A Spatial Memory Deficit in Male But Not Female Rats After Neonatal Diazepam Exposure: A New Model for Developmental Sedative Neurotoxicity.

BACKGROUND: Developmental anesthetic neurotoxicity is well described in animal models for GABAergic, sedating drugs. Here we investigate the role of the benzodiazepine, diazepam on spatial and recognition memory of young adult rats after neonatal exposure. METHODS: On postnatal day 7, male (n = 30) and female (n = 30) rats were exposed to diazepam (30 mg/kg intraperitoneally) or vehicle. On postnatal day 42, animals started a series of behavioral tests including Barnes maze (spatial memory), object recognition battery (recognition memory), and open field and elevated plus maze (anxiety). In a separate cohort, blood gases were obtained from diazepam-exposed animals and compared to isoflurane-exposed animals (1 MAC for 4 hours). RESULTS: Male animals exposed to diazepam had impaired performance in the Barnes maze and were unable to differentiate the goal quadrant from chance (1-sample t test; tdiazepam/male (14) = 1.49, P = .158). Female rats exposed to diazepam performed the same as the vehicle controls ( tdiazepam/female (12) = 3.4, P = .005, tvehicle/female (14) = 3.62, P = .003, tvehicle/male (13) = 4.76, P < .001). There were no statistical differences in either males or females in measures of recognition memory, anxiety, or locomotor activity in other behavioral tests. Physiologic measurements of arterial blood gases taken from animals under sedation with diazepam were much less aberrant than those exposed to the volatile anesthetic isoflurane by t test (pH diazepam [M = 7.56, standard deviation {SD} = 0.11] versus pH Isoflurane [M = 7.15, SD = 0.02], t (10) = 8.93, P < .001; Paco 2diazepam [M = 32.8 mm Hg, SD = 10.1] versus Paco 2Isoflurane [M = 91.8 mm Hg, SD = 5.8], t (10) = 8.93, P < .001). CONCLUSIONS: The spatial memory results are consistent with volatile anesthetic suggesting a model in which development of the GABA system plays a critical role in determining susceptibility to behavioral deficits.

Effects of chronic diazepam exposure on the behaviors and oxidative stress homeostasis in the eyes and brains of female Japanese medaka.

Diazepam (DZP) residue has been frequently detected in wastewater, surface water, and groundwater due to its extensive use over the decades. In this study, we exposed female Japanese medaka (Oryzias latipes) to environmentally relevant doses of DZP (800 and 8000 ng/L) for 4 weeks, aimed to investigate their behavioral responses and possible links with ocular and brain oxidative stress homeostasis. As a result, DZP exposure could significantly reduce swimming activity (800 ng/L) and anxiety (800 and 8000 ng/L), indicating a sedative effect on medaka. The DZP exposure also significantly increased the social interaction in medaka at 8000 ng/L. Furthermore, exposure to DZP could alter the ocular and brain oxidative stress homeostasis in medaka. The ocular CAT activities significantly increased in the 800 ng/L-DZP groups, and the brain SOD, CAT, GST and MDA levels also significantly increased in both DZP exposure groups. Correlation analysis revealed that the ocular and brain oxidative stress induced by DZP exposure might play an important role in their behavioral toxicity to medaka. Our findings highlight the necessity to clarify the exact link between DZP exposure-induced oxidative stress in the neural and sensor systems and its behavioral toxicity to better assess the risks on nontarget aquatic species.

Diazepam at environmentally relevant concentrations disturbed social interactions and brain neurotransmitters in adult Japanese medaka (Oryzias latipes).

Pollution by diazepam (DZP) is increasingly recognized as a major threat to aquatic organisms, but knowledge about its potential risk to fish is still limited. In this study, we exposed female and male Japanese medaka (Oryzias latipes) to environmentally relevant DZP (0.8 and 8 µg/L) for 28 days and investigated variation in their behavior (on days 7, 14, and 28) and brain neurotransmitter levels (on day 28). The results showed that DZP could be accumulated in the brain and gonads in Japanese medaka. When two fish of the same sex were placed in an aquarium, DZP exposure exhibited typical sedative effects on females (on day 7) and males (on days 7 and 14). However, these sedative effects on both sexes were no longer present after 28 days of exposure. Exposure to DZP induced sex-specific impacts on the social interactions of medaka on days 7, 14, and 28 of exposure in a time-dependent manner. When both sexes were placed into an aquarium in a ratio of 1:1, DZP could significantly alter their locomotor activity and social interaction on days 14 and 28 of the exposure. After 28 days of exposure, DZP significantly altered the levels of several neurotransmitters in the brain of medaka, also in sex-specific manners. The alterations in dopamine and serotonin levels exhibited significant correlations with the increased social interaction between females. At the same time, that of γ-aminobutyric acid significantly correlated to the decreased social interaction between males. Our findings suggest that chronic exposure to DZP, even at environmentally relevant concentrations, can accumulate in the brains and gonads of fish, and alter their behaviors by mediating brain neurotransmitter levels, which may further disturb their reproduction and population dynamics.

A psychiatric drug found in waste-water plant effluents alters the migratory behavior of critically endangered Anguilla anguilla juveniles.

Migratory fishes cross or settle in several environments potentially polluted. Psychiatric drugs, which represent one growing pollution and are found in discharges from waste-water treatment plants, may alter individual behaviors. Here, we assessed behavioral alterations in the upstream migratory behavior of Anguilla anguilla caused by diazepam, an anxiolytic. We monitored the swimming activity, swimming behavior, and boldness to assess whether diazepam impacts them or not. Our 7-day behavioral follow-up allowed us to test the kinetics of the potential effects of diazepam. We found diazepam reduced swimming activity and altered individual swimming behavior, with fewer individuals swimming against the current, so swimming upstream. Those effects varied over time and were stronger at the end of our monitoring, suggesting chemical pollutants encountered in estuaries may act as a chemical burden for individuals, despite metabolisation. We also found diazepam favored bolder behavior in glass eels. Our results provide new knowledge on chemical pollution and psychiatric drugs inducing behavioral alterations. Those alterations may have ecological and evolutionary consequences for glass eels, by diminishing predator avoidance and impacting spatial colonization, and thus, local density.

Behavioral and electrophysiological (ECoG) effects of haplophyllum robustum and TRPA1 antagonist in adult male wistar rats.

This article aimed to investigate the effects of Haplophyllum robustum hydroalcoholic extract on animals' behavioral and electrocorticographic changes. This plant is mainly found in Turkey, Iran, and Central Asia, and is reported to have convulsive effects. In this article, we worked on the effects of its hydroalcoholic extract on electrocorticography (ECoG), along with changes induced by intracerebroventricular administration of GABAA antagonists. Furthermore, the effects of low doses of this extract on behavioral depression were examined. Four animal sets were used to compare ECoG in Wistar rats. A group of negative control, a group of positive control (PTZ), and two groups received an injection of plant extract (500 mg/kg, ip), with or without administration of Diazepam (5 mg/kg). Also, three sets were applied to compare receiving and not receiving intracerebroventricular (icv) injection of Transient receptor potential ankyrin 1 antagonist (HC-030031) (2 µg/kg) on plant-induced seizure delay and animal death. Two groups of control and a group with plant extract together with TRPA1 antagonist were administrated. Furthermore, in the present study, the forced swimming test (FST) was used as a model of depression. The behaviors of animals in three groups of negative control and positive control (Fluoxetine) and plant extract (200 mg/kg, ip) were compared. According to the ECoG, high doses of extract of plants led to seizures similar to PTZ, which were then reduced by diazepam injection. At this dose, injection of TRPA1 antagonist did not significantly delay the onset of seizures or the death of the animals. Further, a subconvulsive dose of hydroalcoholic plant extracts was equally effective in treating depression as Fluoxetine injections.

Peek-A-Boo Test: A Simple Test for Assessing the Effect of Anxiolytics on Fish Behavior.

The potential of pharmaceuticals and personal care products to alter the behavior of aquatic organisms is a growing concern. To assess the actual effect of these substances on aquatic organisms, a simple but effective behavioral test is required. We devised a simple behavioral (Peek-A-Boo) test to assess the effect of anxiolytics on the behavior of a model fish (medaka, Oryzias latipes). In the Peek-A-Boo test, we investigated the response of medaka to an image of a predator fish (donko fish, Odontobutis obscura). The test revealed that the time taken for test medaka exposed to diazepam (0.8, 4, 20, or 100 µg/L) to approach the image was shorter by a factor of 0.22 to 0.65, and the time spent in the area close to the image was longer by a factor of 1.8 to 2.7 than in the solvent control group for all diazepam exposure groups (p < 0.05). Hence, we confirmed that the test could detect changes in medaka behavior caused by diazepam with high sensitivity. The Peek-A-Boo test we devised is a simple behavioral test with high sensitivity for fish behavioral alteration. Environ Toxicol Chem 2023;42:2358-2363. © 2023 SETAC.

Neuroprotective anticonvulsant and anxiolytic effects of octreotide in wistar rats.

Somatostatin interneurons exhibited anti-epileptic activity. As a result, somatostatin agonists appear to be a promising target for antiepileptic drug development (AEDs). In this regard, we investigated the effects of octreotide, a somatostatin analog, on pentylenetetrazol (PTZ)-induced seizures in male Wistar rats. Animals were given octreotide at doses of 50 or 100 µg/kg for seven days. The anxiolytic effects of octreotide were then evaluated using open field and elevated plus-maze tests. Following that, mice were intraperitoneally given a single convulsive dosage of PTZ (60 mg/kg) and then monitored for 30 min for symptoms of seizures. Finally, the antioxidant capacity of brain tissue and histopathological changes in the hippocampus were investigated. Octreotide therapy for seven days at 50 or 100 µg/kg was more effective than diazepam in preventing acute PTZ-induced seizures (P < 0.05). Furthermore, both octreotide dosages revealed substantial anxiolytic effects in open-field and elevated plus-maze tests compared to untreated rats. Nonetheless, octreotide's anxiolytic impact was less effective than diazepam's. On the other hand, octreotide also suppressed neuronal apoptosis and attenuated oxidative stress. Our results suggest that chronic administration of octreotide has anticonvulsant, anxiolytic, and antioxidant activity in the male Wistar rat model.

Combined effect of diazepam and polystyrene microplastics on the social behavior of medaka (Oryzias latipes).

The combined effect of microplastics and pharmaceuticals on aquatic organisms is an issue of concern. In this laboratory study, we evaluated the combined effect of polystyrene microplastics (2-µm diameter) and diazepam on the social behavior of medaka (Oryzias latipes) by using the shoaling behavior test with five treatment groups: solvent control, polystyrene microplastics exposure (0.04 mg/L), low-concentration diazepam exposure (0.03 mg/L), high-concentration diazepam exposure (0.3 mg/L), and polystyrene microplastics and low-concentration diazepam co-exposure. After 7 days of exposure, the shoal-leaving behavior of the high-concentration diazepam exposure group (8.9 ± 8.3 counts/medaka) and the co-exposure group (6.8 ± 6.7 counts/medaka) was significantly greater than that in the solvent control group (1.8 ± 2.6 counts/medaka). Even after 5 days of recovery, medaka in the co-exposure group left the shoal more often (7.3 ± 5.0 counts/medaka) than those in the solvent control group (2.6 ± 2.6 counts/medaka), whereas the shoal-leaving behavior in other exposure groups, except for the high-concentration diazepam exposure group, was restored. Our findings show that the combined effects of diazepam and polystyrene microplastics suppressed medaka social behavior, suggesting that the presence of microplastics can enhance the adverse effects of pollutants on the social behavior of aquatic organisms.

Evaluation of nootropic activity of telmisartan and metformin on diazepam-induced cognitive dysfunction in mice through AMPK pathway and amelioration of hippocampal morphological alterations.

Cognitive impairments such as dementia are considered the biggest challenges for public health. Benzodiazepines are often prescribed for treatment of anxiety disorder but they are associated with elevated risk of dementia. The present study has been designed to evaluate the neuroprotective effect of telmisartan and metformin on diazepam-induced cognitive dysfunction in mice. Piracetam was used as an established nootropic agent. Mice were divided into 8 groups, group1; control group which received normal saline. groups 2, 3 and 4 were received telmisartan 0.3 mg/kg/day, metformin 100 mg/kg/day and piracetam 200 mg/kg/day respectively. group 5; DZP group that injected with diazepam 2.5 mg/kg, groups 6, 7 and 8 were received diazepam 2.5 mg/kg + telmisartan 0.3 mg/kg/day, metformin 100 mg/kg/day and piracetam 200 mg/kg/day respectively. All drugs were administrated for 15 successive days. Cognitive skills of the animals were examined with Elevated plus maze and Passive Shock Avoidance tests. Investigations of oxidative stress markers were performed. Gene expression levels of TNF-α, NFκB, Caspase 3 and AMPK were analyzed using RT-PCR. Histological and immunohistochemical techniques were performed in hippocampus using H&E, cresyl violet stain, anti GFAP and anti COX-2 immunostain. The study revealed that administration of diazepam increased initial and retention transfer latency as well as it decreased step down latency that means it caused memory impairment. There was a significant increase in hippocampal expression levels of TNF-α, NFκB, and Caspase 3 and downregulation of AMPK expression levels associated with increased neurodegeneration, astrocytes activation and COX-2 immunohistochemical staining. This study indicates that diazepam caused a decline in cognitive function depending on hippocampal activity. Telmisartan, a common antihypertensive agent and metformin, a traditional antidiabetic drug improved this cognitive dysfunction through their anti-oxidant and anti-inflammatory effect as they decreased initial and retention transfer latency as well as it increased step down latency. Also they decreased TNF-α, NFκB, and Caspase 3 and upregulated AMPK expression, moreover they ameliorated the hippocampal morphological alterations, GFAP and COX-2 immunoexpression.

Quality by design (QbD) optimization of diazepam-loaded nanostructured lipid carriers (NLC) for nose-to-brain delivery: Toxicological effect of surface charge on human neuronal cells.

Diazepam is commonly used in the management of epileptic seizures, although it has limitations that can be overcome by using formulations that are easier to administer and capable of directing the drug to the brain. In this field, it has been reported that the use of nanostructured lipid carriers (NLC) via intranasal (or via nose-to-brain) promotes the targeting of drugs to the brain, improving the effectiveness of therapy. The aim of this work was to optimize two diazepam-loaded NLC formulations for nose-to-brain delivery, one with positive surface charge and one with negative surface charge. The quality by design (QbD) approach was used to design the experiments, where the quality target product profile (QTPP), the risk assessment and the critical quality attributes (CQAs) were defined to ensure safety, efficacy and quality of the final formulations. The experiments started with the optimization of critical material attributes (CMAs), related to the ratios of lipids and emulsifiers, followed by the selection of critical process parameters (CPPs), related to the production methods of the diazepam-loaded NLC formulation (ultrasound technique and high-pressure homogenization - HPH). Afterwards, the positive surface charge of the diazepam-loaded NLC was optimized. Finally, the biocompatibility with human neuronal cells of the formulation with a negative surface charge and of the formulation with a positive surface charge was evaluated. The results of the optimization of the CMAs showed that the ratios of lipids and emulsifiers more adequate were 6.7:2.9 and 4.2:0.3 (% w,w), respectively. Regarding the CPPs, HPH was considered the most suitable production method, resulting in an optimized diazepam-loaded NLC formulation (F1C15) with negative surface charge, showing particle size of 69.59 ± 0.22 nm, polydispersity index (PDI) of 0.19 ± 0.00, zeta potential (ZP) of -23.50 ± 0.24 mV and encapsulation efficiency (EE) of 96.60 ± 0.03 %. The optimized diazepam-loaded NLC formulation (F2A8) with positive surface charge had particle size of 124.40 ± 0.84 nm, PDI of 0.17 ± 0.01, ZP of 32.60 ± 1.13 mV and EE of 95.76 ± 0.24 %. In addition, the incorporation of diazepam in NLC resulted in a sustained release of the drug. No significant changes in particle size, PDI, ZP and EE were observed for the formulation F1C15, after 3 months of storage, whereas for formulation F2A8, particle size increased significantly. Biocompatibility studies showed that the formulation F2A8 was more cytotoxic than the formulation F1C15. Thereby, we conclude that the formulation F1C15 is more suitable for targeting the brain, when compared with the formulation F2A8. From the results of these studies, it can be confirmed that the QbD approach is an adequate and central tool to optimize NLC formulations.

Chlorinated disinfection byproducts of diazepam perturb cell metabolism and induce behavioral toxicity in zebrafish larvae.

Numerous byproducts resulting from chlorinated disinfection are constantly being generated during water treatment processes. The potential risks of these new emerging pollutions remain largely unknown. Here, we determined the risks of chlorinated disinfection byproducts of diazepam (DZP) in the cellular and zebrafish exposure experiments. The cytotoxicity of disinfection byproducts (MACB and MBCC) was greater than DZP in macrophage raw 264.7 cells at 10 mg/L. We further found that the effects of MBCC on the metabolism of glycine, serine, threonine and riboflavin were far greater than DZP by the targeted metabolomics methods. Moreover, MBCC significantly decreased the peak amplitude of neuronal action potential in primary embryonic rat (Spragu-Dawley SD) hippocampal neurons. We finally determined behavioral toxicity of DZP and byproducts in zebrafish larvae. MBCC significantly decreased the maximal swim-activity and peak duration of zebrafish after 72 h exposure. Altogether, these findings indicate the MBCC pose serious pressures on public health.

Bumetanide prevents diazepam-modified anxiety-like behavior in lipopolysaccharide-treated mice.

Benzodiazepine receptor agonists are widely prescribed therapeutic agents that alter gamma-aminobutyric acid (GABA)A receptor activity and have anxiolytic effects. Post-operative use of benzodiazepines is a risk factor of delirium. Inflammatory conditions alter the anxiolytic effects of benzodiazepine. We investigated the effect of diazepam, a typical benzodiazepine anxiolytic, on changes in the emotional behavior of mice in a hole-board test after lipopolysaccharide (LPS) treatment. Diazepam dose-dependently increased the number of head-dips at doses that did not alter locomotor activity; however, diazepam dose-dependently significantly decreased the number of head-dips at doses that did not alter locomotor activity in LPS-treated mice. Flumazenil, a benzodiazepine receptor antagonist, normalized the decrease in head-dipping behavior caused by diazepam treatment in normal and LPS-treated mice. The decrease of the head-dipping effect caused by diazepam was attenuated by minocycline in LPS-treated mice. We further found that the decrease in head-dipping behavior caused by diazepam was blocked by bumetanide, a Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) antagonist, in LPS-treated mice. These findings suggest that diazepam induces the anxiety-like behavior under inflammation conditions, and may cause the GABAA receptor dysfunction associated with the chloride plasticity mediated by NKCC1, which contributes to benzodiazepine-induced delirium after surgery.

Perampanel, a potent AMPA receptor antagonist, protects against tetramethylenedisulfotetramine-induced seizures and lethality in mice: comparison with diazepam.

Tetramethylenedisulfotetramine (TETS), a noncompetitive GABAA receptor antagonist, is a potent, highly lethal convulsant that is considered to be a chemical threat agent. Here, we assessed the ability of the AMPA receptor antagonist perampanel to protect against TETS-induced seizures and lethality in mice when administered before or after treatment with the toxicant. For comparison, we conducted parallel testing with diazepam, which is a first-line treatment for chemically induced seizures in humans. Pre-treatment of mice with either perampanel (1-4 mg/kg, i.p.) or diazepam (1-5 mg/kg, i.p.) conferred protection in a dose-dependent fashion against tonic seizures and lethality following a dose of TETS (0.2 mg/kg, i.p.) that rapidly induces seizures and death. The ED50 values for protection against mortality were 1.6 mg/kg for perampanel and 2.1 mg/kg for diazepam. Clonic seizures were unaffected by perampanel and only prevented in a minority of animals by high-dose diazepam. Neither treatment prevented myoclonic body twitches. Perampanel and diazepam also conferred protection against tonic seizures and lethality when administered 15 min following a 0.14 mg/kg, i.p., dose of TETS and 5 min following a 0.2 mg/kg, i.p., dose of TETS. Both posttreatments were highly potent at reducing tonic seizures and lethality in animals exposed to the lower dose of TETS whereas greater doses of both treatments were required in animals exposed to the larger dose of TETS. Neither treatment was as effective suppressing clonic seizures. In an experiment where 0.4 mg/kg TETS was administered by oral gavage and the treatment drugs were administered 5 min later, perampanel only partially protected against lethality whereas diazepam produced nearly complete protection. We conclude that perampanel and diazepam protect against TETS-induced tonic seizures and lethality but have less impact on clonic seizures. Both drugs could have utility in the treatment of TETS intoxication but neither eliminates all seizure activity.

Behavioral Battery for Testing Candidate Analgesics in Mice. I. Validation with Positive and Negative Controls.

This study evaluated a battery of pain-stimulated, pain-depressed, and pain-independent behaviors for preclinical pharmacological assessment of candidate analgesics in mice. Intraperitoneal injection of dilute lactic acid (IP acid) served as an acute visceral noxious stimulus to produce four pain-related behaviors in male and female ICR mice: stimulation of 1) stretching, 2) facial grimace, 3) depression of rearing, and 4) depression of nesting. Additionally, nesting and locomotion in the absence of the noxious stimulus were used to assess pain-independent drug effects. These six behaviors were used to compare effects of two mechanistically distinct but clinically effective positive controls (ketoprofen and oxycodone) and two negative controls that are not clinically approved as analgesics but produce either general motor depression (diazepam) or motor stimulation (amphetamine). We predicted that analgesics would alleviate all IP acid effects at doses that did not alter pain-independent behaviors, whereas negative controls would not. Consistent with this prediction, ketoprofen (0.1-32 mg/kg) produced the expected analgesic profile, whereas oxycodone (0.32-3.2 mg/kg) alleviated all IP acid effects except depression of rearing at doses lower than those that altered pain-independent behaviors. For the negative controls, diazepam (1-10 mg/kg) failed to block IP acid-induced depression of either rearing or nesting and only decreased IP acid-stimulated behaviors at doses that also decreased pain-independent behaviors. Amphetamine (0.32-3.2 mg/kg) alleviated all IP acid effects but only at doses that also stimulated locomotion. These results support utility of this model as a framework to evaluate candidate-analgesic effects in a battery of complementary pain-stimulated, pain-depressed, and pain-independent behavioral endpoints. SIGNIFICANCE STATEMENT: Preclinical assays of pain and analgesia often yield false-positive effects with candidate analgesics. This study used two positive-control analgesics (ketoprofen, oxycodone) and two active negative controls (diazepam, amphetamine) to validate a strategy for distinguishing analgesics from nonanalgesics by profiling drug effects in a battery of complementary pain-stimulated, pain-depressed, and pain-independent behaviors in male and female mice.

Impacts of chronic exposure to sublethal diazepam on behavioral traits of female and male zebrafish (Danio rerio).

Residues of the psychoactive drug diazepam (DZP) may pose potential risks to fish in aquatic environments, especially by disrupting their behavioral traits. In this study, female and male zebrafish were subjected to chronic exposure (21 days) to sublethal doses (120 and 12 µg/L) of DZP, aimed to compare the characteristics of their behavioral responses to DZP exposure, and to investigate the possible links between those behavioral responses and variations in their brain γ-aminobutyric acid (GABA) and acetylcholinesterase (AChE) levels. Chronic exposure to DZP significantly decreased the swimming velocity and locomotor activity of both genders, indicating a typical sedative effect. Compared with males, whose locomotor activity was only significantly decreased by exposure to DZP for 21 days, females became hypoactive on day 14 (i.e., more sensitive), and they developed tolerance to the hypoactive effect induced by 120 µg/L DZP by day 21. Exposure to DZP significantly disturbed the behavioral traits related to social interactions in females but not in males. Those results indicate that DZP exhibits sex-dependent effects on the behaviors of fish. Moreover, exposure to DZP for 21 days significantly disturbed almost all of the tested behavioral traits associated with courtship when both genders were put together. Sex-dependent responses in brain GABA and AChE levels due to DZP exposure were also identified. Significant relationships between the brain GABA/AChE levels and some behavioral parameters related to locomotor activity were detected in females, but not in males.

Stability of Diazepam Solution for Injection Following Long-Term Storage in an Ambient Temperature of the Mediterranean Climate.

PURPOSE: Diazepam is utilized as a convulsion antidote following nerve gas attacks. As an emergency medicine, it requires storage at ambient temperatures which often doesn't meet manufacturers' requirements, leading to an early invalidation of the product. Current work investigated this issue. METHODS: Long-term stability of diazepam ampoules for injection stored in an ambient temperature of the Mediterranean climate for ~10 years vs storage at room temperature was studied. RESULTS: Diazepam assay and pH remained within pharmacopeial specifications irrespective of storage conditions. A major degradation product 2-methylamino-5-chlorobenzophenone (MACB) showed a clear trend of accumulation as a function of storage time, exceeding the permitted limit at ~2 years, irrespective of storage conditions. A strong correlation between the discoloration of the solutions and the concentration of MACB was obtained. Intravenous administration of MACB to rats at doses ~2200-fold higher than permissible specification levels caused neither mortality nor any toxicological nor post-mortem findings. CONCLUSIONS: Regarding the parameters tested: diazepam assay, MACB assay, and pH, storing ampoules of diazepam solution for injection in field conditions of high temperatures of the Mediterranean climate did not cause accelerated degradation as compared to room temperature. These findings open an option for the usage of expired ampoules in special scenarios.

Optimized Photo-Fenton degradation of psychoactive pharmaceuticals alprazolam and diazepam using a chemometric approach-Structure and toxicity of transformation products.

The objectives of the present study were: a) to evaluate the photocatalytic degradation of two benzodiazepine pharmaceuticals, alprazolam and diazepam, using Photo-Fenton, b) to optimize the experimental parameters through a central composite experimental design, c) to assess their mineralization and toxicity variations and d) to identify the transformation products during the process and to propose transformation pathways. Response Surface Methodology proved to be a useful tool for the optimization of the degradation process as the statistical coefficients (R2 = 0.967 for alprazolam and R2 = 0.929 for diazepam) showed satisfactory values confirming the adequate correlation between the predicted models and experimental values. Two sets of experimental conditions were proposed taking into consideration criteria related to the reaction rate and the minimum use of iron. Toxicity of the system varied with time after the treatment, indicating the gradual production of transformation products which differ in their toxic potential. Fifteen and twenty-three photocatalytic degradation products were identified for ALP and DZP respectively using LC-(ESI)MS/MS. In the case of ALP, the main degradation reactions included, phenyl-group removal and the opening of the 7-membered ring, while for DZP, degradation occurred through hydroxylation, formation of benzophenone and the opening of the 7-membered cyclic group.

Acute and chronic effects of diazepam on the polychaete Hediste diversicolor: Antioxidant, metabolic, pharmacologic, neurotoxic and behavioural mechanistic traits.

Pharmaceutical drugs are widespread environmental contaminants, but data about their adverse effects are still limited to a few compounds. This study analyzed the acute (96 h) and chronic (28 days) impacts of environmentally realistic levels of diazepam (acute exposure: 0.001, 0.01, 0.1, 1, 10 µg/L; chronic exposure: 0.1, 1, 10, 100, 1000 ng/L), in the polychaete Hediste diversicolor, by measuring behavioral and biochemical (catalase [CAT], glutathione-S-transferases [GSTs], cholinesterases [ChEs], glutathione peroxidase [GPx], lipid peroxidation [TBARS]) parameters. Acute exposure to diazepam altered behavioral traits, decreasing burrowing times and causing hyperactivity, whilst burrowing time increased and hypoactivity resulted after chronic exposure. All biomarkers were affected after the chronic exposure, with the exception of lipid peroxidation. Our data demonstrate that realistic levels of diazepam may impair behavioral and biochemical traits in polychaetes, suggesting that diazepam exposure presents a significant challenge to the environment that supports these organisms.

Toxicological outcome of exposure to psychoactive drugs carbamazepine and diazepam on non-target insect Nauphoeta cinerea.

The continuous detection of human pharmaceuticals during environmental biomonitoring is a global concern because of the menaces they may exert on non-target organisms. Carbamazepine (CBZ) and diazepam (DZP) are commonly prescribed psychotropic drugs which have been reported to coexist in the environment globally. Nauphoeta cinerea is a common insect with high ecological impact. This study elucidated the influence of co-exposure to DZP (0.5 and 1.0 µg kg-1 diet) and CBZ (1.5 and 3.0 µg kg-1 diet) for 42 days on the behavior and biochemical responses in Nauphoeta cinerea. Results showed that DZP alone did not induce adverse effect on the behavior and antioxidant status in the exposed insects. However, exposure to CBZ alone and binary mixtures of DZP and CBZ significantly decreased locomotor and exploratory accomplishments evidenced by decreased mobile episodes, total mobile time, maximum speed, total distance traveled, absolute turn angle, body rotation and path efficiency in comparison with control. The decline observed in the exploratory activities of insects fed with CBZ alone and the mixtures was confirmed by track plots and heat maps. Further, acetylcholinesterase and antioxidant enzyme activities decreased significantly whereas reactive oxygen and nitrogen species, nitric oxide and lipid peroxidation levels increased significantly in the hemolymph, head and midgut of insects exposed to CBZ alone and the mixtures. Collectively, CBZ alone and binary mixtures of CBZ and DZP caused neurotoxicity via induction of inflammatory and oxidative stress in insects. Nauphoeta cinerea may be a potential non-target insect model for monitoring ecotoxicological hazard of pharmaceuticals.

Diazepam, metformin, omeprazole and simvastatin: a full discussion of individual and mixture acute toxicity.

High consumption of drugs, combined with their presence in the environment, raises concerns about its consequences. Even though researches are often engaged in analyzing substances separately, that is not the environmental reality. Therefore, the aim of this study was to investigate the acute toxicity of the pharmaceuticals simvastatin, metformin, omeprazole and diazepam, and all possible mixtures between them, to the organism Aliivibrio fischeri, verifying possible synergistic or antagonistic effects and assessing byproducts formation. In terms of individual toxicity, omeprazole is the most toxic of the active ingredients, followed by simvastatin, diazepam and, finally, metformin. When the toxicity of mixtures was tested, synergism, antagonism and hormesis were perceived, most probably generated due to byproducts formation. Moreover, it was observed that even when compounds are at concentrations below the non-observed effect concentration (NOEC), there may be toxicity to the mixture. Hence, this work points to the urgent need for more studies involving mixtures, since chemicals are subject to interactions and modifications, can mix, and potentiate or nullify the toxic effect of each other.