Semisynthesis of radiolabeled amino acid and lipid brevetoxin metabolites and their blood elimination kinetics in C57BL/6 mice.

Brevetoxin B (BTX-B), produced by dinoflagellates of the species Karenia, is a highly reactive molecule, due in part to an α,ß-unsaturated aldehyde group at the terminal side chain, leading to the production of metabolites in shellfish by reduction, oxidation, and conjugation. We have investigated in mice the blood elimination of three common bioactive brevetoxin metabolites found in shellfish, which have been semisynthesized from BTX-B in radioactive forms. BTX-B was reduced at C42 to yield [(3)H] dihydro-BTX-B. [(3)H] S-desoxy-BTX-B2 (cysteine brevetoxin B) was semisynthesized from BTX-B by the conjugation of cysteine at the C50 olefinic group then [(3)H] radiolabeled by C42 aldehyde reduction. [(14)C] N-Palmitoyl-S-desoxy-BTX-B2 was prepared using S-desoxy-BTX-B2 as the starting material with addition of the [(14)C] radiolabeled fatty acid via cysteine-amide linkage. The elimination of intravenously administered [(3)H] S-desoxy-BTX-B2, [(14)C] N-palmitoyl-S-desoxy-BTX-B2, or [(3)H] dihydro-BTX-B was measured in blood collected from C57BL/6 mice over a 48 h period. Each brevetoxin metabolite tested exhibited biexponential elimination kinetics and fit a two-compartment model of elimination that was applied to generate toxicokinetic parameters. The rate of transfer between the central compartment (i.e., blood) and the peripheral compartment (e.g., tissue) for each brevetoxin differed substantially, with dihydro-BTX-B exchanging rapidly with the peripheral compartment, S-desoxy-BTX-B2 eliminating rapidly from the central compartment, and N-palmitoyl-S-desoxy-BTX-B2 eliminating slowly from the central compartment. Toxicokinetic parameters were analyzed in the context of the unique structure of each brevetoxin metabolite resulting from a reduction, amino acid conjugation, or fatty acid addition to BTX-B.

Platelet uptake of GABA and glutamate in patients with bipolar disorder.

OBJECTIVES: Gamma aminobutyric acid (GABA) and glutamate (Glu) are the major neurotransmitters of the human central nervous system, and their actions are determined by specific transporters. Several studies suggest that GABA- and Glu-uptake mechanisms are modified in patients with bipolar disorder (BD). We explored the functionality of the GABA and Glu transporters in three groups of patients with BD, each with a different polarity of index episode (manic, depressive, or euthymic) at the time of blood draw. METHODS: Forty patients with a diagnosis of BD, according to DSM-IV-TR criteria, and 15 healthy subjects were enrolled in the study. GABA and Glu uptake were evaluated in freshly prepared platelets using [(3) H]GABA or [(3) H]glutamate. RESULTS: Compared to controls, GABA uptake was significantly increased in patients with depressive episodes and significantly decreased in subjects with manic episodes. Glu uptake was significantly increased in patients with index manic episodes and in euthymic patients compared to healthy controls. Moreover, a positive correlation was found between GABA platelet uptake and Hamilton Depression Rating Scale scores and between Glu platelet uptake and Young Mania Rating Scale scores in patients with manic episodes. CONCLUSIONS: We found a relationship between GABA- and Glu-uptake levels and the polarity of episodes in patients with BD. Our data suggest that the functionality of both GABA and Glu transporters could represent a useful neurobiological marker to characterize the real polarity of an index episode of illness in patients with BD.

Hydrogen-rich water attenuates the radiotoxicity induced by tritium exposure in vitro and in vivo.

Radionuclide tritium is widely used in the nuclear energy production industry and creates a threat to human health through radiation exposure. Herein, the radioactive elimination and radioprotective effect of hydrogen-rich water (HRW), a potential antioxidant with various medical applications, on tritiated water (HTO) exposure, was studied in vitro and in vivo. Results showed that intragastric administration of HRW effectively promoted the elimination of urinary tritium, decreased the level of serum tritium and tissue-bound tritium (OBT), and attenuated the genetic damage of blood cells in mice exposed to HTO (18.5 MBq/kg). Pretreatment with HRW effectively reduces tritium accumulation in HTO-treated human blood B lymphocyte AHH-1 cells. In addition, the anti-oxidative properties of HRW could attenuate the increased intracellular ROS (such as O2•-, •OH and ONOO-), resulting in reversing the exhaustion of cellular endogenous antioxidants (reduced GSH and SOD), decreasing lipid peroxidation (MDA), relieving DNA oxidative damage, and depressing cell apoptosis and cytotoxicity induced by HTO exposure. In conclusion, HRW is expected to be an effective radioactive elimination agent through the competition effect of isotope exchange or a radioprotective agent by scavenging free radicals induced by HTO exposure.

CFSE flow cytometric quantification of lymphocytic proliferation in extracorporeal photopheresis: use for quality control.

Quality control is essential to validate extracorporeal photopheresis (ECP) processes. There is just one protocol based on PHA-induced proliferation. Since it involves the use of radioactive thymidine, we developed another technique using CFSE labeling. We compared the two tests in a paired series including 18 procedures. The thymidine test was valid. Once proliferation was obtained (10 patients out of 13), the CFSE test was in close agreement with it. In particular, two cases of residual proliferation after ECP were simultaneously detected by both techniques. Only the CFSE test allows targeted analysis of lymphocytes, thus identifying a surviving lymphocytic sub-population.

Peripheral endocannabinoid signaling controls hyperphagia in western diet-induced obesity.

The endocannabinoid system in the brain and periphery plays a major role in controlling food intake and energy balance. We reported that tasting dietary fats was met with increased levels of the endocannabinoids, 2-arachidonoyl-sn-glycerol (2-AG) and anandamide, in the rat upper small intestine, and pharmacological inhibition of this local signaling event dose-dependently blocked sham feeding of fats. We now investigated the contribution of peripheral endocannabinoid signaling in hyperphagia associated with chronic consumption of a western-style diet in mice ([WD] i.e., high fat and sucrose). Feeding patterns were assessed in male C57BL/6Tac mice maintained for 60days on WD or a standard rodent chow (SD), and the role for peripheral endocannabinoid signaling at CB1Rs in controlling food intake was investigated via pharmacological interventions. In addition, levels of the endocannabinoids, 2-AG and anandamide, in the upper small intestine and circulation of mice were analyzed via liquid chromatography coupled to tandem mass spectrometry to evaluate diet-related changes in endocannabinoid signaling and the potential impact on food intake. Mice fed WD for 60days exhibited large increases in body weight, daily caloric intake, average meal size, and rate of feeding when compared to control mice fed SD. Inhibiting peripheral CB1Rs with the peripherally-restricted neutral cannabinoid CB1 receptor antagonist, AM6545 (10mg/kg), significantly reduced intake of WD during a 6h test, but failed to modify intake of SD in mice. AM6545 normalized intake of WD, average meal size, and rate of feeding to levels found in SD control mice. These results suggest that endogenous activity at peripheral CB1Rs in WD mice is critical for driving hyperphagia. In support of this hypothesis, levels of 2-AG and anandamide in both, jejunum mucosa and plasma, of ad-libitum fed WD mice increased when compared to SC mice. Furthermore, expression of genes for primary components of the endocannabinoid system (i.e., cannabinoid receptors, and endocannabinoid biosynthetic and degradative enzymes) was dysregulated in WD mice when compared to SC mice. Our results suggest that hyperphagia associated with WD-induced obesity is driven by enhanced endocannabinoid signaling at peripheral CB1Rs.

Inhalational model of cocaine exposure in mice: neuroteratological effects.

We developed a novel inhalation-based mouse model of prenatal cocaine exposure. This model approximates cocaine abuse via smoking, the preferred route of cocaine administration by heavy drug users. The model is also characterized by (i) absence of procedural stress from drug administration, (ii) long-term drug exposure starting weeks before pregnancy and continuing throughout the entire gestation, and (iii) self-administration of cocaine in multi-hour daily sessions reminiscent of drug binges, which allows animals to set up the levels of their own drug consumption. The offspring of female mice inhaling cocaine in our model displayed no gross alterations in their cortical cytoarchitecture. These offspring, however, showed significant impairments in sustained attention and spatial working memory. We hope that the introduction of the present model will lead to a significant increase in our understanding of outcomes of prenatal cocaine exposure.

The uptake, excretion, and radiation hazards of tritiated thymidine in humans.

Nine patients with malignant disease were given i.v. injections of tritiated thymidine, 0.2 mCi/kg, for tumor cell kinetics studies. Serial plasma, urine, saliva, and air vapor samples were collected variously for up to 79 days, and tritium activity was measured. The initial half-life of plasma activity was rapid. After 1 day, the activity decayed with a half-life of 10.8 days, indicating equilibration of activity with the total body water. Urine activity was over 100 times the plasma activity within 1 hr, with equilibration approaching the plasma activity after 2 days, and then decayed at a similar rate. Saliva and air vapor activity increased to plasma levels and then decayed at the same rate as did plasma activity. In the first 24 hr, approximately one-third of the total injected activity was excreted in the urine. During the first 12 days there were 54.2% urinary and 10.6% insensible losses. Maximum losses determined by extrapolation of observed data were 68% urinary and 19.5% insensible losses, or a total of 87.5%. Approximately 7% of the injected activity may represent material initially incorporated into DNA but later metabolized and excreted. The radiation dose from total body water is estimated at 0.69 rad. The estimated dose absorbed by cell nuclei from incorporated material is a maximum of 20.5 rads. These radiation doses would not seem to contraindicate injection of 0.2 mCi tritiated thymidine per kg to patients in this clinical and experimental setting. Measurements of activity in personnel and room air indicate that the use of such doses is not hazardous if appropriate precautions are followed.

Quantitating isotopic molecular labels with accelerator mass spectrometry.

Accelerator mass spectrometry (AMS) traces isotopically labeled biochemicals and provides significant new directions for understanding molecular kinetics and dynamics in biological systems. AMS traces low-abundance radioisotopes for high specificity but detects them with MS for high sensitivity. AMS reduces radiation exposure doses to levels safe for use in human volunteers of all ages. Total radiation exposures are equivalent to those obtained in very short airplane flights, a commonly accepted radiation risk. Waste products seldom reach the Nuclear Regulatory Commission (NRC) definition of radioactive waste material for (14)C and (3)H. Attomoles of labeled compounds are quantified in milligram-sized samples, such as 20 microl of blood. AMS is available from several facilities that offer services and new spectrometers that are affordable. Detailed examples of designing AMS studies are provided, and the methods of analyzing AMS data are outlined.

Immune cell activity during the initial stages of withdrawal from chronic exposure to cocaine or morphine.

The immunosuppression accompanying illicit drug use has been shown to contribute to a decreased resistance to a variety of pathogens; however, there is relatively little information on how long these effects persist following withdrawal from chronic drug exposure. To begin to address this question, Sprague-Dawley male rats were administered either cocaine (10 mg/kg, i.p., b.i.d.) for 7 days or morphine (escalating doses up to 40 mg/kg, s.c., b.i.d.) for a 10-day period. Control groups of animals received similar saline injections for equivalent time periods. Drug administration was abruptly discontinued and animals were sacrificed at 2, 24, 72 or 96 h following the last dose. At these time points, proliferation responses of peripheral blood T-lymphocytes stimulated by concanavalin A (Con A) and plasma levels of corticosterone were measured. Plasma corticosterone levels of cocaine- or morphine-treated animals were found to be significantly elevated 24 h following drug cessation as compared to saline animals. At this time, proliferation responses were significantly decreased and were further suppressed during cocaine and morphine withdrawal at 96 and 72 h, respectively. These results suggest that abrupt cessation of cocaine or morphine administration leads to activation of stress-related pathways that may contribute to an increased susceptibility of infection during the initial withdrawal phase.

Entry of [3H]norepinephrine, [125I]albumin and Evans blue from blood into brain following unilateral osmotic opening of the blood-brain barrier.

The blood-brain barrier in adult rats was opened unilaterally by infusing 1.58 M L (+)-arabinose in 0.9% NaCl solution into the internal carotid artery, via a catheter in the external carotid. The common carotid remained patent during the procedure. Osmotic barrier opening allowed entry into the brain of three intravascularly administered tracers--a visual tracer Evans blue (pulsely injected) and radioactive tracers [3H]norepinephrine (continuously infused) and [125I]albumin (pulsely injected). In osmotically perfused brain tissue, uptake of both 3H and 125I from blood was increased 2-5-fold above control, with maximal increases observed in the caudate nucleus, hippocampus and thalamus. In control brain regions, Evans blue and albumin remained intravascular, whereas norepinephrine was taken up, possibly by sympathetic nerve endings in cerebral vessels, as a function of blood plasma concentration and duration of exposure. The barrier closed within 4 h after intracarotid arabinose infusion, and barrier opening was not associated with edema as measured two days after infusion.

Evaluation of [3H]LY341495 for labeling group II metabotropic glutamate receptors in vivo.

New glutamatergic drugs are being developed as potential therapies for neurodegenerative disorders, anxiety disorders, and psychoses. The development of effective mGluR radiotracers would provide essential tools with which to probe these sites in living humans, providing critical information about certain disease processes involving the glutamaterigic system and its regulation in humans. As a first step towards this goal, the tritiated form of the high affinity group II metabotropic glutamate receptor (mGluR) antagonist LY341495 [K(D) (mGluR(2)) = 1.67 +/- 0.20 nM, K(D) (mGluR(3)) = 0.75 +/- 0.43 nM] was evaluated to determine its potential to label mGluRs in vivo. Dissection analysis of the regional brain distribution over time of [(3)H]LY341495 in male rats revealed low brain uptake and no significant demonstrable saturable binding of this tracer. A group II mGluR tracer possessing higher affinity than [(3)H]LY341495 and an absence of carboxylic acid groups is likely required for in vivo PET imaging purposes.

Mechanism of covalent adduct formation of aucubin to proteins.

The iridoid glucoside aucubin can irreversibly bind to proteins through the formation of its aglycone. In view of a possible involvement of these protein adducts in the toxicity of aucubin, we investigated the mechanism of binding of aucubin to proteins. [3H]aucubin in itself did not result in binding to protein whereas it covalently bound to rat serum albumin as a function of exposure time and dose in the presence of beta-glucosidase. The rate and extent of protein binding were significantly increased in the presence of the imine-trapping agent sodium cyanide. Oral administration of [3H]aucubin to rats showed that the total radioactivity in plasma remained at a similar level for up to 6 h once peak level was reached, suggesting that a considerable amount of radioactivity might be covalently associated with plasma proteins. The levels of radioactivity in the liver and kidney after oral dosing were higher than those after i.v. dosing. These results indicate that the open-chain aglycone of aucubin can form an imine bond with a nucleophilic site of the protein and these irreversible bindings may partially contribute to its biological and toxic effects.

Assessing the plasma pharmacokinetics, tissue distribution, excretion and effects on cholesterol pharmacokinetics of a novel hydrophilic compound, FM-VP4, following administration to rats.

PURPOSE: The purpose of this project was to 1) assess the disposition kinetics of [3H]-cholesterol following co-administration with a novel hydrophilic compound, FM-VP4, and 2) determine the pharmacokinetics, tissue distribution and excretion of [3H]FM-VP4 following single oral (150 mg/kg which includes 100 mCi of radiolabel) and intravenous (15 mg/kg which includes 10 mCi of radiolabel) doses. METHODS: Following an overnight fast (12-16 h) and 48 h post-surgery, adult male Sprague Dawley rats were divided into six treatment groups (n=4/group). Groups received single oral doses of 25 mCi/ml [3H]cholesterol alone or with 5, 10, 20, 50 and 100 mg/kg FM-VP4 at 0700 h. Ten percent Intralipid was used to solubilize and co-administer [3H]-cholesterol and FM-VP4. LC-MS analysis confirmed minimal cholesterol and vegetable stanol content within 10% Intralipid. Thin layer chromatography was used to confirm that the majority of radioactivity measured in plasma was associated with either esterified or unesterified cholesterol. In a second study pharmacokinetics of [3H]FM-VP4 were studied following intravenous or orally gavaged doses (n=8). Tissues, urine and feces were also collected in FM-VP4 kinetics study to measure tissue distribution of radioactivity. Plasma [3H]-cholesterol and [3H]FM-VP4 were tested for radioactivity. RESULTS: FM-VP4 co-administration significantly decreased [3H]-cholesterol AUC0-48h and Cmax, and increased CL/F and Vd/F of [3H]-cholesterol as compared to controls in a dose-dependent manner. Following oral administration of [3H]FM-VP4, the majority of radioactivity following was recovered in the feces and gastrointestinal (GI) tract. The compound exhibited an oral bioavailability of 6.5%. Following IV administration, a two-compartment pharmacokinetic model was observed and the majority of the radioactivity was recovered in the GI tract. CONCLUSIONS: FM-VP4 reduces plasma concentration of [3H]-cholesterol in fasting rats. [3H]FM-VP4 has a very low oral bioavailability.

High level of tritium-remaining in brain of rats exposed to tritiated water as infants.

Newborn CD/Crj rats were given tritiated water (HTO) saline i.p. at the dose of 220 microCi (8.14 MBq)/g of B.W. We monitored residual tritium in the sera and organs of male rats up to 26 weeks of age. The total absorbed doses in serum, liver, spleen, testis and brain were estimated to be 3.10, 2.35, 2.43, 2.38 and 2.84 Gy, respectively. Over four weeks of age, a 3-19 times higher tritium content was noted in brain compared to the values of other organs. At 26 weeks of age, testis atrophy was predominant with 20% of control value by relative organ weight in association with decreased spermatogonia and increased Leydig cells. The serum testosterone level was 67% and relative adrenal weight was significantly increased in comparison with the control values. The results indicated that the high accumulation of tritium in brain, destruction of spermatogenesis and feedback of androgenic response were caused by exposure to HTO as infants.

Blood clearance and organ localization of Candida albicans and E coli following dual infection in rats.

Immunosuppressed prematures, cancer patients, and transplant recipients are susceptible to bacterial or fungal sepsis or both. This report evaluates whether the ability of the reticuloendothelial system (RES) to remove blood-borne viable radiolabeled 35S Escherichia coli and 3H-Leucine Candida albicans is adversely affected by a dual intravenous challenge of these organisms. Male Sprague Dawley rats (n = 150) weighing 175 to 180 g were placed in 5 experimental groups (n = 30). Group I received intravenous (IV) C albicans (10(7)/mL), group II received E coli (10(9)/mL), group III received a dual injection of C albicans and E coli, group IV received Candida 1 hour prior to E coli, and group V received E coli 1 hour prior to fungi. At 1, 4, and 24 hours, tissue samples (50 to 100 mg) of liver, spleen, kidneys, and lungs were processed for liquid scintillation counting. Organ distribution of bacteria and fungi was calculated and expressed as mean percent +/- SD of labeled organisms. The liver trapped 72% +/- 10% and the lungs 1.1% +/- 0.3% of E coli (group II) (P < .001). The organ distribution of Candida (group I), however, was similar in liver and lungs (42.5% +/- 10% and 41.4% +/- 6.4%, respectively). Liver localization of E coli was unaffected by simultaneous or staggered fungal injection (groups III, 4, and V). Lung distribution of E coli following dual injection (group III) was significantly higher than controls (group II) (3.6% +/- 0.7% v 1.1% +/- 0.3%; P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Tritium concentrations of blood samples collected throughout Japan.

Tritium concentrations were measured for blood samples collected from 20 cities throughout Japan during 1989-1990. The mean 3H concentration was found to be 1.4 +/- 0.4 Bq L-1 and 1.0 +/- 0.4 Bq L-1 (combustion water) for free water 3H and organically-bound 3H, respectively, excluding the abnormally high data of one city. The organically-bound 3H contents clearly depended on the latitudes of sampling locations, although the free water 3H concentrations showed no correlation with the latitudes. Organically-bound 3H is considered to be more suitable than free water 3H as an indicator of long time 3H exposure to human.

Blood tritium level as a conservative estimate of soft tissue dose after tritium injection in mice.

The usefulness of the determination of the tritium concentration in blood for estimating the average soft tissue dose after tritium intake was tested. Tritiated water and a mixture of tritiated amino acids dissolved in saline solution were separately administered to adult male mice by intraperitoneal injection. The tritium levels in the blood, urine, whole body, spleen, liver, testis, and brain were then determined at various periods. With both types of tritium administration, the results suggested that the accumulated dose in the blood pool gave a conservative estimate of the accumulated dose to the soft tissue.

Dose absorbed by mouse red bone marrow after oral administration of tritiated water.

Tritiated water (HTO) was given to mice orally. Dose accumulation patterns in various organs and tissues after oral HTO intake were compared with those after intraperitoneal injection. The accumulated dose was 10-20% higher after intraperitoneal injection than after oral administration. A new technique was developed to isolate mouse red bone marrow from tibia. The absorbed beta-ray dose in the red bone marrow after oral tritium intake was lower than the body-averaged dose that was estimated based on the tritium concentration in the urine or the blood and higher than the absorbed dose in the liver and testis.

Radioactive waste minimization implications of clinically-indicated exsanguination procedures.

Exsanguination is a method of animal euthanasia approved for use in specific circumstances. Animals undergoing exsanguination are fully anesthetized, and the blood is removed resulting in hypovolemia. In situations where radioactive materials are used as part of a research protocol that remain predominantly suspended in the blood, the exsanguination procedure can result in a significant lowering of residual radioactivity content. This reduction can greatly affect the types of waste management and minimization options that can be subsequently applied. In this study, data were collected from 20 rabbits injected with approximately 29.6 MBq (0.8 mCi) of tritiated thymidine as part of a percutaneous transluminal carotid artery angioplasty study. Residual concentrations of radioactivity were consistently reduced by an average of 88%. The reduction was very significant in this instance, since the residual activities were below the established exemption limit of 1.85 kBq g(-1) (0.05 microCi g(-1)) for disposal of these wastes as non-radioactive. Although the exsanguination procedure can result in significant waste minimization opportunities in certain circumstances, this should not be the rationale for its use. Rather, the method of euthanasia should be based exclusively on sound animal care and use principles, and waste management strategies should then be made following that decision. Health Phys. 79(3):291-293; 2000 Key words: waste, low-level; waste management; radiation protection; blood

Free water 3H concentrations in serum samples collected during 1969-1992 in Akita, Japan.

The measurements for human and environmental samples from the 1960's and 1970's are important to understand the long-term transfer of 3H from the environment to the human body. The authors have previously reported 3H concentrations in diet samples collected in Akita Prefecture during 1969-1988. Serum samples from persons living in Akita Prefecture during 1969-1992 were recently obtained. The samples were originally gathered for medical examinations and stored in freezers at -20 degrees C. Composite samples from 100 persons on average were made for analysis. The free water 3H (FWT) concentrations in those samples were determined and compared with 3H concentrations in diet samples and precipitation. The long-term variation pattern of the FWT concentrations in the serum samples was similar to patterns in the diet samples and precipitation, but the FWT concentrations in the serum samples were slightly higher than those in the latter two. A single compartment model calculation showed that the apparent mean residence time of serum FWT was 1.4 y using precipitation as an input to the compartment.