Formulation and pharmacokinetics of multi-layered matrix tablets: Biphasic delivery of diclofenac.

The rapid availability of the drug at the site of action followed by maintaining its effect for a long period of time is of great clinical importance. Thus, the purpose of the present study was to prepare and evaluate multi-layered matrix tablets of diclofenac using Eudragit RL/RS blend to achieve both immediate and sustained therapeutic effects. Diclofenac potassium (25 mg) was incorporated in an outer immediate release layer to provide immediate pain relief whereas diclofenac sodium (75 mg) was incorporated in the inner core to provide extended drug release. Wet granulation was employed to prepare the inner core of the tablets that were further layered with an immediate release drug layer in the perforated pan coater. The in-vitro and in-vivo performance of the developed formulation was compared with the marketed products Voltaren® SR 75 mg and Cataflam® 25 mg. The in-vitro drug release of the prepared formulation showed similarity (f2 = 66.19) to the marketed product. The pharmacokinetic study showed no significant difference (p > 0.05) in AUC0-24 and Cmax between the test and reference formulations. The AUC0-24 values were 105.36 ± 83.3 and 92.87 ± 55.53 µg h/ml whereas the Cmax values were 11.25 ± 6.87 and 12.97 ± 8.45 µg/ml, for the test and reference, respectively. The multi-layered tablets were proved to be bioequivalent with the commercially available tablets and were in agreement with the observed in-vitro drug release results. Stable physical characteristics and drug release profiles were observed in both long term and accelerated conditions stability studies.

Multiple response optimization of processing and formulation parameters of Eudragit RL/RS-based matrix tablets for sustained delivery of diclofenac.

CONTEXT: Multiple response optimization is an efficient technique to develop sustained release formulation while decreasing the number of experiments based on trial and error approach. OBJECTIVE: Diclofenac matrix tablets were optimized to achieve a release profile conforming to USP monograph, matching Voltaren®SR and withstand formulation variables. The percent of drug released at predetermined multiple time points were the response variables in the design. Statistical models were obtained with relative contour diagrams being overlaid to predict process and formulation parameters expected to produce the target release profile. MATERIALS AND METHODS: Tablets were prepared by wet granulation using mixture of equivalent quantities of Eudragit RL/RS at overall polymer concentration of 10-30%w/w and compressed at 5-15KN. RESULTS AND DISCUSSION: Drug release from the optimized formulation E4 (15%w/w, 15KN) was similar to Voltaren, conformed to USP monograph and found to be stable. Substituting lactose with mannitol, reversing the ratio between lactose and microcrystalline cellulose or increasing drug load showed no significant difference in drug release. Using dextromethorphan hydrobromide as a model soluble drug showed burst release due to higher solubility and formation of micro cavities. CONCLUSION: A numerical optimization technique was employed to develop a stable consistent promising formulation for sustained delivery of diclofenac.

The Liver MicroRNA Expression Profiles Associated With Chronic Hepatitis C Virus (HCV) Genotype-4 Infection: A Preliminary Study.

BACKGROUND: MicroRNAs (miRNAs) have been repeatedly shown to play important roles in liver pathologies, including hepatitis, liver cirrhosis, and liver cancer. Egypt has the highest hepatitis C virus (HCV) infection rate worldwide, predominantly involving genotype-4. OBJECTIVES: In this study, we attempted to characterize the miRNA profile of the poorly studied genotype 4 of HCV in chronically infected Egyptian patients to obtain a better understanding of the disease and its complications and help in the design of better management protocols. PATIENTS AND METHODS: We analyzed the expression levels of a selected panel of 94 miRNAs in fresh liver biopsies collected from 50 Egyptian patients diagnosed with chronic HCV infection using quantitative real-time polymerase chain reaction (PCR) assay. Non-parametric tests were used to analyze the expression level of each miRNA and association with the clinicopathological features of enrolled patients in this study. RESULTS: Our results revealed differential expression levels of the analyzed miRNAs compared to the normal controls. Twenty-seven miRNAs (including miR-105, miR-147, miR-149-3p, and miR-196b) showed up-regulation, while 17 miRNAs (including miR-21, miR-122, miR-199a-3p, and miR-223) showed down-regulation. An inverse correlation was observed between levels of miR-95, miR-130a, and miR-142-5p with the blood albumin level. Increased expression levels of seven miRNAs (miR-29c, miR-30c, miR-126, miR-145, miR-199a, miR-199a-3p, and miR-222) were observed with severe chronic hepatic inflammation. Several deregulated miRNAs found in this study have been previously linked to chronic liver inflammation and the risk of hepatocellular carcinoma (HCC) development. CONCLUSIONS: The identified expression profiles of some examined miRNAs might offer important points to consider for the treatment of naive patients and the management of chronically infected HCV patients in Egypt and around the world.

STUDYING THE IMPACT OF FORMULATION AND PROCESSING PARAMETERS ON THE RELEASE CHARACTERISTICS FROM HYDROXYPROPYL METHYLCELLULOSE MATRIX TABLETS OF DICLOFENAC.

Hydrophilic matrices, especially HPMC based, are widely used to provide sustained delivery where drug release occurs mainly by diffusion. A 3(2) full factorial design was used to develop and evaluate HPMC matrix tablet for sustained delivery of diclofenac. The influences of polymer concentration/viscosity, diluent type/ratio, drug load/solubility, compression force and pH change on drug release were investigated. Ten tablet formulations were prepared using wet granulation. HPMC K15M (10-30% w/w) was used as the polymer forming matrix. The release kinetics, compatibility studies, lot reproducibility and effect on storage were discussed. Increasing polymer concentration and compression force showed antagonistic effect on release rate. Mannitol tends to increase release rate more than lactose. Reversing diluent ratio between lactose and MCC did not affect drug release. Changing pH resulted in burst release whereas drug solubility is pH independent. F1 showed similar release to Voltaren SR and followed Higuchi model. Drug and polymer were compatible to each other. The formulation is stable at long and intermediate conditions with a significant increase in release rate at accelerated conditions due to water uptake and polymer swelling. The developed formulation was successful for a sustained delivery of diclofenac.

Monochromatic Infrared Photo Energy Versus Low Level Laser Therapy in Chronic Low Back Pain.

INTRODUCTION: Low back pain (LBP) is the most common musculoskeletal disease. Monochromatic infrared photo energy (MIPE) and low level laser therapy (LLLT) are light modalities used to reduce pain and increase blood flow. The aim of this study was to compare the effects of the MIPE and LLLT in reducing functional disability and pain as well as improving lumbar range of motion (ROM) in patients with chronic LBP. METHODS: Seventy participants with LBP completed the program and were randomly assigned into 2 groups. Group 1 (n = 35) received MIPE and therapeutic exercises. Group 2 (n = 35) received LLLT and therapeutic exercises. Both groups received 2 visits per week for 6 weeks. Outcome measures were functional rating index (FRI), visual analogue scale (VAS) and modified-modified Schober test at baseline and after 6 weeks. RESULTS: There were statistically significant improvements in functional disability, pain and lumbar ROM (P < .05) in each group. However, no significant differences were recorded between the groups (P > .05). CONCLUSION: Therefore, MIPE and LLLT may play a role in treating chronic LBP and there are no differences between the two modalities in improving functional disability, pain and lumbar ROM in patients with chronic LBP.

Monochromatic Infrared Photo Energy versus Low Level Laser Therapy in Patients with Knee Osteoarthritis.

INTRODUCTION: Knee osteoarthritis (KO) is the most common joint disease for which there is no optimal treatment. Monochromatic infrared photo energy (MIPE) is a relatively new light modality used to reduce pain and increase circulation. Low Level Laser Therapy (LLLT) is another light modality used to reduce pain in KO. METHODS: The aim of this study was to compare the effects of the MIPE and LLLT in improving pain and function in KO. Sixty participants with KO completed the program and were randomly assigned into two groups. Group 1 (experimental, n=30) received MIPE and exercises. Group 2 (control, n=30) received LLLT and exercises. Both groups received two visits per week for six weeks. Outcome included pain intensity measured on a visual analogue scale and physical function measured with the lower extremity functional scale, before and after the 12 therapy sessions (6 weeks after the start of the intervention). RESULTS: There were statistically significant improvements in pain intensity and lower extremity functional scale scores (p<0.05) in each group. However, no significant differences were recorded between the groups (p>0.05). CONCLUSION: Therefore, MIPE and LLLT reduce pain and improve function in KO; however, there are no differences between the two modalities in reducing pain and increasing physical function in KO.

Cardiovascular toxicity of Citrus aurantium in exercised rats.

When safety concerns forced the removal of ephedra from the market, other botanicals, including Citrus aurantium or bitter orange (BO) were used as replacements. A major component of the BO extract is synephrine, a chemical that is structurally similar to ephedrine. Because ephedrine has cardiovascular effects that may be exacerbated during physical exercise, the purpose of this study was to determine whether extracts containing synephrine produced adverse effects on the cardiovascular system in exercising rats. Sprague-Dawley rats were dosed daily by gavage for 28 days with 10 or 50 mg of synephrine/kg body weight from one of two different extracts; caffeine was added to some doses. The rats ran on a treadmill for 30 min/day, 3 days/week. Heart rate, blood pressure, body temperature, and QT interval were monitored. Both doses of both extracts significantly increased systolic and diastolic blood pressure for up to 8 h after dosing. Effects on heart rate and body temperature appeared to be due primarily to the effects of caffeine. These data suggest that the combination of synephrine, caffeine, and exercise can have significant effects on blood pressure and do not appear to be effective in decreasing food consumption or body weight.

Physiological effects following administration of Citrus aurantium for 28 days in rats.

BACKGROUND: Since ephedra-containing dietary supplements were banned from the US market, manufacturers changed their formulations by eliminating ephedra and replacing with other botanicals, including Citrus aurantium, or bitter orange. Bitter orange contains, among other compounds, synephrine, a chemical that is chemically similar to ephedrine. Since ephedrine may have cardiovascular effects, the goal of this study was to investigate the cardiovascular effects of various doses of bitter orange extract and pure synephrine in rats. METHOD: Female Sprague-Dawley rats were dosed daily by gavage for 28 days with synephrine from two different extracts. One extract contained 6% synephrine, and the other extract contained 95% synephrine. Doses were 10 or 50mg synephrine/kg body weight from each extract. Additionally, caffeine was added to these doses, since many dietary supplements also contain caffeine. Telemetry was utilized to monitor heart rate, blood pressure, body temperature and QT interval in all rats. RESULTS AND CONCLUSION: Synephrine, either as the bitter orange extract or as pure synephrine, increased heart rate and blood pressure. Animals treated with 95% synephrine showed minimal effects on heart rate and blood pressure; more significant effects were observed with the bitter orange extract suggesting that other components in the botanical can alter these physiological parameters. The increases in heart rate and blood pressure were more pronounced when caffeine was added. None of the treatments affected uncorrected QT interval in the absence of caffeine.

The safety and regulation of natural products used as foods and food ingredients.

The use of botanicals and dietary supplements derived from natural substances as an adjunct to an improved quality of life or for their purported medical benefits has become increasingly common in the United States. This review addresses the safety assessment and regulation of food products containing these substances by the U.S. Food and Drug Administration (FDA). The issue of safety is particularly critical given how little information is available on the toxicity of some of these products. The first section uses case studies for stevia and green tea extracts as examples of how FDA evaluates the safety of botanical and herbal products submitted for consideration as Generally Recognized as Safe under the Federal Food, Drug, and Cosmetics Act. The 1994 Dietary Supplement Health Education Act (DSHEA) created a regulatory framework for dietary supplements. The article also discusses the regulation of this class of dietary supplements under DSHEA and addresses the FDA experience in analyzing the safety of natural ingredients described in pre-market safety submissions. Lastly, we discuss an ongoing interagency collaboration to conduct safety testing of nominated dietary supplements.

Splenda alters gut microflora and increases intestinal p-glycoprotein and cytochrome p-450 in male rats.

Splenda is comprised of the high-potency artificial sweetener sucralose (1.1%) and the fillers maltodextrin and glucose. Splenda was administered by oral gavage at 100, 300, 500, or 1000 mg/kg to male Sprague-Dawley rats for 12-wk, during which fecal samples were collected weekly for bacterial analysis and measurement of fecal pH. After 12-wk, half of the animals from each treatment group were sacrificed to determine the intestinal expression of the membrane efflux transporter P-glycoprotein (P-gp) and the cytochrome P-450 (CYP) metabolism system by Western blot. The remaining animals were allowed to recover for an additional 12-wk, and further assessments of fecal microflora, fecal pH, and expression of P-gp and CYP were determined. At the end of the 12-wk treatment period, the numbers of total anaerobes, bifidobacteria, lactobacilli, Bacteroides, clostridia, and total aerobic bacteria were significantly decreased; however, there was no significant treatment effect on enterobacteria. Splenda also increased fecal pH and enhanced the expression of P-gp by 2.43-fold, CYP3A4 by 2.51-fold, and CYP2D1 by 3.49-fold. Following the 12-wk recovery period, only the total anaerobes and bifidobacteria remained significantly depressed, whereas pH values, P-gp, and CYP3A4 and CYP2D1 remained elevated. These changes occurred at Splenda dosages that contained sucralose at 1.1-11 mg/kg (the US FDA Acceptable Daily Intake for sucralose is 5 mg/kg). Evidence indicates that a 12-wk administration of Splenda exerted numerous adverse effects, including (1) reduction in beneficial fecal microflora, (2) increased fecal pH, and (3) enhanced expression levels of P-gp, CYP3A4, and CYP2D1, which are known to limit the bioavailability of orally administered drugs.

Imidacloprid induces neurobehavioral deficits and increases expression of glial fibrillary acidic protein in the motor cortex and hippocampus in offspring rats following in utero exposure.

Imidacloprid, a neonicotinoid, is one of the fastest growing insecticides in use worldwide because of its selectivity for insects. The potential for neurotoxicity following in utero exposure to imidacloprid is not known. Timed pregnant Sprague-Dawley rats (300-350 g) on d 9 of gestation were treated with a single intraperitoneal injection (i.p.) of imidacloprid (337 mg/kg, 0.75 x LD50, in corn oil). Control rats were treated with corn oil. On postnatal day (PND) 30, all male and female offspring were evaluated for (a) acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity, (b) ligand binding for nicotinic acetylcholine receptors (nAChR) and muscarinic acetylcholine receptors (m2 mAChR), (c) sensorimotor performance (inclined plane, beam-walking, and forepaw grip), and (d) pathological alterations in the brain (using cresyl violet and glial fibrillary acidic protein [GFAP] immunostaining). The offspring of treated mothers exhibited significant sensorimotor impairments at PND 30 during behavioral assessments. These changes were associated with increased AChE activity in the midbrain, cortex and brainstem (125-145% increase) and in plasma (125% increase). Ligand binding densities for [3H]cytosine for alpha4beta2 type nAchR did not show any significant change, whereas [3H]AFDX 384, a ligand for m2mAChR, was significantly increased in the cortex of offspring (120-155% increase) of imidacloprid-treated mothers. Histopathological evaluation using cresyl violet staining did not show any alteration in surviving neurons in various brain regions. On the other hand, there was a rise in GFAP immunostaining in motor cortex layer III, CA1, CA3, and the dentate gyrus subfield of the hippocampus of offspring of imidacloprid-treated mothers. The results indicate that gestational exposure to a single large, nonlethal, dose of imidacloprid produces significant neurobehavioral deficits and an increased expression of GFAP in several brain regions of the offspring on PND 30, corresponding to a human early adolescent age. These changes may have long-term adverse health effects in the offspring.

In utero exposure to nicotine and chlorpyrifos alone, and in combination produces persistent sensorimotor deficits and Purkinje neuron loss in the cerebellum of adult offspring rats.

This study was carried out to investigate the effect of in utero exposure to the cholinotoxicants, nicotine and chlorpyrifos, alone or in combination on neurobehavioral alterations and neuronal morphology latter in adult age. In the present study, 90 days old (corresponding to a human adult age) male and female offspring rats were evaluated for neurobehavioral, and neuropathological alterations following maternal, gestational exposure to nicotine and chlorpyrifos (O,O-diethyl-O-3,5,6-trichloro-2-pyridinyl phosphorothioate), alone and in combination. Female Sprague-Dawley rats (300-350 g) with timed-pregnancy were treated with nicotine (3.3 mg/kg/day, in bacteriostatic water via s.c. implantation of mini osmotic pump), chlorpyrifos (1.0 mg/kg, daily, dermal, in 75% ethanol, 1.0 ml/kg) or a combination of both chemicals, on gestational days (GD) 4-20. Control animals received bacteriostatic water via s.c. implantation of mini osmotic pump and dermal application of 70% ethanol. The offspring at postnatal day (PND) 90 were evaluated for neurobehavioral performance, changes in the activity of plasma butyrylcholinesterase (BChE) and acetylcholinesterase (AChE), and neuropathological alterations in the brain. Neurobehavioral evaluations included beam-walk score, beam-walk time, incline plane performance and forepaw grip time. Male and female offspring from mothers treated with nicotine and CPF, alone or in combination showed impairments in the performance of neurobehavioral tests, indicating sensorimotor deficits. Female offspring from mothers treated with a combination of nicotine and chlorpyrifos showed significant increase in plasma BChE activity. Brain regional AChE activity showed differential increases in male and female offspring. Brainstem and cerebellum of female offspring from mothers treated with nicotine or chlorpyrifos, alone or in combination showed increased AChE activity, whereas brainstem of male offspring from mothers treated with nicotine alone or a combination of nicotine and chlorpyrifos showed increase in AChE activity. Also, male offspring exposed in utero to nicotine exhibited increased AChE activity. Histopathological evaluations using cresyl violet staining showed a decrease in surviving Purkinje neurons in the cerebellum in offspring of all treatments groups. An increase in glial fibrillary acidic protein (GFAP) immuno-staining was observed in cerebellum white matter as well as granular cell layer (GCL) of cerebellum following all exposures. These results indicate that in utero exposure to nicotine and chlorpyrifos, alone and in combination produced significant sensorimotor deficits in male and female offspring, differential increase in brain AChE activity, a decrease in the surviving neurons and an increased expression of GFAP in cerebellum in adult offspring rats at a corresponding human adult age. Collectively, this study demonstrates that maternal exposure to environmental neurotoxic chemicals, i.e., nicotine and chlorpyrifos leads to developmental abnormalities in the offspring that persist latter into adulthood.

Maternal exposure of rats to nicotine via infusion during gestation produces neurobehavioral deficits and elevated expression of glial fibrillary acidic protein in the cerebellum and CA1 subfield in the offspring at puberty.

Maternal smoking during pregnancy is known to be a significant contributor to developmental neurological health problems in the offspring. In animal studies, nicotine treatment via injection during gestation has been shown to produce episodic hypoxia in the developing fetus. Nicotine delivery via mini osmotic pump, while avoiding effects due to hypoxia-ischemia, it also provides a steady level of nicotine in the plasma. In the present study timed-pregnant Sprague-Dawley rats (300-350 g) were treated with nicotine (3.3 mg/kg, in bacteriostatic water via s.c. implantation of mini osmotic pump) from gestational days (GD) 4-20. Control animals were treated with bacteriostatic water via s.c. implantation of mini osmotic pump. Offspring on postnatal day (PND) 30 and 60, were evaluated for changes in the ligand binding for various types of nicotinic acetylcholine receptors and neuropathological alterations. Neurobehavioral evaluations for sensorimotor functions, beam-walk score, beam-walk time, incline plane and grip time response were carried out on PND 60 offspring. Beam-walk time and forepaw grip time showed significant impairments in both male and female offspring. Ligand binding densities for [3H]epibatidine, [3H]cytisine and [3H]alpha-bungarotoxin did not show any significant changes in nicotinic acetylcholine receptors subtypes in the cortex at PND 30 and 60. Histopathological evaluation using cresyl violet staining showed significant decrease in surviving Purkinje neurons in the cerebellum and a decrease in surviving neurons in the CA1 subfield of hippocampus on PND 30 and 60. An increase in glial fibrillary acidic protein (GFAP) immuno-staining was observed in cerebellum white matter as well as granular cell layer of cerebellum and the CA1 subfield of hippocampus on PND 30 and 60 of both male and female offspring. These results indicate that maternal exposure to nicotine produces significant neurobehavioral deficits, a decrease in the surviving neurons and an increased expression of GFAP in cerebellum and CA1 subfield of hippocampus of the offspring on PND 30 and 60. The results show that although 60-day-old male and female rat offspring of mothers exposed to nicotine during gestation did not differ from control in body weight gain or nicotinic acetylcholine receptors ligand binding, they exhibited significant sensorimotor deficits that were consistent with the neuropathological alterations seen in the brain. These neurobehavioral and pathological deficits indicate that maternal nicotine exposure may produce long-term adverse health effects in the offspring.

Newly born cells in the ageing dentate gyrus display normal migration, survival and neuronal fate choice but endure retarded early maturation.

Addition of new granule cells to the dentate gyrus (DG) from stem or progenitor cells declines considerably during ageing. However, potential age-related alterations in migration, enduring survival and neuronal fate choice of newly born cells, and rate of maturation and dendritic growth of newly differentiated neurons are mostly unknown. We addressed these issues by analysing cells that are positive for 5'-bromodeoxyuridine (BrdU), doublecortin (DCX), BrdU and DCX, and BrdU and neuron-specific nuclear antigen (NeuN) in the DG of young adult, middle-aged and aged F344 rats treated with daily injections of BrdU for 12 consecutive days. Analyses performed at 24 h, 10 days and 5 months after BrdU injections reveal that the extent of new cell production decreases dramatically by middle age but exhibits no change thereafter. Interestingly, fractions of newly formed cells that exhibit appropriate migration and prolonged survival, and fractions of newly born cells that differentiate into neurons, remain stable during ageing. However, in newly formed neurons of the middle-aged and aged DG, the expression of mature neuronal marker NeuN is delayed and early dendritic growth is retarded. Thus, the presence of far fewer new granule cells in the aged DG is not due to alterations in the long term survival and phenotypic differentiation of newly generated cells but solely owing to diminished production of new cells. The results also underscore that the capability of the DG milieu to support neuronal fate choice, migration and enduring survival of newly born cells remains stable even during senescence but its ability to promote rapid neuronal maturation and dendritic growth is diminished as early as middle age.

Nestin expression in hippocampal astrocytes after injury depends on the age of the hippocampus.

Analysis of the expression of nestin in reactive astrocytes facilitates quantification of the extent of activation of astrocytes after injury in the mature CNS. We hypothesize that the capability of astrocytes for re-expressing nestin in response to CNS injury diminishes as a function of age. We quantified astrocytes positive for S-100beta protein, glial fibrillary acidic protein (GFAP) and nestin in the hippocampus of young adult, middle-aged, and aged Fischer 344 rats after an intracerebroventricular kainic acid (KA) administration. In all age groups, KA administration induced degeneration of CA3 pyramidal neurons, which led to a significant deafferentation in the CA1 region. The KA-induced neurodegeneration and deafferentation resulted in an increased population of astrocytes positive for S-100beta and glial fibrillary acidic protein (GFAP) in all age groups. Interestingly, these increases were highly comparable across the three age groups. However, in areas of both neurodegeneration and deafferentation, the overall numerical density of nestin-positive reactive astrocytes varied depending on the age at the time of injury with noticeably decreased numerical density in the injured middle-aged and aged hippocampus. In contrast, nestin-immunoreactive radial glia framework after lesion is not impaired with aging in the ependymal lining of the CA3 region.

Maternal exposure to nicotine and chlorpyrifos, alone and in combination, leads to persistently elevated expression of glial fibrillary acidic protein in the cerebellum of the offspring in late puberty.

We previously showed that maternal exposure to nicotine, alone or in combination with chlorpyrifos, caused an increase in glial fibrillary acidic protein (GFAP) immunostaining in the CA1 subfield of hippocampus and cerebellum in postnatal day (PND) 30 offspring. In the present study, PND 60 offspring were evaluated for histopathological and cholinergic effects following maternal exposure to nicotine and chlorpyrifos, alone and in combination. Timed-pregnant Sprague-Dawley rats (300-350 g) were treated daily with nicotine (1 mg/kg, s.c., in normal saline) or chlorpyrifos (0.1 mg/kg, dermal, in ethanol) or a combination of nicotine and chlorpyrifos from gestational days (GD) 4 to 20. Control animals were treated with saline and ethanol. On PND 60, the offspring were evaluated for cholinergic changes and pathological effects. Plasma butyrylcholinesterase (BChE) activity in the female offspring from chlorpyrifos treated mothers showed a significant increase (approximately 183% of control). Male offspring from mothers treated with either chlorpyrifos or nicotine alone showed a significant increase in the acetylcholinesterase (AChE) activity in the brainstem while female offspring from mothers treated with either nicotine or a combination of nicotine and chlorpyrifos showed a significant increase (approximately 134 and 126% of control, respectively) in AChE activity in the brainstem. No significant changes were observed in the ligand binding densities for alpha4beta2 and alpha7 nicotinic acetylcholine receptors in the cortex. Histopathological evaluation using cresyl violet staining showed a significant decrease in surviving Purkinje neurons in the cerebellum of the offspring from nicotine treated mothers. An increase in GFAP immunostaining in cerebellar white matter was observed in the offspring from the mothers treated with nicotine. These results suggest that maternal exposure to real-life levels of nicotine and/or chlorpyrifos causes differential regulation of brainstem AChE activity. Also, nicotine caused a decrease in the surviving neurons and an increased expression of GFAP in cerebellar white matter of the offspring on PND 60. These changes can lead to long-term neurological adverse health effects later in life.

Neurological deficits induced by malathion, DEET, and permethrin, alone or in combination in adult rats.

Malathion (O,O-dimethyl-S-[1,2-carbethoxyethyl]phosphorodithionate), DEET (N,N-diethyl-m-toluamide), and permethrin [(+/-)-cis/trans-3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropane carboxylic acid (3-phenoxyphenyl) methyl ester] are commonly used pesticides. To determine the effects of the dermal application of these chemicals, alone or in combination, the sensorimotor behavior, central cholinergic system, and histopathological alterations were studied in adult male Sprague-Dawley rats following a daily dermal dose of 44.4 mg/kg malathion, 40 mg/kg DEET, and 0.13 mg/kg permethrin, alone and in combination for 30 d. Neurobehavioral evaluations of sensorimotor functions included beam-walking score, beam walk time, inclined plane, and grip response assessments. Twenty-four hours after the last treatment with each chemical alone or in combination all behavioral measures were impaired. The combination of DEET and permethrin, malathion and permethrin, or the three chemicals together resulted in greater impairments in inclined performance than permethrin alone. Only animals treated with a combination of DEET and malathion or with DEET and permethrin exhibited significant increases in plasma butyrlcholinesterase (BChE) activity. Treatment with DEET or permethrin alone, malathion and permethrin, or DEET and permethrin produced significant increases in cortical acetylcholinesterase (AChE) activity. Combinations of malathion and permethrin or of DEET and permethrin produced significant decreases in midbrain AChE activity. Animals treated with DEET alone exhibited a significant increase in cortical m2 muscarinic ACh receptor binding. Quantification of neuron density in the dentate gyrus, CA1 and CA3 subfields of the hippocampus, midbrain, brainstem, and cerebellum revealed significant reductions in the density of surviving neurons with various treatments. These results suggest that exposure to real-life doses of malathion, DEET, and permethrin, alone or in combination, produce no overt signs of neurotoxicity but induce significant neurobehavioral deficits and neuronal degeneration in brain.

Co-exposure to pyridostigmine bromide, DEET, and/or permethrin causes sensorimotor deficit and alterations in brain acetylcholinesterase activity.

Military personnel deployed in the Persian Gulf War (PGW) were exposed to a combination of chemicals, including pyridostigmine bromide (PB), DEET, and permethrin. We investigated the dose-response effects of these chemicals, alone or in combination, on the sensorimotor performance and cholinergic system of male Sprague-Dawley rats. Animals were treated with a daily dermal dose of DEET and/or permethrin for 60 days and/or PB (gavage) during the last 15 days. Neurobehavioral performance was assessed on day 60 following the beginning of the treatment with DEET and permethrin. The rats were sacrificed 24 h after the last treatment for biochemical evaluations. PB alone, or in combination with DEET, or DEET and permethrin resulted in deficits in beam-walk score and longer beam-walk times compared to controls. PB alone, or in combination with DEET, permethrin, or DEET and permethrin caused impairment in incline plane performance and forepaw grip strength. PB alone at all doses slightly inhibited plasma butyrylcholinesterase activity, whereas combination of PB with DEET or permethrin increased its activity. Brainstem acetylcholinesterase (AChE) activity significantly increased following treatment with combinations of either DEET or permethrin at all doses, whereas the cerebellum showed a significant increase in AChE activity following treatment with a combination of PB/DEET/permethrin. Co-exposure to PB, DEET, and permethrin resulted in significant inhibition in AChE in midbrain. PB alone or in combination with DEET and permethrin at all doses increased ligand binding for m2 muscarinic acetylcholine receptor in the cortex. In addition, PB and DEET together or a combination of PB, DEET, and permethrin significantly increased ligand binding for nicotinic acetylcholine receptor. These results suggest that exposure to various doses of PB, alone and in combination with DEET and permethrin, leads to sensorimotor deficits and differential alterations of the cholinergic system in the CNS.

Terbutaline is a developmental neurotoxicant: effects on neuroproteins and morphology in cerebellum, hippocampus, and somatosensory cortex.

Beta(2)-adrenoceptor agonists, especially terbutaline, are widely used to arrest preterm labor, but they also cross the placenta to stimulate fetal beta-adrenoceptors that control neural cell differentiation. We evaluated the effects of terbutaline administration in neonatal rats, a stage of neurodevelopment corresponding to human fetal development. Terbutaline administered on postnatal days PN2 to 5 elicited neurochemical changes indicative of neuronal injury and reactive gliosis: immediate increases in glial fibrillary acidic protein and subsequent induction of the 68-kDa neurofilament protein. Quantitative morphological evaluations carried out on PN30 indicated structural abnormalities in the cerebellum, hippocampus, and somatosensory cortex. In the cerebellum, PN2 to 5 terbutaline treatment reduced the number of Purkinje cells and elicited thinning of the granular and molecular layers. The hippocampal CA3 region also displayed thinning, along with marked gliosis, effects that were restricted to females. In the somatosensory cortex, terbutaline evoked a reduction in the proportion of pyramidal cells and an increase in smaller, nonpyramidal cells; again, females were affected more than males. Although abnormalities were obtained with later terbutaline treatment (PN11 to 14), in general the effects were smaller than those seen with PN2 to 5 exposure. Our results indicate that terbutaline is a neurotoxicant that elicits biochemical alterations and structural damage in the immature brain during a critical period. These effects point to a causal relationship between fetal terbutaline exposure and the higher incidence of cognitive and neuropsychiatric disorders reported for the offspring of women receiving terbutaline therapy for preterm labor.