Effect of Hepatic Impairment on the Pharmacokinetics of Itacitinib.

Itacitinib is a potent, selective JAK-1 inhibitor currently in development for the treatment of chronic graft-vs-host-disease in combination with corticosteroids. Itacitinib is primarily eliminated via cytochrome P450 3A metabolism with minimal renal elimination. The purpose of this open-label study was to investigate the effect of hepatic impairment, as determined by Child-Pugh grade, on itacitinib pharmacokinetics. All participants received a single 300-mg dose of itacitinib orally in the fasted state. Blood samples were collected serially through 96 hours after dosing; 4 hours after dosing, an additional sample was collected for protein binding determination. Participants with moderate hepatic impairment (N = 8) had an approximate 2.5-fold increase in total exposure (area under the plasma concentration-time curve from time 0 to infinity [AUC0-∞ ]) and an approximate 2-fold increase in maximal exposure (Cmax ) compared to those with normal hepatic function (N = 8) (geometric mean ratio, 2.51 [90% confidence interval (CI), 1.54-4.08] for AUC0-∞ and 1.95 [90%CI, 1.14-3.35] for Cmax ). Participants with severe hepatic impairment (N = 6) had an approximate 4-fold increase in total exposure (AUC0-∞ ) and an approximate 3.5-fold increase in maximal exposure compared to participants with normal hepatic function (geometric mean ratio, 4.08 [90%CI, 2.41-6.89] for AUC0-∞ and 3.48 [90%CI, 1.94-6.23] for Cmax ). Protein binding was similar between participants with moderate or severe hepatic impairment and participants with normal hepatic function, with average unbound fractions (percent free) of 25.7%, 31.5%, and 25.6%, respectively. There were no serious or fatal treatment-related adverse events. The results of this study combined with exposure, efficacy, and safety data from the pivotal study in the relevant patient population will inform final dosing recommendations.

Marine biomaterials: Biomimetic and pharmacological potential of cultivated Aplysina aerophoba marine demosponge.

Marine demosponges of the Verongiida order are considered a gold-mine for bioinspired materials science and marine pharmacology. The aim of this work was to simultaneously isolate selected bromotyrosines and unique chitinous structures from A. aerophoba and to propose these molecules and biomaterials for possible application as antibacterial and antitumor compounds and as ready-to-use scaffolds for cultivation of cardiomyocytes, respectively. Among the extracted bromotyrosines, the attention has been focused on aeroplysinin-1 that showed interesting unexpected growth inhibition properties for some Gram-negative clinical multi-resistant bacterial strains, such as A. baumannii and K. pneumoniae, and on aeroplysinin-1 and on isofistularin-3 for their anti-tumorigenic activity. For both compounds, the effects are cell line dependent, with significant growth inhibition activity on the neuroblastoma cell line SH-SY5Y by aeroplysinin-1 and on breast cancer cell line MCF-7 by isofistularin-3. In this study, we also compared the cultivation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) on the A. aerophoba chitinous scaffolds, in comparison to chitin structures that were pre-coated with Geltrex™, an extracellular matrix mimetic which is used to enhance iPSC-CM adhesion. The iPSC-CMs on uncoated and pure chitin structures started contracting 24 h after seeding, with comparable behaviour observed on Geltrex-coated cell culture plates, confirming the biocompatibility of the sponge biomaterial with this cell type. The advantage of A. aerophoba is that this source organism does not need to be collected in large quantities to supply the necessary amount for further pre-clinical studies before chemical synthesis of the active compounds will be available. A preliminary analysis of marine sponge bioeconomy as a perspective direction for application of biomaterials and secondary bioactive metabolites has been finally performed for the first time.

The Effect of Renal Impairment on the Pharmacokinetics and Safety of Itacitinib.

Itacitinib is a novel, selective, Janus kinase 1 inhibitor in development for treatment of graft-versus-host disease. The objective of this study was to assess pharmacokinetics and safety of 300-mg itacitinib dosed in participants with normal renal function (n = 10), severe renal impairment (n = 8), and end-stage renal disease (ESRD) on hemodialysis (n = 8). Serial plasma and urine samples (urine from normal and severe groups only) were collected before dosing until 72 hours after dosing. In the ESRD group, itacitinib was evaluated in 2 periods, when dosed before (period 1) and after (period 2) a hemodialysis session. Geometric mean ratios (90% confidence interval) in participants with severe renal impairment, ESRD period 1 and ESRD period 2 relative to participants with normal renal function were 1.65 (1.13-2.39), 0.71 (0.49-1.03), and 0.83 (0.57-1.20) for maximum plasma drug concentration and 2.23 (1.56-3.18), 0.81 (0.57-1.16), and 0.95 (0.66-1.35) for area under the plasma concentration-time curve from time zero to infinity. Itacitinib was well tolerated, and 3 grade 1 treatment-emergent adverse events were reported over the course of the study. Given the magnitude of exposure changes in participants with severe renal impairment or ESRD and the historic risk-benefit profile, no dose adjustment is recommended for itacitinib in patients with impaired renal function, although the final dosage recommendation will be based on cumulative pharmacokinetics and safety from this study and from the pivotal graft-versus-host disease trial. Additionally, itacitinib may be administered to patients undergoing dialysis regardless of the time of dialysis.

The oral Janus kinase/spleen tyrosine kinase inhibitor ASN002 demonstrates efficacy and improves associated systemic inflammation in patients with moderate-to-severe atopic dermatitis: results from a randomized double-blind placebo-controlled study.

BACKGROUND: ASN002 is an oral dual inhibitor of Janus kinase and spleen tyrosine kinase, which are involved in the pathogenesis of atopic dermatitis (AD) through their regulatory role on T helper (Th)1, Th2 and Th17/Th22 pathways. OBJECTIVES: The objectives of this study were to evaluate the efficacy, safety, pharmacokinetics and effects on systemic biomarkers of ASN002 in patients with moderate-to-severe AD. Methods A total of 36 patients with moderate-to-severe AD were randomized (3 : 1) to ASN002 or placebo in the phase Ib study. Three dosage cohorts were studied over a 28-day period (20 mg, 40 mg and 80 mg once daily). RESULTS: ASN002 was superior to placebo for the proportion of patients achieving Eczema Area and Severity Index (EASI) 50 (20 mg 20%, P = 0·93; 40 mg 100%, P = 0·003; 80 mg 83%, P = 0·03; placebo 22%), EASI 75 (20 mg 0%, P = 0·27; 40 mg 71%, P = 0·06; 80 mg 33%, P = 0·65; placebo 22%) and in change from baseline in pruritus (20 mg -1·3 ± 2·1, P = 0·81; 40 mg -3·1 ± 2·7, P = 0·27; 80 mg -4·7 ± 2·1, P = 0·01; placebo -1·6 ± 1·8). Adverse events were generally mild and similar across all groups. ASN002 showed dose-dependent plasma exposure with low interpatient variability, significantly downregulated several serum biomarkers involved in Th1, Th2 and Th17/Th22 immunity, and decreased the atherosclerosis-associated biomarker E selectin/SELE. CONCLUSIONS: In patients with moderate-to-severe AD, ASN002 showed strong efficacy with rapid onset of action and associated improvements in systemic inflammation.

Pharmacokinetics and metabolism of GW9508 in rat by liquid chromatography/electrospray ionization tandem mass spectrometry.

In this study, a simple, fast and sensitive LC/MS/MS method was developed and validated for the determination of GW9508 in rat plasma. The sample was precipitated with acetonitrile and subsequently separated on ZORBAX Eclipse XDB C18 column (50 mm × 2.1 mm, 5 µm). Mobile phase was composed of 0.1% formic acid in water and acetonitrile with gradient elution, at a flow rate of 0.4 mL/min. The analyte and internal standard were quantitatively monitored with precursor-to-product transitions of m/z 348.2→183.1 and m/z 397.2→260.2, respectively. The linearity of the assay was evident in the range of 1-1000 ng/mL with correlation coefficient more than 0.998. The validation parameters were all within the acceptable limits. The validated method has been successfully applied to the pharmacokinetics study of GW9508 in rat plasma, and our results demonstrated that GW9508 showed low clearance, moderate half-life and ideal bioavailability (54.88%). Furthermore, metabolites stemmed from rat plasma, rat hepatocytes and human hepatocytes were analyzed by an LC-Q-Exactive-Orbitrap-MS assay, resulting in the identification of seven metabolites based on the accurate mass and fragment ions. Acylglucuronide conjugate (M6) was found as the most abundant metabolite in all tested matrices. The metabolic pathways were proposed as hydroxylation and glucuronidation. This study provided an overview of disposition of GW9508, which is highly instructive for better understanding the effectiveness and toxicity of this drug.

Diverse Excretion Pathways of Benzyl Glucosinolate in Humans after Consumption of Nasturtium (Tropaeolum majus L.)-A Pilot Study.

SCOPE: Different metabolic and excretion pathways of the benzyl glucosinolate breakdown products benzyl isothiocyanate and benzyl cyanide are investigated to obtain information about their multiple fate after ingestion. Detailed focus is on the so far underestimated transformation/excretion pathways-protein conjugation and exhalation. METHODS AND RESULTS: Metabolites, protein conjugates, and non-conjugated isothiocyanates are determined in plasma, urine, and breath of seven volunteers after consuming freeze-dried nasturtium or bread enriched with nasturtium. Samples are collected up to 48 h at selected time points. The metabolites of the mercapturic acid pathway are detectable in plasma up to 24 h after consumption. Additionally, mercapturic acid is the main metabolite in urine, but non-conjugated benzyl isothiocyanate is detectable as well. Protein conjugates show high amounts in plasma even 48 h after consumption. In breath, benzyl isothiocyanate and benzyl cyanide are detectable up to 48 h after consumption. CONCLUSION: Isothiocyanates are not only metabolized via the mercapturic acid pathway, but also form protein conjugates in blood and are exhaled. To balance intake and excretion, it is necessary to investigate all potential metabolites and excretion routes. This has important implications for the understanding of physiological and pharmacological effects of isothiocyanate-containing products.

Microwave assisted synthesis of binary grafted psyllium and its utility in anticancer formulation.

A binary grafted copolymer of Psyllium mucilage (Psy) with acrylic acid (AA) and acrylonitrile (An) has been successfully synthesized under microwave conditions for in vitro drug release study. The grafting was confirmed by FTIR spectroscopy, XRD, SEM, EDX, TGA analytical techniques and the intrinsic viscosity study. The swelling behavior of grafted material has been studied in solution of different pH and time. We also prepare Psy-g-Poly (AA-co-An) based beads with anti-cancer drug [(2-Chloro-3-(4-hydroxyphenylamino) naphthalene-1, 4-dione)]. The drug release behavior of Psy-g-Poly (AA-co-An) based beads has been determined in aqueous medium at different pH. It has been observed that highest drug release at pH 1.6. The drug release kinetics was analysed using the different models. This study demonstrates that the release of drug depends on the composition of beads and pH of release medium. Kinetics of drug release from beads is best fitted by zero order and first order model.

Suicide attempt with acetonitrile ingestion in a nursing mother.

CONTEXT: Acetonitrile (ACN) is a solvent rapidly absorbed through lungs and intestinal tract, and is slowly metabolized to cyanide (CN) by enzymatic processes mediated by CYP2E1. OBJECTIVE: To describe the clinical and laboratory evolution, ACN elimination half-life, and its presence in breast milk in a nursing mother who attempted suicide. CASE DETAILS: A 25-year-old 2-month nursing mother ingested an estimated dose of 2.1 g/kg of ACN. Blood and urine samples were collected 24 h later for ACN, CN and thiocyanate analysis, and 12.5 g sodium thiosulfate i.v. in 1-h infusion was started and repeated every 24 h for 4 days. ACN results showed 200 mg/L in blood and 235 mg/L in urine. ACN analysis in the breast milk at Day 6 showed level of 21 mg/L compared to 27 mg/L in blood collected at the same time, suggesting a possible relationship of 1.3:1.0 ratio. An elimination half-life of 40.4 h was calculated, compared to 32 and 36 h showed in other studies. DISCUSSION: The clinical management must involve the use of CN antidotes for more than 24 h depending on the symptoms and blood levels of ACN. Furthermore, our data showed the possible existence of a close relationship between plasma and breast milk levels.

Effects of temperature, surfactants and skin location on the dermal penetration of haloacetonitriles and chloral hydrate.

Dermal exposure has been recognized as an important contributor to the total internal dose to disinfection-by-products (DBPs) in water. However, the effect of the use of surfactants, water temperature and area of the body exposed to DBPs on their dermal flux has not been characterized and was the focus of the present study using an in-vitro system. The dermal flux of mg/l concentrations of haloacetonitriles and chloral hydrate (CH), important cytotoxic DBPs, increased by approximately 50% to 170% with increasing temperature from 25 °C to 40 °C. The fluxes for the torso and dorsum of the hand were much higher than that of palm and scalp skin. An increase in flux was observed for chloroacetonitrite and dichloroacetonitrile, two less lipophilic HANs, but not for trichloroacetonitrile or CH, with the addition of 2% sodium lauryl sulfate or 2% sodium laureth sulfate, two surfactants commonly used in soaps and shampoos used in showering and bathing. Thus, factors such as temperature, surfactants and skin location affect dermal penetration and should be considered when evaluating dermal absorption.

Percutaneous absorption of haloacetonitriles and chloral hydrate and simulated human exposures.

Disinfection-by-products (DBPs) have long been a human health concern and many are known carcinogens and teratogens. Skin is exposed to DBPs in water through bathing and swimming; however, dermal uptake of many DBPs has not been characterized. The present studies were initiated to measure the permeation coefficients (K(p) ) for haloacetonitriles (HANs) and chloral hydrate (CH), important cytotoxic DBPs. The K(p) values measured using fully hydrated dermatomed torso skin at 37 °C for the HANs ranged from 0.099 to 0.17 cm h⁻¹, and was 0.0039 cm h⁻¹ for CH. Of the HANs, dibromoacetonitrile had the highest permeability while chloroacetonitrile had the lowest permeability and a direct relationship was observed between their K(p) and their octanol/water partition coefficients (K(ow) ). The K(p) values of the HANs were also approximately 30 times that of CH. The monthly dermal and ingestion doses of HANs and CH of an average American population were estimated using Monte Carlo simulations. The dermal doses of HANs from showering and bathing ranged from 0.39 to 0.78 times their ingestion doses but only approximately 0.02 times their ingestion doses for CH, assuming that the K(p) values determined are applicable to shorter water contact times. However, that ratio can vary markedly with chlorinated swimming pool exposures, with a range of 0.30-2.3 for HANs and 0.19-0.25 for CH. Dermal exposure to HANs and CH seems to be a significant route of exposure and should be considered when evaluating their total exposure during the routine usage of water for bathing and swimming.

Heated indoor swimming pools, infants, and the pathogenesis of adolescent idiopathic scoliosis: a neurogenic hypothesis.

BACKGROUND: In a case-control study a statistically significant association was recorded between the introduction of infants to heated indoor swimming pools and the development of adolescent idiopathic scoliosis (AIS). In this paper, a neurogenic hypothesis is formulated to explain how toxins produced by chlorine in such pools may act deleteriously on the infant's immature central nervous system, comprising brain and spinal cord, to produce the deformity of AIS. PRESENTATION OF THE HYPOTHESIS: Through vulnerability of the developing central nervous system to circulating toxins, and because of delayed epigenetic effects, the trunk deformity of AIS does not become evident until adolescence. In mature healthy swimmers using such pools, the circulating neurotoxins detected are chloroform, bromodichloromethane, dibromochloromethane, and bromoform. Cyanogen chloride and dichloroacetonitrile have also been detected. TESTING THE HYPOTHESIS: In infants, the putative portals of entry to the blood could be dermal, oral, or respiratory; and entry of such circulating small molecules to the brain are via the blood-brain barrier, blood-cerebrospinal fluid barrier, and circumventricular organs. Barrier mechanisms of the developing brain differ from those of adult brain and have been linked to brain development. During the first 6 months of life cerebrospinal fluid contains higher concentrations of specific proteins relative to plasma, attributed to mechanisms continued from fetal brain development rather than immaturity. IMPLICATIONS OF THE HYPOTHESIS: The hypothesis can be tested. If confirmed, there is potential to prevent some children from developing AIS.

Metabolism and disposition of [14C]dibromoacetonitrile in rats and mice following oral and intravenous administration.

1. Tissue distribution, metabolism, and disposition of oral (0.2-20 mg/kg) and intravenous (0.2 mg/kg) doses of [2-(14)C]dibromoacetonitrile (DBAN) were investigated in male rats and mice. 2. [(14)C]DBAN reacts rapidly with rat blood in vitro and binds covalently. Prior depletion of glutathione (GSH) markedly diminished loss of DBAN. Chemical reaction with GSH readily yielded glutathionylacetonitrile. 3. About 90% of the radioactivity from orally administered doses of [(14)C]DBAN was absorbed. After intravenous administration, 10% and 20% of the radioactivity was recovered in mouse and rat tissues, respectively, at 72 h. After oral dosing, three to four times less radioactivity was recovered, but radioactivity in stomach was mostly covalently bound. 4. Excretion of radioactivity into urine exceeded that in feces; 9-15% was exhaled as labeled carbon dioxide and 1-3% as volatiles in 72 h. 5. The major urinary metabolites were identified by liquid chromatography-mass spectrometry, and included acetonitrile mercaptoacetate (mouse), acetonitrile mercapturate, and cysteinylacetonitrile. 6.The primary mode of DBAN metabolism is via reaction with GSH, and covalent binding may be due to reaction with tissue sulphydryls.

Haloacetonitriles: metabolism and toxicity.

The haloacetonitriles (HANs) exist in drinking water exclusively as byproducts of disinfection. HANs are found in drinking water more often, and in higher concentrations, when surface water is treated by chloramination. Human exposure occurs through consumption of finished drinking water; oral and dermal contact also occurs, and results from showering, swimming and other activities. HANs are reactive and are toxic to gastrointestinal tissues following oral administration. Such toxicity is characterized by GSH depletion, increased lipid peroxidation, and covalent binding of HAN-associated radioactivity to gut tissues. The presence of GSH in cells is an important protective mechanism against HAN toxicity; depletion of cellular GSH results in increased toxicity. Some studies have demonstrated an apparently synergistic effect between ROS and HAN administration, that may help explain effects observed in GI tissues. ROS are produced in gut tissues, and in vitro evidence indicates that ROS may contribute to the degradation and formation of reactive intermediates from HANs. The rationale for ROS involvement may involve HAN-induced depletion of GSH and the role of GSH in scavenging ROS. In addition to effects on GI tissues, studies show that HAN-derived radiolabel is found covalently bound to proteins and DNA in several organs and tissues. The addition of antioxidants to biologic systems protects against HAN-induced DNA damage. The protection offered by antioxidants supports the role of oxidative stress and the potential for a threshold in han-induced toxicity. However, additional data are needed to substantiate evidence for such a threshold. HANs are readily absorbed from the GI tract and are extensively metabolized. Elimination occurs primarily in urine, as unconjugated one-carbon metabolites. Evidence supports the involvement of mixed function oxidases, the cytochrome P450 enzyme family and GST, in HAN metabolism. Metabolism represents either a detoxification or bioactivation process, depending on the particular HAN and the enzyme involved. HANs can inhibit CYP2E1-mediated metabolism, an effect which may be dependent on a covalent interaction with the enzyme. In addition, HAN compounds inhibit GST-mediated conjugation, but this effect is reversible upon dialysis, indicating that the interaction does not represent covalent binding. No subchronic studies of HAN toxicity are available in the literature. However, studies show that HANs produce developmental toxicity in experimental animals. The nature of developmental toxicity is affected by the type of administration vehicle, which renders interpretation of results more difficult. Skin tumors have been found following dermal application of HANs, but oral studies for carcinogenicity are negative. Pulmonary adenomas were increased following oral administration of HANs, but the A/J strain of mice employed has a characteristically high background rate of such tumors. HANs interact with DNA to produce unscheduled DNA repair, SCE and reverse mutations in Salmonella. HANs did not induce micronuclei or cause alterations in sperm head morphology in mice, but did induce micronuclei in newts. Thus, there is concern for the potential carcinogenicity of HANs. It would be valuable to delineate any relationship between the apparent threshold for micronuclei formation in newts and the potential mechanism of toxicity involving HAN-induced oxidative stress. Dose-response studies in rodents may provide useful information on toxicity mechanisms and dose selection for longer term toxicity studies. Additional studies are warranted before drawing firm conclusions on the hazards of HAN exposure. Moreover, additional studies on HAN-DNA and HAN-protein interaction mechanisms, are needed. Such studies can better characterize the role of metabolism in toxicity of individual HANs, and delineate the role of oxidative stress, both of which enhance the capacity to predict risk. Most needed, now, are new subchronic (and chronic) toxicity studies; the results of such well-planned, controlled, conducted, interpreted and published investigations would be valuable in establishing margins of safety for HANs in human health risk assessment.

Pharmacokinetic study of clazuril (Appertex) in eggs and plasma from laying hens after single or multiple treatments, using a new HPLC method for detection.

1. Anticoccidials are widely used as food additives to prevent and treat coccidiosis. They are licensed for use in a prescribed concentration and during a specific time interval with broilers and pullets, but not for laying hens. 2. This study was set up to develop a new high pressure liquid chromatography (HPLC) method to detect clazuril (CZ: (+/-)-2-chloro-alpha-(4-chlorophenyl)-4-(4,5-dihydro-3,5-dioxo-1,2,4-triazin-2(3H)-yl)-benzeneacetonitrile) in egg yolk and albumen and in plasma; to investigate both the presence of residues of CZ in eggs and its pharmacokinetic behaviour in laying hens. 3. A single oral dose (3 mg/kg BW) and multiple oral doses (3 mg/kg BW for 5 d) were investigated. The analytical method gave very good recovery (64 to 74%) in the three different matrices (yolk, albumen and plasma); precision and accuracy were within 11%. 4. After a single dose no residue was detected in eggs collected for up to 10 d, while following multiple dose treatment, CZ residues were detected until 10 d after the end of treatment. The concentration of the drug was higher in yolk than in albumen with a maximum ratio of 10 : 1. 5. Pharmacokinetics of CZ in laying hens after a single dose showed a detectable concentration of the drug up to 24 h. It reached a steady state after the third administration in multiple dosing. 6. Although further studies are necessary, these results indicate that a single oral dose of CZ could be used as an anticoccidial for laying hens due to the lack of residues in eggs.

Estudio cinético de la fotodegradación del ión p-hidroxibencenodiazonio en medio polar / Kinetic study of the photodegradation of p-hydroxybenzenediazonium ion in polar media

Se ha realizado un estudio sobre la fotodescomposición del ión p-hidroxibencenodiazonio (PDQ) basado en los datos espectrofotométricos y cromatográfi cos obtenidos con disoluciones de PDQ expuestas a irradiación UV (254 nm) en medio de acetonitrilo y agua. Los resultados de HPLC y HPLC-masa (HPLC/MS) indican que el 4-acetamidofenol es el principal producto que se forma tras la irradiación de PDQ en acetonitrilo. Esto se explica como consecuencia de la formación inicial del catión arilo, que posteriormente participa en una reacción de Ritter. El análisis cinético de los datos espectrofotométricos revela que la fotodegradación de PDQ es más rápida en acetonitrilo (constante de velocidad observada, kobs, = 0,1442 s-1) que en acetonitrilo acidifi cado (kobs = 0,009 s-1), lo que indica una mayor fotoestabilidad de la especie protonada derivada de PDQ. La constante de segundo orden (0,062 M s-1) encontrada para la fotodescomposición de PDQ en tampón fosfato (pH 7) se justifi ca por el establecimiento de un equilibrio entre las especies protonada y no protonada procedentes de la disociación ácida de PDQ

A study on the photodecomposition of p-hydroxybenzenediazonium ion (PDQ) has been made using chromatographic and spectrophotometric data obtained from UV-irradiated (254 nm) PDQ solutions in acetonitrile and aqueous media. The HPLC and HPLC-mass results indicate that 4-acetamidophenol is the main product formed after the irradiation of PDQ in acetonitrile. This is explained as a consequence of the initial formation of the aryl cation which is later involved in a Ritter’s reaction. A kinetic analysis of the spectrophotometric data reveals that PDQ photodegradation is faster in acetonitrile (observed rate constant (kobs) = 0.1442 s-1) than in acidifi ed acetonitrile (kobs = 0.009 s-1) indicating a higher photostability of the protonated species derived from PDQ. The second order constant (0.062 M s-1) found for the PDQ photodecomposition in phosphate buffer (pH 7) is explained in term of the equilibrium between protonated and non-protonated species coming from the acid dissociation of PDQ

Fetal origin of adverse pregnancy outcome: the water disinfectant by-product chloroacetonitrile induces oxidative stress and apoptosis in mouse fetal brain.

Epidemiological studies indicate a relationship between water disinfectant by-products (DBP) and adverse pregnancy outcomes (APO) including neural tube defects. These studies suggest that fetal brain may be vulnerable to DBP during early stages of development. Therefore, we examined several molecular markers commonly known to indicate chemical-induced neurotoxicity during fetal brain development following prenatal exposure to the DBP; chloroacetonitrile (CAN). Pregnant mice, at gestation day 6 (GD6), were treated with a daily oral dose of CAN (25 mg/kg). At GD12, two groups of animals were treated with an i.v. tracer dose of [2-14C]-CAN. These animals were sacrificed at 1 and 24 h after treatment and processed for quantitative in situ micro-whole-body autoradiography. The remaining groups of animals continued to receive CAN. At GD18, control and treated animals were weighed, anesthetized, and fetuses were obtained and their brains were removed for biochemical and immunohistochemical analyses. Whole-body autoradiography studies indicate a significant uptake and retention of [2-14C]-CAN/metabolites (M) in fetal brain (cerebral cortex, hippocampus, cerebellum) at 1 and 24 h. There was a 20% reduction in body weight and a 22% reduction in brain weight of fetuses exposed to CAN compared to controls. A significant increase in oxidative stress markers was observed in various fetal brain regions in animals exposed to CAN compared to controls. This was indicated by a 3- to 4-fold decrease in the ratio of the reduced to oxidized form of glutathione (GSH/GSSG), increased lipid peroxidation (1.3-fold), and increased 8-hydroxy-2-deoxyguanosine levels (1.4-fold). Cupric silver staining indicated a significant increase in the number of degenerating neurons in cortical regions in exposed animals. In animals exposed to CAN there was increase in nuclear DNA fragmentation (TUNEL staining) detected in the cerebral cortex and cerebellum (2-fold increase in apoptotic indices). Caspase-3 activity in cerebral cortex and cerebellum of treated animals were also increased (1.7- and 1.5-fold, respectively). In conclusion, this study indicates that CAN/M crossed the placenta and accumulated in fetal brain tissues where it caused oxidative stress and neuronal apoptosis. These events could predispose the fetus to altered brain development leading to APO as well as behavioral and learning and memory deficits.

Determination of acetonitrile and cyanide in rat blood: application to an experimental study.

Methods were developed for the analysis of acetonitrile and its metabolite cyanide in the blood of rats exposed to acetonitrile. Acetonitrile was analyzed by the headspace technique coupled to gas chromatography with detection by flame ionization, and cyanide was analyzed by high-performance liquid chromatography with fluorescence detection (lambdaex = 418 nm and lambdaem = 460 nm) after derivatization of the ion with naphthalene 2,3-dicarboxyaldehyde and taurine. The quantitation limits of the methods for the analysis of acetonitrile and cyanide were 4.875 microg/mL and 0.025 microg/mL, respectively. The coefficients of variation of 10% or less obtained for intra- and interassay precision indicate the precision of these analytical methods and the systematic errors, all less than 5%, indicate that the methods are quite accurate. The methods were applied to an experimental study after the animals received acetonitrile at the doses of 2 mmol/kg or 5 mmol/kg.

Glucose production, gluconeogenesis, and hepatic tricarboxylic acid cycle fluxes measured by nuclear magnetic resonance analysis of a single glucose derivative.

A triple-tracer method was developed to provide absolute fluxes contributing to endogenous glucose production and hepatic tricarboxylic acid (TCA) cycle fluxes in 24-h-fasted rats by (2)H and (13)C nuclear magnetic resonance (NMR) analysis of a single glucose derivative. A primed, intravenous [3,4-(13)C(2)]glucose infusion was used to measure endogenous glucose production; intraperitoneal (2)H(2)O (to enrich total body water) was used to quantify sources of glucose (TCA cycle, glycerol, and glycogen), and intraperitoneal [U-(13)C(3)] propionate was used to quantify hepatic anaplerosis, pyruvate cycling, and TCA cycle flux. Plasma glucose was converted to monoacetone glucose (MAG), and a single (2)H and (13)C NMR spectrum of MAG provided the following metabolic data (all in units of micromol/kg/min; n = 6): endogenous glucose production (40.4+/-2.9), gluconeogenesis from glycerol (11.5+/-3.5), gluconeogenesis from the TCA cycle (67.3+/-5.6), glycogenolysis (1.0+/-0.8), pyruvate cycling (154.4+/-43.4), PEPCK flux (221.7+/-47.6), and TCA cycle flux (49.1+/-16.8). In a separate group of rats, glucose production was not different in the absence of (2)H(2)O and [U-(13)C]propionate, demonstrating that these tracers do not alter the measurement of glucose turnover.

Urinary thiocyanate levels as a biomarker for the generation of inorganic cyanide from benzyl cyanide in the rat.

A colorimetric procedure was developed and validated for the determination of thiocyanate in rat urine over the concentration range of 7-7000 microg/ml. It was applied to the determination of thiocyanate following its oral administration to male and female rats. The mean percentage urinary recoveries of sodium thiocyanate given by oral gavage at 10 and 100 mg/kg were 60 and 39%, respectively, for male rats and 89 and 73% for females over a period of 3 days. Most of the elimination occurred in the 0-48-h period post-dosing but significant amounts were still being excreted in the 48-72-h period. It was concluded from these results that the recoveries of urinary thiocyanate were such that this anion was suitable for use as a biomarker for the release of cyanide from organonitriles such as benzyl cyanide. Benzyl cyanide (150 mg/kg) administered orally to rats led to markedly increased urinary thiocyanate levels; for male rats this was equivalent to 54% of the dose and for females this was 65% over a period of 3 days. When adjusted for incomplete recoveries of the marker, thiocyanate, these values equated to 61 and 89%, respectively. It was concluded that this validated assay could be used to assess cyanide release from topically applied fragrance organonitriles (Potter, J., Smith, R.L., Api, A.M., 2000. An assessment of the release of inorganic cyanide from the fragrance materials, benzyl cyanide, geranyl nitrile and citronellyl nitrile applied dermally to the rat. Food and Chemical Toxicology 39, 147-151).

An assessment of the release of inorganic cyanide from the fragrance materials benzyl cyanide, geranyl nitrile and citronellyl nitrile applied dermally to the rat.

Organonitriles are widely used as components of fragrances that are incorporated into consumer products, many of which are for human topical use. Some organontriles are readily broken down metabolically to potentially toxic inorganic cyanide. Studies were therefore undertaken to assess whether this occurs with three representative fragrance nitriles, namely, benzyl cyanide, geranyl nitrile and citronellyl nitrile when applied dermally to the rat. The nitriles (benzyl cyanide, 150 mg/kg; geranyl and citronellyl nitriles, 400 mg/kg) were applied to the shaved backs of rats and maintained under occlusion for 24 h. Urine samples were collected for 0-24 h, 24-48 h and 48-72 h from the time of first application. These samples were analysed for thiocyanate, a biomarker for cyanide formation in vivo, as described previously (Potter, J., Smith, R.L., Api, A.M., 2000. Urinary thiocyanate levels as a biomarker for the generation of inorganic cyanide from benzyl cyanide in the rat. Food and Chemical Toxicology 39, 141-146). In the case of benzyl cyanide, there was a marked increase in urinary thiocyanate levels attributable to the release of cyanide in vivo. The amount of thiocyanate recovered was equivalent to 37% of the dose for males and 32% for females. For geranyl nitrile there was no significant increase in urinary thiocyanate excretion and there was only a marginal increase in the case of citronellyl nitrile that was equivalent to 0.40% of the applied dose for males and 0.29% for females.