Exposure level monitor of a carcinogenic glutamic acid pyrolysis product in rabbits

In order to determine a suitable indicator for estimating the exposure levels of the dietary carcinogen 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), a carcinogenic glutamic acid pyrolysis product, the levels of Glu-P-1 bound to blood components were monitored for 8 weeks by a high-performance liquid chromatography method after the dietary carcinogen was administered as single oral doses (0.2-50 mg) to rabbits. In all rabbits dosed with Glu-P-1, Glu-P-1 in erythrocytes was detectable even on day 42 after administration. Glu-P-1 in plasma disappeared faster than did Glu-P-1 in erythrocytes. Glu-P-1 levels in rabbit hemoglobin were linearly related to administered doses at all points of time investigated. The results suggest that Glu-P-1 covalently bound to hemoglobin is very suitable for monitoring long-term exposure levels.     

Detection of Trp-P-1 and Trp-P-2, carcinogenic tryptophan pyrolysis products, in dialysis fluid of patients with uremia

In order to estimate the exposure levels of mutagenic and carcinogenic heterocyclic amines in humans, we developed a high-performance liquid chromatography method to detect 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in dialysis fluid of patients with uremia. Using this methods, dialysis fluid of 12 patients who had received hemodialysis treatment or continuous ambulatory peritoneal dialysis was examined. Trp-P-1 was detected in dialysate of all uremic patients (727 +/- 282 pmoles, n = 12). In patients who had been treated with continuous ambulatory peritoneal dialysis, the average amount of Trp-P-1 found in whole dialysate (6 l) per day was 710 +/- 203 pmoles (mean +/- S.D., n = 8). Moreover, Trp-P-2 could be detected in 5 out of 12 patients (206 +/- 85 pmoles, n = 5). These results indicate that patients with uremia are actually exposed to carcinogenic tryptophan pyrolysis products. The average exposure level of Trp-P-1 in uremic patients apparently exceeded 710 pmoles (150 ng) per day.     

Attempts to use the HPRT-assay as an automated short-term monitor for an acute exposure to mutagens

Attempts have been made to use the hypoxanthine-guanine-phospho-ribosyl-transferase-assay as a method for automated screening of agent-induced phenotypic variants of human peripheral lymphocytes reflecting 6-thioguanine resistance and assumed to indicate genotoxic action. Different protocols of the hypoxanthine-guanine-phospho-ribosyl-transferase-system were used in this study in order to investigate whether the system can be a candidate for a short-term test for a rapid and reliable identification of biological systems exposed to agents. The current protocols were based on: 1) fluoresceinated monoclonal antibodies against bromodeoxyuridine-DNA for labelling of 6-thioguanine-resistant human lymphocytes and direct flow-cytometric enumeration of bromodeoxyuridine-positive events and: 2) indirect flow-cytometric enrichment of 6-thioguanine-resistant cells labelled with ³H-thymidine followed by autoradiographic enumeration of positive events. Both the direct and the indirect enumeration method yielded similar results down to the range 10^–4 with respect to frequency of variants. For the less time-consuming direct enumeration method the resolution was limited due to non-specific binding of the antibody and false positives. It was, nevertheless, sufficient to score variants induced in vitro with the mutagens EMS, MMC and TT in the same range as e.g. that of cancer patients during and after chemotherapy or radiotherapy, or that of psoriasis patients during and after PUVA (8-methoxypsoralen and long range UV light)-therapy. We conclude that the direct enumeration protocol can be used for a rapid screening of so called outliers, but a more sensitive test, such as the more time-consuming enrichment protocol based on autoradiography, must be used in order to score variants in the range 10^–5–10^–6 Abbreviations ³H-TdR Tritiated thymidine - BrdU 5-2-bromodeoxyuridine - DNA Deoxyribonucleic acid - EMS Ethyl-methanesulphonate - FBS Fetal bovine serum - FITC Fluorescein Isothiocyanate - G₀ Cell cycle stage - G₁ Cell cycle stage - G₂ Cell cycle stage - HPRT Hypoxanthine Guanine Phospho Ribosyl Transferase - LI Labelling index - MMC Mitomycin C - PBL Peripheral blood lymphocytes - PBS Phosphate buffered saline - PHA Phytohemagglutinin - PI Propidium iodide - PUVA 8-Methoxypsoralen and long range UV-light therapy - RNA Ribonucleic acid - RPMI RPMI-1640 culturing medium - S_e Early S-stage in the cell-cycle - S_l Late S-stage in the cell-cycle - SCE Sister chromatid exchange - TG 6-Thioguanine - TT Thiotepa - UV Ultraviolet light - V_f Variant frequency     

Determination of DNA level in human plasma

It has been first demonstrated that DNA concentration in the plasma of healthy donors was 5 to 30 mu kg per I ml of plasma or 10-50% of DNA content in leukocytes contained in the same blood volume. DNA plasma concentration was determined by registering DNA-bisbenzimide complex fluorescence in supernatant derived from plasma containing 10% NaCl upon heating for 2-3 min at 100 degrees C. The method is simple, specific and permits the determination of DNA concentrations in 0.05-0.1 ml of plasma samples.     

Cholesterol ozonation products as biomarkers for ozone exposure in rats.

Cholesterol, 1, which is present in both the lung lining fluid and cell membranes of lung tissue, reacts with ozone in aqueous systems to give 3 beta-hydroxy-5-oxo-5,6-secocholestan-6-al (2) as the major product. Reaction of 2 with 2,4-dinitrophenyl hydrazine (DNPH) in aqueous solutions, liposomes or lung extracts affords the anti and syn DNPH derivatives of 2 (3b and 3c) and of the rearrangement product 3,5-dihydroxy-B-norcholestane-6-carboxaldehyde (3a). These derivatives also are detected in lung tissue extracts from rats exposed to 1.3 ppm ozone for 12 hr.     

Soft-Talker: A sound level monitor for the hard-of-hearing using an improved tactile transducer

We describe a small wearable device which enables deaf people to monitor the volume of their voices; it consists of a microphone, amplifier, signal rectifier, smoothing and a level detector connected to a wrist-worn vibrator, and provides vibrotactile feedback of voice level.     

Cultured human monocytes require exposure to bacterial products to maintain an optimal oxygen radical response

Freshly isolated human blood monocytes displayed a vigorous oxygen radical response, measured as release of superoxide anion (O2-), after stimulation with phorbol myristate acetate (PMA) or opsonized zymosan. High O2- release was observed with cells isolated by using a variety of procedures. Monocytes cultured in endotoxin-free medium M199 with or without 5% heat-inactivated autologous serum gradually lost this ability to produce O2- in response to PMA over the course of 4 days. The decreased responsiveness to PMA was accompanied by decreased adherence and viability. The loss of function, adherence, and viability was prevented by supplementing the culture medium with either bacterial lipopolysaccharide (LPS) or muramyl dipeptide (MDP). The O2- response of monocytes cultured for several days without bacterial products could be partially restored by the addition of LPS on day 2 or 3 of culture. Partial restoration could be detected in monocytes after only 1 hr of exposure to LPS, although a maximal response required a 2-day exposure. The minimum effective concentration of MDP was 1 ng/ml; stereoisomers of MDP, which are inactive as adjuvants, had no effect at 1 micrograms/ml. The minimum effective concentration of LPS was 1 pg/ml, corresponding to fewer than 10 molecules of LPS per monocyte. These results suggest that exposure to LPS or other bacterial products, represented here by MDP, may be required to preserve the microbicidal potential of human monocyte-macrophages in vivo.     

Gene expression as a biomarker for human radiation exposure

Accidental exposure to ionizing radiation can be unforeseen, rapid, and devastating. The detonation of a radiological device leading to such an exposure can be detrimental to the exposed population. The radiation-induced damage may manifest as acute effects that can be detected clinically or may be more subtle effects that can lead to long-term radiation-induced abnormalities. Accurate identification of the individuals exposed to radiation is challenging. The availability of a rapid and effective screening test that could be used as a biomarker of radiation exposure detection is mandatory. We tested the suitability of alterations in gene expression to serve as a biomarker of human radiation exposure. To develop a useful gene expression biomonitor, however, gene expression changes occurring in response to irradiation in vivo must be measured directly. Patients undergoing radiation therapy provide a suitable test population for this purpose. We examined the expression of CC3, MADH7, and SEC PRO in blood samples of these patients before and after radiotherapy to measure the in vivo response. The gene expression after ionizing radiation treatment varied among different patients, suggesting the complexity of the response. The expression of the SEC PRO gene was repressed in most of the patients. The MADH7 gene was found to be upregulated in most of the subjects and could serve as a molecular marker of radiation exposure.     

Rat lungs as affected by an acute exposure to an increased level of carbon dioxide in the ambient air

Changes in lung characteristics of healthy adult male rats were studied following a 24-hr exposure to 15% CO₂ in air. The animals were weighed before and after the exposure period. Following the CO₂-exposure, the lungs of the rats were removed and their weights were determined. Continuous pressurevolume curves including inflation and deflation first with air and then with 0.9% saline were recorded from the excised lungs. The average gain in body weight of the control rats during the 24-hr period was 9 g while the CO₂-exposed rats lost 24 g. The mean values for wet and dry lung weights and trapped gas volume in the lung were essentially the same in the two groups of rats. Similarly the lung tissue compliance both during inflation and deflation was not significantly different between the two groups of animals. One characteristic feature of the total lung compliance for 69% of the CO₂-exposed rats was a progressive decrease in compliance on successive runs associated with a decrease in the area described by the compliance curves. These would suggest instability of lung alveoli in these rats with a significant alteration of their surfactant activity.

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Zusammenfassung In gesunden, erwachsenen männlichen Ratten wurden die Lungeneigenschaften nach 24-Stunden Haltung in 15% CO₂ haltiger Luft untersucht. Die Tiere wurden vor und nach der Belastung gewogen. Anschliessend wurden die Lungen entfernt, gewogen und fortlaufende Druck-Volumen Kurven mit Aufblasen und Ablassen zuerst von Luft dann von 0.9% Kochsalzlösung aufgenommen. Die Kontrolltiere nahmen in Mittel 9 g zu, während die CO₂-belasteten Tiere 24 g abnahmen. Die Gewichte der feuchten und trockenen Lungen und der eingeschlossenen Luftmenge waren in beiden Gruppen gleich. Die Nachgiebigkeit der Lungen beim Aufblasen und Ausblasen war nicht signifikant verschieden. Die Lungen der CO₂-belasteten Tiere wiesen in 69% ein progressives Nachlassen der Nachgiebigkeit bei wiederholten Füllungen auf, die mit einer Verkleinerung der Fläche der Retraktions-Schleife verbunden war. Dies weist auf eine Schwäche der Alveolarauskleidung mit einer signifikanten Veränderung ihrer oberflächlichen Aktivität hin.

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Resume On a étudié les modifications survenues aux caractéristiques des poumons de rats mâles adultes placés durant 24 heures dans une atmosphère contenant 15% de CO₂. Les animaux furent pesés avant et après l'expérience. On a en outre extrait et pesé les poumons des animaux après qu'ils aient subi le traitement au CO₂. On a ensuite établi les courbes continues du rapport pression/volume (gonflement et vidange inclus) en utilisant d'abord de l'air, puis une solution de sel de cuisine à 0,9%. Le gain de poids des rats de contrôle a été de 9 g en 24 heures, alors que les animaux soumis au CO₂ perdaient 24 g en moyenne. Le poids des poumons humides ou secs ainsi que la quantité d'air qu'ils pouvaient contenir ont été égaux dans les 2 groupes. L'élasticité des poumons n'a pas présenté de différence significative lors de leur gonflement ou de leur vidange. Pourtant, un trait caractéristique des poumons de rats soumis au traitement par CO₂ a été, dans 69% des cas, une diminution progressive de l'élasticité après avoir été gonflés et dégonflés plusieurs fois. Cette diminution est en relation avec un rétrécissement de la surface de la boucle de rétractation. Ce résultat laisse à penser qu'on a affaire à une altération du revêtement alvéolaire accompagnée d'une modification significative de son activité superficielle.

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This study was supported by the Air Force Office of Scientific Research, Office of Aerospace Research, US Air Force (Contract F44620-68-C-0014).     

The effect of exposure to carcinogenic metals on histone tail modifications and gene expression in human subjects

The precise mechanisms by which nickel and arsenic compounds exert their carcinogenic properties are not completely understood. In recent years, alterations of epigenetic mechanisms have been implicated in the carcinogenesis of compounds of these two metals. In vitro exposure to certain nickel or arsenic compounds induces changes in both DNA methylation patterns, as well as, in the levels of posttranslational modifications of histone tails. Changes in DNA methylation patterns have been reported in human subjects exposed to arsenic. Here we review our recent reports on the alterations in global levels of posttranslational histone modifications in peripheral blood mononuclear cells (PBMCs) of subjects with occupational exposure to nickel and subjects exposed to arsenic in their drinking water. Occupational exposure to nickel was associated with an increase in H3K4me3 and decrease in H3K9me2. A global increase in H3K9me2 and decrease in H3K9ac was found in subjects exposed to arsenic. Additionally, exposure to arsenic resulted in opposite changes in a number of histone modifications in males when compared with females in the arsenic population. The results of these two studies suggest that exposure to nickel or arsenic compounds, and possibly other carcinogenic metal compounds, can induce changes in global levels of posttranslational histone modifications in peripheral blood mononuclear cells.     

Protein-source tryptophan as an efficacious treatment for social anxiety disorder: a pilot study

Until recently, intact protein that is rich in tryptophan was not seen as an alternative to pharmaceutical-grade tryptophan because protein also contains large neutral amino acids (LNAAs) that compete for transport sites across the blood-brain barrier. Recent evidence indicates that when deoiled gourd seed (a rich source of tryptophan with approximately 22 mg/g protein) is combined with glucose (a carbohydrate that reduces serum levels of competing LNAAs) a clinical effect similar to that of pharmaceutical-grade tryptophan is achieved. Objective and subjective measures of anxiety in those suffering from social phobia (also known as social anxiety disorder) were employed to measure changes in anxiety in response to a stimulus as part of a double-blind, placebo-controlled, crossover study with a wash-out period of 1 week between study sessions. Subjects were randomly assigned to start with either (i) protein-source tryptophan (deoiled gourd seed) in combination with carbohydrate or (ii) carbohydrate alone. One week after the initial session, subjects returned for a follow-up session and received the opposite treatment of that received at the first session. All 7 subjects who began the study completed the 2-week protocol. Protein-source tryptophan with carbohydrate, but not carbohydrate alone, resulted in significant improvement on an objective measure of anxiety. Protein-source tryptophan combined with a high glycemic carbohydrate is a potential anxiolytic to those suffering from social phobia.     

Analysis of the mechanism by which tryptophan analogs inhibit human myeloperoxidase

Myeloperoxidase (MPO) catalyzes the formation of oxidants that have been implicated in the pathogenesis of various diseases, including cardiovascular and pulmonary diseases and cancer. Inhibition of MPO oxidant-generating activity represents an attractive target for preventing the progression of inflammatory conditions. Peroxidation and chlorination catalytic activity were utilized to screen for the most effective tryptophan analog that inhibits MPO. Rapid kinetic measurements were performed to determine the mechanisms through which these compounds inhibit the catalytic activity of MPO. Substituents on the amino and carboxyl termini of tryptophan enhance its affinity toward MPO compared to a substituent on the indole ring. Hydrogen-bond donor capabilities and a positive charge on the amino group are not required for MPO inhibition. Hydroxyl-containing indoles did not inhibit MPO H₂O₂-consumption activity. Elimination of the negative charge from the carboxyl terminus by introducing a hydrophobic character significantly enhanced tryptophan analog affinity for MPO and improved its inhibitory properties. Further mechanistic studies indicated that indole compounds inhibited MPO activity through the accumulation of compound II, an inactive MPO intermediate. Our results show that specific structural features of tryptophan analogs are involved in increasing the affinity for MPO and providing a significantly greater inhibition of MPO's catalytic activities.     

Issues in the development of medical products based on human plasma

Product development and process validation are shown in the case of several products obtained from human plasma. These are virus-inactivated plasma, intravenous immunoglobulins and the clotting factors VIII and IX. Different analytical methods are presented, which are used for product control and in-process control. For the production of virus-inactivated human plasma a down-scale protocol is presented, allowing a simulation of the production on a laboratory scale. Virus validation has shown that the reduction of transfusion-relevant viruses in the process was higher than six log steps. Determination of leachables from the RP-column, which was used in this production, proved that they appear in the final product in quantities below the detection limits only. It was also shown that the chemicals used for virus inactivation could be quantitatively removed from the product. For the isolation of other products, here intravenous gamma globulins and the clotting factors VIII and IX, similar validation steps had to be taken. In the case of clotting factor VIII the following data were determined, the reduction of viruses, the amount of leachables from the column, the residues of chemicals from the solvent/detergent treatment for virus inactivation. Virus reduction was successfully performed as well as the removal of chemicals used for virus inactivation. The amount of leachables from the columns used for chromatographic purification was found to be far below the permissible levels.