effect of alpha tocopherol as a possible ameliorating agent. The current study evaluated the brain neurotransmitters; dopamine, glutamate, and norepinephrine. The study also assessed the expression of the apoptosis-related markers Bax, Bcl2, and caspase 3. Western-blotted analysis of the three major mitogen-activated protein kinases (MAPKs), the c-JUN N-terminal kinase (JNK), the p38 MAPK, and the extracellular signal-regulated kinase (ERK), has also been performed. The study also evaluated oxidative stress via assessment of the cortical tissue levels of reduced glutathione and malondialdehyde and the activity of superoxide dismutase. Histopathological examination and histomorphometric evaluation of the darkly degenerated cortical neurons have also been performed. Pregabalin in high doses (150 mg/kg/day and 300 mg/kg/day) disrupted the ERK/JNK/p38-MAPK signaling, reversed the bax/bcl2 ratio, and induced oxidative stress. It also diminished the release of dopamine, glutamate, and norepinephrine and increased the count of degenerated neurons. Alpha tocopherol treatment significantly attenuated the deleterious effects induced by pregabalin. The role of alpha tocopherol in ameliorating the oxidative stress injury, and apoptosis induced by pregabalin, along with its role in normalizing neurotransmitters, modulating the ERK/JNK/p38-MAPK signaling pathways and improving the histopathological cortical changes, offers alpha tocopherol as a promising adjunctive therapy in patients undergoing prolonged pregabalin therapy as those suffering from prolonged seizures and neuropathies.

Assessment of Pregabalin-Induced Cardiotoxicity in Rats: Mechanistic Role of Angiotensin 1-7.

Pregabalin (PRG) possesses great therapeutic benefits in the treatment of epilepsy, neuropathic pain, and fibromyalgia. However, clinical data have reported incidence or exacerbation of heart failure following PRG administration. Experimental data exploring cardiac alterations and its underlying mechanisms are quite scarce. The aim of the present work was to investigate the effect of PRG on morphometric, echocardiographic, neurohumoral, and histopathological parameters in rats. It was hypothesized that alterations in cardiac renin angiotensin system (RAS) might be involved in PRG-induced cardiotoxicity. To further emphasize the role of RAS in the mechanism of PRG-induced cardiotoxicity, the protective potential of diminazene aceturate (DIZE), an ACE2 activator, was investigated. Results showed 44% decrease in ejection fraction and sevenfold increase in plasma N-terminal pro-brain natriuretic peptide. Histopathological examination also showed prominent vacuolar changes and edema in cardiomyocytes. In addition, PRG significantly increased angiotensin II (Ang II), angiotensin converting enzyme (ACE) and angiotensin II type 1 receptor (AT1R) levels, while decreased angiotensin 1-7 (Ang 1-7), angiotensin converting enzyme 2 (ACE2), and Mas receptor (MasR) cardiac levels. DIZE co-administration showed prominent protection against PRG-induced echocardiographic, neurohumoral, and histopathological alterations in rats. In addition, downregulation of ACE/Ang II/AT1R and upregulation of ACE2/Ang 1-7/MasR axes were noted in DIZE co-treated rats. These findings showed, for the first time, the detailed cardiac deleterious effects of PRG in rats. The underlying pathophysiological mechanism is probably mediated via altered balance between the RAS axes in favor to the ACE/Ang II/AT1R pathway. Accordingly, ACE2 activators might represent promising therapeutic agents for PRG-induced cardiotoxicity.

Teratogenic Effects of Third-Generation Antiepileptic Drug, Pregabalin: An In vivo Study.

BACKGROUND: In the recent past, many third-generation antiepileptic drugs (AEDs) including Pregabalin (PGB) were launched for the treatment of diverse forms of epilepsy with better efficacy and safety profile than first-and-second-generation AEDs, but their teratogenic safety has not been established so far. OBJECTIVE: The present study has been undertaken to evaluate the reproductive and teratogenic potential (external and skeletal) of a novel and third generation AED, PGB in pregnant albino rats. METHODS: In this study, pregnant subjects were exposed to clinically relevant doses (41, 82 and 123 mg) of PGB from gestation days 6-20, and sacrificed on GD-21, and their fetuses were collected and examined to identify the birth defects and skeletal anomalies. RESULTS: This study revealed that prenatal exposure to PGB induced dose-dependent substantial fetal resorptions, litter size, fetal length and weight; and variety of minor external and internal malformations in fetuses predominant with limbs, tail, eyes, abdomen including hemorrhages, and poor skeletal ossification. CONCLUSION: Thus, PGB was found to be teratogenic in rats at equivalent therapeutic doses, hence precaution should be taken before prescribing PGB to pregnant women with epilepsy.

A novel therapeutic with two SNAP-25 inactivating proteases shows long-lasting anti-hyperalgesic activity in a rat model of neuropathic pain.

A pressing need exists for long-acting, non-addictive medicines to treat chronic pain, a major societal burden. Botulinum neurotoxin type A (BoNT/A) complex - a potent, specific and prolonged inhibitor of neuro-exocytosis - gives some relief in several pain disorders, but not for all patients. Our study objective was to modify BoNT/A to overcome its inability to block transmitter release elicited by high [Ca2+]i and increase its limited analgesic effects. This was achieved by fusing a BoNT/A gene to that for the light chain (LC) of type/E. The resultant purified protein, LC/E-BoNT/A, entered cultured sensory neurons and, unlike BoNT/A, inhibited release of calcitonin gene-related peptide evoked by capsaicin. Western blotting revealed that this improvement could be due to a more extensive truncation by LC/E of synaptosomal-associated protein of Mr = 25 k, essential for neuro-exocytosis. When tested in a rat spared nerve injury (SNI) model, a single intra-plantar (IPL) injection of LC/E-BoNT/A alleviated for ∼2 weeks mechanical and cold hyper-sensitivities, in a dose-dependent manner. The highest non-paralytic dose (75 U/Kg, IPL) proved significantly more efficacious than BoNT/A (15 U/Kg, IPL) or repeated systemic pregabalin (10 mg/Kg, intraperitoneal), a clinically-used pain modulator. Effects of repeated or delayed injections of this fusion protein highlighted its analgesic potential. Attenuation of mechanical hyperalgesia was extended by a second administration when the effect of the first had diminished. When injected 5 weeks after injury, LC/E-BoNT/A also reversed fully-established mechanical and cold hyper-sensitivity. Thus, combining advantageous features of BoNT/E and/A yields an efficacious, locally-applied and long-acting anti-hyperalgesic.

Embryo-Fetal Developmental Toxicity Studies with Pregabalin in Mice and Rabbits.

Pregabalin was evaluated for potential developmental toxicity in mice and rabbits. Pregabalin was administered once daily by oral gavage to female albino mice (500, 1250, or 2500 mg/kg) and New Zealand White rabbits (250, 500, or 1250 mg/kg) during organogenesis (gestation day 6 through 15 [mice] or 6 through 20 [rabbits]). Fetuses were evaluated for viability, growth, and morphological development. Pregabalin administration to mice did not induce maternal or developmental toxicity at doses up to 2500 mg/kg, which was associated with a maternal plasma exposure (AUC0-24 ) of 3790 µg•hr/ml, ≥30 times the expected human exposure at the maximum recommended daily dose (MRD; 600 mg/day). In rabbits, treatment-related clinical signs occurred at all doses (AUC0-24 of 1397, 2023, and 4803 µg•hr/ml at 250, 500, and 1250 mg/kg, respectively). Maternal toxicity was evident at all doses and included ataxia, hypoactivity, and cool to touch. In addition, abortion and females euthanized moribund with total resorption occurred at 1250 mg/kg. There were no treatment-related malformations at any dose. At 1250 mg/kg, compared with study and historical controls, the percentage of fetuses with retarded ossification was significantly increased and the mean number of ossification sites was decreased, which correlated with decreased fetal and placental weights, consistent with in utero growth retardation. Therefore, the no-effect dose for developmental toxicity in rabbits was 500 mg/kg, which produced systemic exposure approximately 16-times human exposure at the MRD. These findings indicate that pregabalin, at the highest dose tested, was not teratogenic in mice or rabbits.

Developmental Toxicity Studies with Pregabalin in Rats: Significance of Alterations in Skull Bone Morphology.

Pregabalin was administered to pregnant Wistar rats during organogenesis to evaluate potential developmental toxicity. In an embryo-fetal development study, compared with controls, fetuses from pregabalin-treated rats exhibited increased incidence of jugal fused to maxilla (pregabalin 1250 and 2500 mg/kg) and fusion of the nasal sutures (pregabalin 2500 mg/kg). The alterations in skull development occurred in the presence of maternal toxicity (reduced body weight gain) and developmental toxicity (reduced fetal body weight and increased skeletal variations), and were initially classified as malformations. Subsequent investigative studies in pregnant rats treated with pregabalin during organogenesis confirmed the advanced jugal fused to maxilla, and fusion of the nasal sutures at cesarean section (gestation day/postmating day [PMD] 21) in pregabalin-treated groups. In a study designed to evaluate progression of skull development, advanced jugal fused to maxilla and fusion of the nasal sutures was observed on PMD 20-25 and PMD 21-23, respectively (birth occurs approximately on PMD 22). On postnatal day (PND) 21, complete jugal fused to maxilla was observed in the majority of control and 2500 mg/kg offspring. No treatment-related differences in the incidence of skull bone fusions occurred on PND 21, indicating no permanent adverse outcome. Based on the results of the investigative studies, and a review of historical data and scientific literature, the advanced skull bone fusions were reclassified as anatomic variations. Pregabalin was not teratogenic in rats under the conditions of these studies.

Effect of pregabalin on contextual memory deficits and inflammatory state-related protein expression in streptozotocin-induced diabetic mice.

Diabetes mellitus is a metabolic disease characterized by hyperglycemia due to defects in insulin secretion or its action. Complications from long-term diabetes consist of numerous biochemical, molecular, and functional tissue alterations, including inflammation, oxidative stress, and neuropathic pain. There is also a link between diabetes mellitus and vascular dementia or Alzheimer's disease. Hence, it is important to treat diabetic complications using drugs which do not aggravate symptoms induced by the disease itself. Pregabalin is widely used for the treatment of diabetic neuropathic pain, but little is known about its impact on cognition or inflammation-related proteins in diabetic patients. Thus, this study aimed to evaluate the effect of intraperitoneal (ip) pregabalin on contextual memory and the expression of inflammatory state-related proteins in the brains of diabetic, streptozotocin (STZ)-treated mice. STZ (200 mg/kg, ip) was used to induce diabetes mellitus. To assess the impact of pregabalin (10 mg/kg) on contextual memory, a passive avoidance task was applied. Locomotor and exploratory activities in pregabalin-treated diabetic mice were assessed by using activity cages. Using Western blot analysis, the expression of cyclooxygenase-2 (COX-2), cytosolic prostaglandin E synthase (cPGES), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), nuclear factor-ĸB (NF-ĸB) p50 and p65, aryl hydrocarbon receptor (AhR), as well as glucose transporter type-4 (GLUT4) was assessed in mouse brains after pregabalin treatment. Pregabalin did not aggravate STZ-induced learning deficits in vivo or influence animals' locomotor activity. We observed significantly lower expression of COX-2, cPGES, and NF-κB p50 subunit, and higher expression of AhR and Nrf2 in the brains of pregabalin-treated mice in comparison to STZ-treated controls, which suggested immunomodulatory and anti-inflammatory effects of pregabalin. Antioxidant properties of pregabalin in the brains of diabetic animals were also demonstrated. Pregabalin does not potentiate STZ-induced cognitive decline, and it has antioxidant, immunomodulatory, and anti-inflammatory properties in mice. These results confirm the validity of its use in diabetic patients. Graphical abstract Effect of pregabalin on fear-motivated memory and markers of brain tissue inflammation in diabetic mice.

Pathogenesis of Pregabalin-Induced Limb Defects in Mouse Embryos.

PURPOSE: It is known that antiepileptic drugs might adversely affect neuronal function and thus influence brain development. However, we have reported that limb deformities are one of the most prominent disturbances caused by pregabalin (PGB) in the developing embryo. The aim of this work is to gain a better understanding of possible molecular mechanisms behind the musculoskeletal injuries and limb deformities associated with PGB. METHODS: Pregnant mice divided into four groups. Each mouse received an intraperitoneal injection (IP) of 0, 20 (group I), 40 (group II) or 80 (group III) mg/kg/day of PGB during the organogenesis period. On gestational day 18, embryos were separated and their limbs were dissected. Levels of apoptotic proteins were analyzed by Western blotting. To establish whether apoptosis is present in the limbs, the specimens were examined by TUNEL. Pathological findings were also reported as a score ranging from 1 to 3 based on the level of differentiation. RESULTS: Western blot analysis demonstrated that PGB in all PGB-treated groups significantly upregulated the levels of cleaved caspase-3, 8 and 9. Also, the results showed that PGB exposure increased the percentage of TUNEL positive cells in different limb tissues especially the mesenchymal tissue. The histopathological findings revealed that PGB administration to pregnant mice inhibited limb tissue differentiation, albeit to varying degrees. CONCLUSIONS: The result of our study revealed that apoptosis and inhibition of limb tissue differentiation play an important role in the pathogenesis of PGB-induced limb malformations. Both intrinsic and extrinsic caspase-dependent pathways of cell death are important in mediating the abnormal limb development triggered by insult with the PGB. Evaluating the effect of PGB on molecules involved in the cross-talk between intrinsic and extrinsic apoptotic pathways and cell adhesion, migration, proliferation, and differentiation during embryonic development can further help to identify and clarify the involved mechanisms.