Modular air-liquid interface aerosol exposure system (MALIES) to study toxicity of nanoparticle aerosols in 3D-cultured A549 cells in vitro.

We present a novel lung aerosol exposure system named MALIES (modular air-liquid interface exposure system), which allows three-dimensional cultivation of lung epithelial cells in alveolar-like scaffolds (MatriGrids®) and exposure to nanoparticle aerosols. MALIES consists of multiple modular units for aerosol generation, and can be rapidly assembled and commissioned. The MALIES system was proven for its ability to reliably produce a dose-dependent toxicity in A549 cells using CuSO4 aerosol. Cytotoxic effects of BaSO4- and TiO2-nanoparticles were investigated using MALIES with the human lung tumor cell line A549 cultured at the air-liquid interface. Experiments with concentrations of up to 5.93 × 105 (BaSO4) and 1.49 × 106 (TiO2) particles/cm3, resulting in deposited masses of up to 26.6 and 74.0 µg/cm2 were performed using two identical aerosol exposure systems in two different laboratories. LDH, resazurin reduction and total glutathione were measured. A549 cells grown on MatriGrids® form a ZO-1- and E-Cadherin-positive epithelial barrier and produce mucin and surfactant protein. BaSO4-NP in a deposited mass of up to 26.6 µg/cm2 resulted in mild, reversible damage (~ 10% decrease in viability) to lung epithelium 24 h after exposure. TiO2-NP in a deposited mass of up to 74.0 µg/cm2 did not induce any cytotoxicity in A549 cells 24 h and 72 h after exposure, with the exception of a 1.7 fold increase in the low exposure group in laboratory 1. These results are consistent with previous studies showing no significant damage to lung epithelium by short-term treatment with low concentrations of nanoscale BaSO4 and TiO2 in in vitro experiments.

New aspects in deriving health-based guidance values for bromate in swimming pool water.

Bromate, classified as a EU CLP 1B carcinogen, is a typical by-product of the disinfection of drinking and swimming pool water. The aim of this study was (a) to provide data on the occurrence of bromate in pool water, (b) to re-evaluate the carcinogenic MOA of bromate in the light of existing data, (c) to assess the possible exposure to bromate via swimming pool water and (d) to inform the derivation of cancer risk-related bromate concentrations in swimming pool water. Measurements from monitoring analysis of 229 samples showed bromate concentrations in seawater pools up to 34 mg/L. A comprehensive non-systematic literature search was done and the quality of the studies on genotoxicity and carcinogenicity was assessed by Klimisch criteria (Klimisch et al., Regul Toxicol Pharmacol 25:1-5, 1997) and SciRAP tool (Beronius et al., J Appl Toxicol, 38:1460-1470, 2018) respectively. Benchmark dose (BMD) modeling was performed using the modeling average mode in BMDS 3.1 and PROAST 66.40, 67 and 69 (human cancer BMDL10; EFSA 2017). For exposure assessment, data from a wide range of sources were evaluated for their reliability. Different target groups (infants/toddlers, children and adults) and exposure scenarios (recreational, sport-active swimmers, top athletes) were considered for oral, inhalation and dermal exposure. Exposure was calculated according to the frequency of swimming events and duration in water. For illustration, cancer risk-related bromate concentrations in pool water were calculated for different target groups, taking into account their exposure using the hBMDL10 and a cancer risk of 1 in 100,000. Convincing evidence was obtained from a multitude of studies that bromate induces oxidative DNA damage and acts as a clastogen in vitro and in vivo. Since statistical modeling of the available genotoxicity data is compatible with both linear as well as non-linear dose-response relationships, bromate should be conservatively considered to be a non-threshold carcinogen. BMD modeling with model averaging for renal cancer studies (Kurokawa et al., J Natl. Cancer Inst, 1983 and 1986a; DeAngelo et al., Toxicol Pathol 26:587-594, 1998) resulted in a median hBMDL10 of 0.65 mg bromate/kg body weight (bw) per day. Evaluation of different age and activity groups revealed that top athletes had the highest exposure, followed by sport-active children, sport-active adults, infants and toddlers, children and adults. The predominant route of exposure was oral (73-98%) by swallowing water, followed by the dermal route (2-27%), while the inhalation route was insignificant (< 0.5%). Accepting the same risk level for all population groups resulted in different guidance values due to the large variation in exposure. For example, for an additional risk of 1 in 100,000, the bromate concentrations would range between 0.011 for top athletes, 0.015 for sport-active children and 2.1 mg/L for adults. In conclusion, the present study shows that health risks due to bromate exposure by swimming pool water cannot be excluded and that large differences in risk exist depending on the individual swimming habits and water concentrations.

Critical evaluation of human health risks due to hydraulic fracturing in natural gas and petroleum production.

The use of hydraulic fracturing (HF) to extract oil and natural gas has increased, along with intensive discussions on the associated risks to human health. Three technical processes should be differentiated when evaluating human health risks, namely (1) drilling of the borehole, (2) hydraulic stimulation, and (3) gas or oil production. During the drilling phase, emissions such as NOx, NMVOCs (non-methane volatile organic compounds) as precursors for tropospheric ozone formation, and SOx have been shown to be higher compared to the subsequent phases. In relation to hydraulic stimulation, the toxicity of frac fluids is of relevance. More than 1100 compounds have been identified as components. A trend is to use fewer, less hazardous and more biodegradable substances; however, the use of hydrocarbons, such as kerosene and diesel, is still allowed in the USA. Methane in drinking water is of low toxicological relevance but may indicate inadequate integrity of the gas well. There is a great concern regarding the contamination of ground- and surface water during the production phase. Water that flows to the surface from oil and gas wells, so-called 'produced water', represents a mixture of flow-back, the injected frac fluid returning to the surface, and the reservoir water present in natural oil and gas deposits. Among numerous hazardous compounds, produced water may contain bromide, arsenic, strontium, mercury, barium, radioactive isotopes and organic compounds, particularly benzene, toluene, ethylbenzene and xylenes (BTEX). The sewage outflow, even from specialized treatment plants, may still contain critical concentrations of barium, strontium and arsenic. Evidence suggests that the quality of groundwater and surface water may be compromised by disposal of produced water. Particularly critical is the use of produced water for watering of agricultural areas, where persistent compounds may accumulate. Air contamination can occur as a result of several HF-associated activities. In addition to BTEX, 20 HF-associated air contaminants are group 1A or 1B carcinogens according to the IARC. In the U.S., oil and gas production (including conventional production) represents the second largest source of anthropogenic methane emissions. High-quality epidemiological studies are required, especially in light of recent observations of an association between childhood leukemia and multiple myeloma in the neighborhood of oil and gas production sites. In conclusion, (1) strong evidence supports the conclusion that frac fluids can lead to local environmental contamination; (2) while changes in the chemical composition of soil, water and air are likely to occur, the increased levels are still often below threshold values for safety; (3) point source pollution due to poor maintenance of wells and pipelines can be monitored and remedied; (4) risk assessment should be based on both hazard and exposure evaluation; (5) while the concentrations of frac fluid chemicals are low, some are known carcinogens; therefore, thorough, well-designed studies are needed to assess the risk to human health with high certainty; (6) HF can represent a health risk via long-lasting contamination of soil and water, when strict safety measures are not rigorously applied.

Critical evaluation of key evidence on the human health hazards of exposure to bisphenol A.

Despite the fact that more than 5000 safety-related studies have been published on bisphenol A (BPA), there seems to be no resolution of the apparently deadlocked controversy as to whether exposure of the general population to BPA causes adverse effects due to its estrogenicity. Therefore, the Advisory Committee of the German Society of Toxicology reviewed the background and cutting-edge topics of this BPA controversy. The current tolerable daily intake value (TDI) of 0.05 mg/kg body weight [bw]/day, derived by the European Food Safety Authority (EFSA), is mainly based on body weight changes in two- and three-generation studies in mice and rats. Recently, these studies and the derivation of the TDI have been criticized. After having carefully considered all arguments, the Committee had to conclude that the criticism was scientifically not justified; moreover, recently published additional data further support the reliability of the two- and three-generation studies demonstrating a lack of estrogen-dependent effects at and below doses on which the current TDI is based. A frequently discussed topic is whether doses below 5 mg/kg bw/day may cause adverse health effects in laboratory animals. Meanwhile, it has become clear that positive results from some explorative studies have not been confirmed in subsequent studies with higher numbers of animals or a priori defined hypotheses. Particularly relevant are some recent studies with negative outcomes that addressed effects of BPA on the brain, behavior, and the prostate in rodents for extrapolation to the human situation. The Committee came to the conclusion that rodent data can well be used as a basis for human risk evaluation. Currently published conjectures that rats are insensitive to estrogens compared to humans can be refuted. Data from toxicokinetics studies show that the half-life of BPA in adult human subjects is less than 2 hours and BPA is completely recovered in urine as BPA-conjugates. Tissue deconjugation of BPA-glucuronide and -sulfate may occur. Because of the extremely low quantities, it is only of minor relevance for BPA toxicity. Biomonitoring studies have been used to estimate human BPA exposure and show that the daily intake of BPA is far below the TDI for the general population. Further topics addressed in this article include reasons why some studies on BPA are not reproducible; the relevance of oral versus non-oral exposure routes; the degree to which newborns are at higher systemic BPA exposure; increased BPA exposure by infusions in intensive care units; mechanisms of action other than estrogen receptor activation; and the current regulatory status in Europe, as well as in the USA, Canada, Japan, New Zealand, and Australia. Overall, the Committee concluded that the current TDI for BPA is adequately justified and that the available evidence indicates that BPA exposure represents no noteworthy risk to the health of the human population, including newborns and babies.

Background of REACH in EU regulations on evaluation of chemicals.

Industrial chemicals are needed for chemical synthesis or technical purposes. These beneficial effects are counterbalanced by the potential health risks for all who come into contact with them. The new chemical legislation of the EU, Registration, Evaluation and Authorization of Chemicals (REACH) will force the responsibility of manufacturers and importers of chemical substances to gather the right information needed to decide on the right circumstances of use and control of chemical substances and products. In order to understand the roots of REACH, experiences gained with regard to existing chemicals legislation, particularly in Germany, will be reviewed. Since Council Directive 67/548/EEC all chemicals placed on the market need a set of standard information and provisions for safe transportation. This directive and its amendments (Council Directive(s) 79/831/EEC and 92/32/EEC) have established for new substances a sound information data basis for classification of dangerous properties. Under Council Regulation 793/93/EEC, regulations and administrative provisions have established the requirement to assess the risk to man and the environment of existing substances. So far, only 119 substances have been evaluated under the forces of this regulation. This separation has led to a substantial imbalance between existing substances and new substances with respect to available data needed to recognize hazards for health. The register of produced and imported chemical substances under REACH should eliminate some of this separation and will also be the key for selection of substances of very high concern by the authorization process to restrict the use and distribution accordingly.

Concept of REACH and impact on evaluation of chemicals.

Industrial chemicals have been in use for many decades and new products are regularly invented and introduced to the market. Also for decades, many different chemical laws have been introduced to regulate safe handling of chemicals in different use patterns. The patchwork of current regulation in the European Union is to be replaced by the new regulation on industrial chemical control, REACH. REACH stands for registration, evaluation, and authorization of chemicals. REACH entered force on June 1, 2007. REACH aims to overcome limitations in testing requirements of former regulation on industrial chemicals to enhance competitiveness and innovation with regard to manufacture safer substances and to promote the development of alternative testing methods. A main task of REACH is to address data gaps regarding the properties and uses of industrial chemicals. Producers, importers, and downstream users will have to compile and communicate standard information for all chemicals. Information sets to be prepared include safety data sheets (SDS), chemical safety reports (CSR), and chemical safety assessments (CSA). These are designed to guarantee adequate handling in the production chain, in transport and in use and to prevent the substances from being released to and distributed within the environment. Another important aim is to identify the most harmful chemicals and to set incentives to substitute them with safer alternatives. On one hand, REACH will have substantial impact on the basic understanding of the evaluation of chemicals. However, the toxicological sciences can also substantially influence the workability of REACH that supports the transformation of data to the information required to understand and manage acceptable and non acceptable risks in the use of industrial chemicals. The REACH regulation has been laid down in the main document and 17 Annexes of more than 849 pages. Even bigger technical guidance documents will follow and will inform about the rules for application and work out of dossiers. The following article gives a comprehensive overview on the concept of REACH to give deeper insight into this document. Members of the scientific community will have to define their own position as researchers, teachers, and experts to support the efforts to protect human health and the environment. The concept of REACH as well as new approaches to adapt standard testing regimes to foster a risk oriented approach in required work load to decrease animal based tests and to strengthen weight of evidence are explained in detail in this article.

Ablation of vitreous tissue with erbium:YAG laser.

PURPOSE: Using a noncontact erbium (Er):yttrium--aluminium--garnet (YAG) laser, ablation of vitreous was compared to distilled water in vitro. METHODS: The porcine vitreous body and distilled water were ablated in vitro at different pulse lengths and pulse energies. Selected pulse energies were 25, 35, 45, 75, and 100 mJ (pulse rate: 1 Hz; laser beam diameter at the surface of the sample: 2 mm). Pulse lengths were at 140 +/- 3 microsec, 190 +/- 4 microsec, and 240 +/- 5 microsec. The loss of weight in vitreous tissue and distilled water was measured using precision scales and corrected for evaporation, respectively. The Mann-Whitney U test was used to assess the significance of differences in ablation rates of water and vitreous. P < 0.05 was considered statistically significant. RESULTS: Reproducible and constant ablation rates were found in both vitreous and distilled water in each of 10 consecutive series of 50 laser pulses at constant laser parameters. Ablation rates per pulse (microg/microsec) of vitreous tissue were as follows: 3.0 microg to 45.8 microg (140 microsec), 10.4 microg to 53.8 microg (190 microsec), and 17.9 microg to 24.2 microg (240 microsec). The ablation rates exhibited a linear correlation with increasing pulse energies and also with decreasing pulse lengths. Considering the pulse lengths of 190 microsec and 240 microsec with all pulse energies tested, the ablation rates of distilled water were significantly higher (P < 0.05) than ablation of vitreous tissue. The ablation rates at a pulse length of 140 microsec were not significantly different. The differences per pulse were as follows: 0.5 microg to 2.1 microg (140 microsec), 1.9 microg to 6.0 microg (190 microsec), and 3.5 microg to 8.7 microg (240 microsec). CONCLUSIONS: Vitreous ablation is possible using Er:YAG laser. The ablation characteristics of vitreous have proved to be similar but not equal to that of water.

Kinetics of melphalan leakage during hyperthermic isolation perfusion in melanoma of the limb.

The kinetics of melphalan leakage into the peripheral blood were studied in 21 patients undergoing hyperthermic isolation perfusion of the upper or lower limb as an adjuvant treatment in high-risk melanoma; in 5 patients cisplatin was added. The melphalan concentrations in the peripheral blood rose predominantly during the first 20 min of perfusion and levelled out to an apparent steady state of about 0.28 micrograms/ml in upper extremity perfusions, and 0.34 (without cisplatin) and 0.37 micrograms/ml (with cisplatin) in lower extremity perfusion. Erythrocytes labelled with technetium Tc 99m, which were added concomitantly with melphalan to the perfusion medium, appeared in the systemic circulation of the patients at an almost constant rate of 0.32% (lower and upper limb perfusions without cisplatin and 0.37% (with cisplatin) of total tracer/min. This perfusate flow rate indicated by labelled erythrocytes completely explained the leakage of melphalan from the perfusion circuit into the peripheral blood. Peak concentrations of melphalan in the peripheral blood were observed immediately after reconstitution of normal hemodynamic conditions once isolation perfusion had been terminated. This fraction of melphalan might originate from tissue-binding sites, but also from vascular compartments; therefore, a thorough washing-out procedure might minimize this effect.

Prilocaine elimination by isolated perfused rat lung and liver.

Prilocaine is assumed to undergo significant elimination by extrahepatic organs and to differ in this respect from other commonly used local anaesthetics. In order to clarify whether the lung may play an important role as a site of elimination of prilocaine, the kinetic parameters were studied in isolated perfused rat lungs and were compared to those of isolated livers. Furthermore, the structurally related compounds bupivacaine and mepivacaine were also investigated in this system. Prilocaine was dispersed into a relatively large apparent distribution volume in perfused rat lung (139 ml versus 97 ml in controls). In single-pass perfused lungs the observed maximum of concentration was decreased by about 60% compared to controls. The mean residence time was prolonged by about 40%. These observations suggest that prilocaine is substantially retained by rat lung and that this effect occurs particularly during first-pass. However, the ability of rat lung to degrade prilocaine was relatively low. The clearance values were about 0.3 ml/min equal to about 20% of the hepatic capacity calculated per g of tissue. Thus it must be assumed that prilocaine is only transiently retained by the lung and will gain systemic availability later on. In rat lungs the kinetics of prilocaine elimination were not substantially different from those of bupivacaine and mepivacaine (16 and 12%). These observations do not support the assumption that especially prilocaine undergoes extrahepatic elimination.

Pulmonary uptake of bupivacaine in isolated perfused rat lung.

The ability of rat lung to remove the local anaesthetic drug bupivacaine from the blood was studied in isolated organs which were perfused either in an open (single-pass mode) or in a closed system (recirculating medium). Isolated perfused rat lungs exhibited a very low capacity to metabolize bupivacaine within 3 h during which the drug circulated continuously through the organ. The clearance values differed only by 0.2 ml/min from the control parameters in sham perfusions. The calculated extraction ratio was 0.2% and the elimination half-life was about 210 min. The volume of distribution of bupivacaine was 133 ml which remarkably surmounted the reference values obtained for sham perfusions. The distribution of bupivacaine into the pulmonary tissue was investigated applying the multiple indicator dilution technique to isolated lungs perfused in the single-pass mode. The mean elimination time of model compounds for distribution into the intravascular space, 14C-insulin, and the total water space, 3H-water, were 68 and 75 s at a flow rate of 6 ml/min. The volume of distribution was 5.9 ml for inulin and 6.5 ml for water. The mean transit time for concomitantly injected bupivacaine was 221 s and the volume of distribution was 14.4 ml. The respective parameters of sham perfusions performed without an isolated organ were substantially lower, i.e. mean elimination time 50, 50 and 61 s and distribution volume 4.9, 5.0 and 6.1 ml for inulin, water and bupivacaine.2+ f1p4

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in isolated rat lung and liver.

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a strong lung carcinogen in all species tested. To elicit its tumorigenic effects NNK requires metabolic activation which is supposed to take place via alpha-hydroxylation, whereas N-oxidation is suggested to be a detoxification pathway. The differences in the organ specific metabolism of NNK may be crucial for the organotropy in NNK-induced carcinogenesis. Therefore, metabolism of NNK was investigated in the target organ lung and in liver of Fischer 344 (F344) rats using the model of isolated perfused organs. High activity to metabolize 35 nM [5-3H]NNK was observed in both perfused organs. NNK was eliminated by liver substantially faster (clearance 6.9 +/- 1.6 ml/min, half-life 14.6 +/- 1.2 min) than by lung (clearance 2.1 +/- 0.5 ml/min, half-life 47.9 +/- 7.4 min). When the clearance is calculated for a gram of organ or for metabolically active cell forms, the risk with respect to carcinogenic mechanisms was higher in lung than in liver. The metabolism of NNK in liver yielded the two products of NNK alpha-hydroxylation, the 4-oxo-4-(3-pyridyl)-butyric acid (keto acid) and 4-hydroxy-4-(3-pyridyl)-butyric acid (hydroxy acid). In lung, the major metabolite of NNK was 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-N-oxide). Substantial amounts of metabolites formed from methyl hydroxylation of NNK, which is one of the two possible pathways of alpha-hydroxylation, were detected in lung but not in liver perfusion. Formation of these metabolites (4-oxo-4-(3-pyridyl)-butanol (keto alcohol), and 4-hydroxy-4-(3-pyridyl)-butanol (diol) can give rise to pyridyloxobutylating of DNA. When isolated rat livers were perfused with 150 microM NNK, equal to a dosage which is sufficient to induce liver tumors in rat, glucuronidation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was increased when compared to the concentration of 35 nM NNK. Nevertheless, the main part of NNK was also transformed via alpha-hydroxylation for this high concentration of NNK.

Effect of nicotine or cotinine on metabolism of 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in isolated rat lung and liver.

The scope of the present study was to investigate whether nicotine or cotinine will affect the metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in isolated perfused rat lungs and livers and to study the effect of starvation on pulmonary metabolism of NNK. NNK metabolism was investigated in isolated perfused liver and lung of male F344 rats perfused with 35 nM [5-3H]NNK in presence of a 1400-fold excess of the main tobacco alkaloid nicotine and its metabolite cotinine. In perfused rat livers, nicotine and cotinine inhibited NNK elimination and metabolism and led to a substantial increase of elimination half-life from 14.6 min in controls to 25.5 min after nicotine and 36.6 min after cotinine co-administration, respectively. In parallel, the pattern of NNK metabolites was changed by nicotine and cotinine. The pathway of alpha-hydroxylation representing the metabolic activation of NNK was decreased to 77% and 85% of control values, whereas N-oxidation of NNK and glucuronidation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was increased 2.6- and 1.2-fold in presence of nicotine and cotinine, respectively. When isolated rat lungs were perfused with 35 nM NNK for 3 h neither the elimination nor the pattern of metabolites were substantially affected due to co-administration of 50 microM nicotine or cotinine. Cytochrome P450 2E1 is known to participate in the activation of NNK and can be induced by starvation. However, isolated rat lungs from male Sprague Dawley rats perfused with [1-14C]NNK at about 2 microM for 3 h, revealed only small differences in pulmonary elimination and pattern of NNK metabolites between fed and starved animals. These results suggest that nicotine and its main metabolite cotinine inhibit the metabolic activation of NNK predominantly in the liver whereas activation in lung, a main target organ of NNK induced carcinogenesis, remained almost unaffected.

Metabolism of benzo[a]pyrene in the combined rat liver--lung perfusion system.

The influence of the insertion of a liver into the perfusion circuit of a lung on the availability of benzo[a]pyrene and benzo[a]pyrene metabolites to the lung was examined. Perfused lungs from 5,6-benzoflavone pretreated rats release high quantities of free benzo[a]pyrene metabolites and conjugates into the perfusion medium. The insertion of a liver taken from an untreated rat reduces the concentration of unmetabolized substrate and of free diol, quinone and phenol metabolites to less than 20% of the concentrations found in the absence of the liver. When the liver of a 5,6-benzoflavone-pretreated rat is used, substrate depletion is not much greater than in the experiments with control livers; however, the concentration of free metabolites is further reduced to one third. In lung tissue, only very low levels of benzo[a]pyrene and greatly reduced levels of free and conjugated metabolites are found when a 5,6-benzoflavone-induced liver had been present during perfusion. These findings can explain the protective effect of the liver on covalent binding of benzo[a]pyrene metabolites to pulmonary macro-molecules observed in previous experiments with the combined liver-lung perfusion model [Klaus et al., Biochem. Biophys. Res. Commun., 105 (1982) 596].

Expression of MRP1 and related transporters in human lung cells in culture.

Multidrug resistance type 1 P-glycoproteins (P-gp) and multidrug resistance associated proteins (MRP) were studied in differentiated primary human lung cells in culture, in comparison with permanent human lung cell lines and primary alveolar type II cells from rat lung. AII cells exhibited low basal levels of mdr1b mRNA, that increased over time and after oxygen radical production induced by paraquat. mRNAs coding for antioxidative enzymes catalase (CAT), maganese superoxide dismutase (Mn-SOD) and copper/zinc superoxide dismutase (Cu/Zn-SOD) were not changed. H358, A549, H322 cells expressed low levels of MDR1 mRNA, but the mdr1 substrate rhodamine 123 (Rh 123) was transported out of H358 and H322 cells in a non-invasive, single cell fluorescence assay. The dye efflux could be inhibited by the chemosensitizer, verapamil. Normal human bronchial epithelial cells (NHBEC) expressed immuno-reactive MDR1 P-gp and the MPR protein that was active in the fluorescence assay using the MRP substrate carboxy-dichlorofluorescein (CDF) and MK-571 as an inhibitor. We did observe inter-individual variation of MRP in both the mRNA and the immunoreactive protein in NHBEC culture. Over time (12 weeks) the protein was relatively stable in NHBEC and epithelial cells from peripheral lung (PLC), but the mRNA level was drastically increased when explant cultures were continued (18 weeks).

Monitoring of cytochrome P-450 1A activity by determination of the urinary pattern of caffeine metabolites in Wistar and hyperbilirubinemic Gunn rats.

Various studies suggest that induction of cytochrome P-450 1A (CYP1A) might be a valuable therapeutic modality for reducing the hyperbilirubinemia of infants with Crigler-Najjar syndrome type I (CNS-I), a severe form of congenital jaundice. To evaluate inducers of CYP1A as possible tools in the treatment of hyperbilirubinemia, a novel assay was established, based on the analysis of the urinary pattern of caffeine metabolites in rats. Wistar rats received [1-Me-(14)C]-caffeine (10 mg/kg i.p.), before and 48h after administration of the potent CYP1A inducer 5,6-benzoflavone (BNF) (80 mg/kg, i.p.). A substantial increase in the fractions of the terminal caffeine metabolites 1-methyluric acid (1-U), 1-methylxanthine (1-X), and a concomitant decrease in the caffeine demethylation product 1,7-dimethylxanthine (1,7-X) was observed after application of BNF. The ratio of the caffeine metabolites (1-U+1-X)/1,7-X may serve as an index of CYP1A activity in rats in vivo. Hyperbilirubinemic, homozygous (jj) Gunn rats are an accepted model for human CNS-I. In male jj Gunn rats treated with BNF or with indole-3-carbinol (I3C, 80 mg/kg, oral gavage), the inducing effect of BNF and 13C on CYP1A activity was confirmed by the urinary pattern of caffeine metabolites, and was parallelled by a decrease in plasma bilirubin levels. These data demonstrate the usefulness of the established caffeine assay for the evaluation of inducers of CYP1A as tools for reducing hyperbilirubinemia and further confirm the potential value of I3C in the treatment of CNS-I.

Photoablation of meningiomas and neurinomas by Holmium Yag laser radiation.

Cranial rat bone was irradiated by 2.1 microns Holmium Yag laser radiation. Quantitative edge rates were calculated. Histologic sections were investigated by light and electron microscopy. Eighteen cases of hard fibrous or calcified spinal and cranial meningiomas and neurinomas were operated upon using pulsed laser beam. In rat cranial bone ablation rate ranged between 0.3-0.5 mm per pulse. Perifocal thermal damage was observed in a zone of 20-90 microns around the lesion. In all human cases tumors could be removed totally without additional neurological deficit. In vivo heat development was measured by an i.r.-camera.

Thermal damage threshold at 633 nm of tympanic membrane of pig.

Doppler vibrometers are used by many research groups to monitor the motion of the tympanic membrane (TM) and of middle ear ossicles for in vivo and in vitro studies. Power densities in these applications reach 80 W/cm(2). To determine the safe limit of exposure, a cw dye laser at a wavelength of 633 nm was used to investigate the threshold of thermal damage of TM of pigs under exposure times of 60 s. To determine the applied power density accurately, the spot size of the laser beam was monitored by an objective lens and a CCD camera. Twenty-six laser exposed samples of TM were stained by haematoxylin and eosin stain and the semi-thin sections were examined microscopically. In none of the sections was any laser induced damage observed with power densities below 7.1 kW/cm(2), whereas serious damage occurred showing coagulation, carbonisation and perforation in all cases with laser powers above 8.2 kW/cm(2). The threshold for damage and the conical shape of the damage zone is explained by photon propagation and absorption in the tissue especially by the increase of the scattering factor at higher tissue temperature. The thermal damage threshold of 8 kW/cm(2) is compared to the maximum permissible exposure given in laser safety standards for skin.

Frequency of HPRT mutants in humans exposed to vinyl chloride via an environmental accident.

The mutant frequency (MF) in the hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) locus of peripheral blood T-lymphocytes was measured in a population environmentally exposed to vinyl chloride - a toxic and carcinogenic substance through an accidental release into the atmosphere. It was compared to MF in a control group of unexposed individuals. Both groups were re-investigated in a follow-up study, 2 years later. No significant difference could be observed in MF between exposed and controls either at the accident nor in the follow-up study. Approximately the same mean HPRT mutant frequencies were observed for both groups in T-lymphocytes from blood samples obtained shortly after the accident and from the follow-up blood samples. Both groups showed a higher mean MF in the re-investigation samples which is most probably due to the significantly lower average cloning efficiency (CE) under non-selective conditions and because of the inverse relationship between CE and MF. The exposed population showed a higher mean T-cell CE at the initial blood sampling as compared to the control group. The concurrent cytogenetic analyses of peripheral lymphocytes showed a significant increase in cells with aberrations in the exposed population. Clastogenic but not mutagenic activity of vinyl chloride was observed in our study.