Diethyldithiocarbamate causes nigral cell loss and dopamine depletion with nontoxic doses of MPTP

Although nontoxic when administered alone, diethyldithiocarbamate (DDC) is known to enhance the dopamine-depleting effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the mouse striatum. The purpose of the present study was twofold: (i) to carefully characterize the effects of DDC on MPTP-induced degeneration of dopaminergic neurons in substantia nigra pars compacta using unbiased, stereological cell counting techniques and (ii) to determine whether or not DDC can convert a nontoxic dose of MPTP into one which is clearly toxic on dopaminergic neurons in the substantia nigra. A single low dose of MPTP (15 mg/kg intraperitoneally (ip)) was used for these studies, which failed to induce any neurochemical or histological effects on the nigrostriatal system of C57BL/6 mice when administered alone. However, when animals were pretreated with DDC (400 mg/kg ip), the same dose of MPTP resulted in a 50% loss of neurons in the substantia nigra pars compacta, as well as a 70% reduction in striatal dopamine (DA). A 31% reduction of DA in the ventral mesencephalon was also seen. This combined regimen of DDC and MPTP was not significantly different from a maximally tolerated "toxic" dose of MPTP alone (15 mg/kg x 4, 1 h apart, ip). As expected, animals receiving DDC alone did not show any dopamine depletion nor nigral neuronal loss. The present study confirms previous work suggesting that DDC enhances MPTP-induced nigral cell loss and shows for the first time that DDC can "unmask" MPTP toxicity. These observations could have implications for theories on the cause of Parkinson's disease.     

Monoamine oxidase-dependent metabolism of dopamine in the striatum and substantia nigra of

Purpose. Oral bioavailability for antisense oligonucleotides has recently been reported but the mechanistic details are not known. The proposed oral delivery of nucleic acids will, therefore, require an understanding of the membrane binding interactions, cell uptake and transport of oligonucleotides across the human gastro-intestinal epithelium. In this initial study, we report on the cell-surface interactions of oligonucleotides with human intestinal cells. Methods. We have used the Caco-2 cell line as an in vitro model of the human intestinal epithelium to investigate the membrane binding interactions of 20-mer phosphodiester (PO) and phosphorothioate (PS) oligonucleotides. Results. The cellular association of both an internally [³H]-labelled and a 5end [³²P]-labelled PS oligonucleotide (3.0% at 0.4 µM extracellular concentration) was similar and was an order of magnitude greater than that of the 5end [³²P]-labelled PO oligonucleotide (0.2%) after 15 minutes incubation in these intestinal cells. The cellular association of PS was highly saturable with association being reduced to 0.9% at 5 µM whereas that of PO was less susceptible to competition (0.2% at 5 µM, 0.1% at 200 µM). Differential temperature-dependence was demonstrated; PS interactions were temperature-independent whereas the cellular association of PO decreased by 75% from 37°C to 17°C. Cell association of oligonucleotides was length and pH-dependent. A decrease in pH from 7.2 to 5.0 resulted in a 2- to 3-fold increase in cell-association for both backbone types. This enhanced association was not due to changes in lipophilicity as the octanol:aqueous buffer distribution coefficients remained constant over this pH range. The ability of NaCl washes to remove surface-bound PS oligonucleotides in a concentration-dependent manner suggests their binding may involve ionic interactions at the cell surface. Cell-surface washing with the proteolytic enzyme, Pronase^®, removed approximately 50% of the cell-associated oligonucleotide for both backbone types. Conclusions. Binding to surface proteins seems a major pathway for binding and internalization for both oligonucleotide chemistries and appear consistent with receptor (binding protein)-mediated endocytosis. Whether this binding protein-mediated entry of oligonucleotides can result in efficient transepithelial transport, however, requires further study.     

Interactions of Phosphodiester and Phosphorothioate Oligonucleotides with Intestinal Epithelial Caco-2 Cells

Purpose. Oral bioavailability for antisense oligonucleotides has recently been reported but the mechanistic details are not known. The proposed oral delivery of nucleic acids will, therefore, require an understanding of the membrane binding interactions, cell uptake and transport of oligonucleotides across the human gastro-intestinal epithelium. In this initial study, we report on the cell-surface interactions of oligonucleotides with human intestinal cells. Methods. We have used the Caco-2 cell line as an in vitro model of the human intestinal epithelium to investigate the membrane binding interactions of 20-mer phosphodiester (PO) and phosphorothioate (PS) oligonucleotides. Results. The cellular association of both an internally [³H]-labelled and a 5end [³²P]-labelled PS oligonucleotide (3.0% at 0.4 µM extracellular concentration) was similar and was an order of magnitude greater than that of the 5end [³²P]-labelled PO oligonucleotide (0.2%) after 15 minutes incubation in these intestinal cells. The cellular association of PS was highly saturable with association being reduced to 0.9% at 5 µM whereas that of PO was less susceptible to competition (0.2% at 5 µM, 0.1% at 200 µM). Differential temperature-dependence was demonstrated; PS interactions were temperature-independent whereas the cellular association of PO decreased by 75% from 37°C to 17°C. Cell association of oligonucleotides was length and pH-dependent. A decrease in pH from 7.2 to 5.0 resulted in a 2- to 3-fold increase in cell-association for both backbone types. This enhanced association was not due to changes in lipophilicity as the octanol:aqueous buffer distribution coefficients remained constant over this pH range. The ability of NaCl washes to remove surface-bound PS oligonucleotides in a concentration-dependent manner suggests their binding may involve ionic interactions at the cell surface. Cell-surface washing with the proteolytic enzyme, Pronase^®, removed approximately 50% of the cell-associated oligonucleotide for both backbone types. Conclusions. Binding to surface proteins seems a major pathway for binding and internalization for both oligonucleotide chemistries and appear consistent with receptor (binding protein)-mediated endocytosis. Whether this binding protein-mediated entry of oligonucleotides can result in efficient transepithelial transport, however, requires further study.     

NTP summary report on the metabolism, disposition, and toxicity of 1,4-butanediol (CAS No. 110-63-4)

1,4-Butanediol is an industrial chemical used in the manufacture of other organic chemicals. It was nominated by the National Cancer Institute and selected for evaluation by the NTP because of high production volume, the potential for worker exposure, the lack of adequate toxicological characterization, and the lack of evaluation for carcinogenic potential. As documented in the scientific literature, 1,4-butanediol is rapidly absorbed and metabolized to gamma-hydroxybutyric acid in animals and humans. A metabolism and disposition study conducted in F344/N rats by the NTP confirmed the rapid and extensive conversion of 1-[14C]-1,4-butanediol to 14CO2. Because of this rapid and extensive conversion, the toxicological profile of 1,4-butanediol reflects that of gamma-hydroxybutyric acid. gamma-Hydroxybutyric acid is a naturally occurring chemical found in the brain and peripheral tissues which is converted to succinate and processed through the tricarboxylic acid cycle. Although the function of gamma-hydroxybutyric acid in peripheral tissues is unknown, in the brain and neuronal tissue it is thought to function as a neuromodulator. gamma-Hydroxybutyric acid readily crosses the blood-brain barrier, and oral, intraperitoneal, or intravenous administration elicits characteristic neuropharmacologic responses. These same responses are observed after administration of 1,4-butanediol. The lactone of gamma-hydroxybutyric acid, gamma-butyrolactone, is also rapidly converted to gamma-hydroxybutyric acid by enzymes in the blood and liver of animals and humans. gamma-Butyrolactone was previously evaluated by the NTP in 14-day and 13-week toxicology studies and 2-year toxicology and carcinogenesis studies in F344/N rats and B6C3F1 mice. No organ-specific toxicity occurred in the toxicology studies. In the carcinogenesis studies, an equivocal response occurred in male mice, based on a marginal increase in the incidence of pheochromocytomas of the renal medulla. Because of the rapid and extensive conversion of gamma-butyrolactone to gamma-hydroxybutyric acid, the evaluation of gamma-butyrolactone was in fact an evaluation of gamma-hydroxybutyric acid. This summary report presents a review of the current literature which documents that both 1,4-butanediol and gamma-butyrolactone are rapidly metabolized to gamma-hydroxybutyric acid, and the pharmacologic and toxicologic responses to these chemicals are due to their metabolic conversion to gamma-hydroxybutyric acid. Because the toxicity and carcinogenicity of gamma-hydroxybutyric acid was fully evaluated in the NTP studies of gamma-butyrolactone, and a lack of organ-specific toxicity or carcinogenic potential was demonstrated, it is concluded that there is a high likelihood that 1,4-butanediol would be negative in a similar set of studies. For these reasons, it is the opinion of the NTP that 1,4-butanediol should be considered not carcinogenic in animals and no further evaluation of 1,4-butanediol is needed at this time.     

Patient maintained alfentanil target-controlled infusion for analgesia during extracorporeal shock wave lithotripsy

Purpose

The purpose of this study was to determine whether alfentanil given by a pharmacokinetic-based target controlled infusion (TCI) system under patient control is a suitable analgesic technique for extracorporeal shock wave lithotripsy (ESWL).

Methods

The design was an open, unblinded, noncomparative, prospective study. Forty outpatients undergoing ESWL were given patient maintained alfentanil TCI. Pain, nausea and sedation were assessed every 300 shocks. Vital signs were recorded every three minutes, pulse oximetry and electrocardiography being monitored continuously. Blood alfentanil concentration was measured for comparison with the predicted value.

Results

Alfentanil consumption (median 1.34 mg, range 0.8–3.6) and measured levels following treatment (median 60 ng·ml^?1, range 15.6–134.3) varied widely. The precision of the TCI system and the median prediction error (bias) were both 49%. The median of pain scores recorded during treatment was 4 (range 0–8). The median respiration rate was 15 bpm (range 10–23), three patients required oxygen (SaO₂ < 92%) Cardiovascular measurements were stable and there was no excessive sedation. The incidence of nausea was 15%. All patients were ready for hospital discharge within one hour following treatment.

Conclusions

Patient maintained alfentanil TCI provides good analgesia for ESWL in the majority of patients with little sedation. Respiratory depression is uncommon but supplementary oxygen should be given prophylactically. There is considerable interindividual variation in demand for alfentanil indicating the usefulness of the patient control method. The TCI system underestimated alfentanil blood concentrations but this did not affect its clinical usefulness.     

Coping in thebarrio: Case studies of Mexican-American families

This paper reports preliminary findings of a study of coping abilities of Mexican-American families. The purpose of the study was to identify variables related to styles of behavior that can be characterized as adaptive. A complex of factors differentiated families who were judged to be dealing effectively with their environment (copers) from those who were not (noncopers). The factors included the health status of the children, various child-rearing attitudes and practices, and patterns of decision making as they related to a more general ability of parents to conceptualize and organize time.The authors wish to thank Bernard R. Cavazos and Maria Rosario de los Santos for their help in data collection and interpretation.     

Evaluation of the toxicity,pathology, and treatment of cyclohexylmethylphosphonofluoridate (CMPF) poisoning in rhesus monkeys

Cyclohexylmethylphosphonofluoridate (CMPF) is an organophosphate cholinesterase inhibitor with military significance. The purpose of these studies was 1) to determine the acute toxicity of CMPF in the male rhesus monkey, 2) to evaluate the efficacy of pyridostigmine (PYR) pretreatment plus atropine and oxime (2-PAM or HI6) treatment, and 3) to evaluate the pathological consequences of acute poisoning. An i. m. LD₅₀ of CMPF was estimated using an up-and-down dose selection procedure and 12 animals. The 48-h and 7-day LD₅₀ was 46.6 g/kg, i.m. In the protection experiments, pyridostigmine (0.3–0.7 mg/kg/24 h) was administered by surgically implanted osmotic minipumps for 3–12 days resulting in 21–65% inhibition of erythrocyte acetylcholinesterase activity. Animals were challenged with 5 × LD₅₀ CMPF (233 g/kg) and treated with atropine (0.4 mg/kg) and either 2-PAM (25.7 mg/kg) or HI6 (37.8 mg/kg) at the onset of signs or 1 min after challenge. Osmotic pumps were removed within 30 min after agent challenge. Pyridostigmine, atropine, and either 2-PAM or HI6 were completely effective against CMPF, saving ten of ten animals in each group. In comparison, three of five animals challenged with 5 × LD₅₀ of soman and treated with atropine and 2-PAM survived 7 days. The primary histologic lesions in the acute toxicity group were neuronal degeneration/necrosis and spinal cord hemorrhage. The CMPF treated groups (total of 20 animals) had minimal nervous system changes with no significant lesion difference resulting from the different oxime therapies. The primary non-neural lesions were degenerative cardiomyopathy and skeletal muscle degeneration which occasionally progressed to necrosis and mineralization. The results indicate that PYR pretreatment in conjunction with atropine and oxime treatment is an effective regimen against battlefield relevant levels (5 × LD₅₀) of CMPF.The experiments reported here were conducted according to the Guide for Care and Use of Laboratory Animals (1985), as prepared by the Committee on Care and Use of Laboratory Animals, National Research Council, NIH Publication No. 85-23. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting views of the Department of the Army or the Department of Defense.     

Tubal sterilization, hysterectomy, and the subsequent occurrence of epithelial ovarian cancer

Several hypotheses predict that tubal sterilization and hysterectomy may influence a woman's risk of developing ovarian cancer. To examine the relation between these surgeries and epithelial ovarian cancer, the authors analyzed data from the Cancer and Steroid Hormone Study, a case-control study of women aged 20-54 years. Eight population-based cancer registries in the United States identified women with newly diagnosed epithelial ovarian cancer during 1980-1982 (n = 494). A comparison sample of female residents of these eight areas (n = 4,238) was identified through random digit dialing. Women who had had tubal sterilization (relative risk (RR) = 0.69, 95% confidence interval (Cl) 0.50-0.95), a hysterectomy only (RR = 0.55, 95% Cl 0.38-0.81), or a hysterectomy with unilateral oophorectomy (RR = 0.60, 95% Cl 0.31-1.17) had lower risks of ovarian cancer than did women who had never had any sterilization surgery. However, the negative associations with tubal sterilization and hysterectomy only appeared to wane after two decades. These findings may be partly explained by the screening for occult ovarian pathology that often accompanies pelvic surgery: Women whose ovaries screen as "negative" may be temporarily at low risk of being diagnosed with ovarian cancer. However, because the decreased risks persisted for so long, it is conceivable that hormonal, mechanical, or circulatory sequelae of these sterilization procedures may act to lower ovarian cancer risk.     

A method for increasing confidence in respiratory gas exchange measurements in mechanically ventilated patients.

Measurement of O2 uptake (VO2) and CO2 output (VCO2) with automated instruments can be used to optimize nutritional management of critically ill patients. However, these measurements may be made infrequently because of calibration problems or suspected inaccuracies, especially when the patient is given supplemental oxygen. A simple method is described for periodic testing of automated gas exchange measurements. While a test lung is mechanically ventilated an accurately known mixture of CO2 and nitrogen is introduced into the inspired gas, simulating VO2 by dilution and VCO2 by addition of CO2. The ratio of VCO2:VO2 (respiratory gas exchange ratio, R) in the "expired gas" should be [FGCO2/(1-FGCO2)] x [(1-FIO2)/FIO2], where FGCO2 is the fraction of CO2 in the diluting gas and FIO2 is the fraction of O2 in the inspired gas. R is independent of the flow rate of the diluting gas or the rate of ventilation of the test lung. Using a mixing chamber-mass spectrometer, we found that R calculated from measurement of CO2 and O2 concentrations in the simulated mixed expired gas closely matched the predicted R for FIO2 = 0.21-0.50. On the other hand, when an automated gas exchange measurement device was tested, R was sometimes excessively high, especially for FIO2 greater than 0.35. This method, using a single diluting gas and without precision flowmeters, may be useful for periodic testing of respiratory gas exchange instruments in the intensive care unit. If a discrepancy is found between measured and predicted R, measurements should not be relied upon until further calibration or repairs can be effected.