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.

In vitro studies of different irradiation conditions for Photodynamic inactivation of Helicobacter pylori.

Helicobacter pylori (HP) infections are considered to be the main cause for chronic gastritis and gastric ulcers, whereby more than half of the world's population is nowadays infected. The increased use of antibiotics is leading to an enhanced resistance. Photodynamic inactivation of bacteria seems to be a potential alternative for antibiotic therapies. In our study we used the photosensitizer Chlorin e6 (Ce6) in combination with red light-emitting diodes to inactivate HP in vitro. Ce6 uptake is determined by spectroscopy. Furthermore diverse experiments of different concentrations in the range of 0-100 µM of the photosensitizer and exposure times up to 300 s are carried out in order to find optimal irradiation parameters (wavelength: 660 nm, power density: 9 mW/cm(2), absorbed dose: up to 2.7 J/cm(2)). The data show a significant reduction after already a few seconds of illumination, even with a low Ce6 concentration in the sub-µM-region. At a concentration of 100 µM a nearly total inactivation (6-log10-reduction) of HP was achieved within 60s of irradiation.

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.

Pharmacokinetic of ALA and h-ALA induced porphyrins in the models Mycobacterium phlei and Mycobacterium smegmatis.

Photodynamic inactivation (PDI) of bacterial strains presents an attractive potential alternative to antibiotic therapies. Success is dependent on the effective accumulation in bacterial cells of photochemical substances called photosensitizers, which are usually porphyrins. It is also important to know the distribution of the photosensitizer in bacteria at the microscopic level. The present results examine the accumulation of photosensitizers by Mycobacterium phlei and Mycobacterium smegmatis, which serve as models for the important pathogens Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium bovis. The kinetics of porphyrin synthesis after treatment with the precursors ALA and h-ALA were studied. The goal was to describe the biosynthesis and the pharmacokinetics of sensitizers in both bacterial strains using fluorescence microscopy and spectroscopy. We could show that both Mycobacterium strains enrich porphyrins after ALA and h-ALA administration detected by fluorescence peaks at about 620nm. By HPLC analyses the major porphyrin could be identified as coproporphyrin. In the future we will apply the new knowledge in in vitro and in vivo experiments to strains of M. tuberculosis, M. leprae and M. bovis and examine cell destruction by PDI.

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.

Alternative methods to safety studies in experimental animals: role in the risk assessment of chemicals under the new European Chemicals Legislation (REACH).

During the last two decades, substantial efforts have been made towards the development and international acceptance of alternative methods to safety studies using laboratory animals. In the EU, challenging timelines for phasing out of many standard tests using laboratory animals were established in the seventh Amending Directive 2003/15/EC to Cosmetics Directive 76/768/EEC. In continuation of this policy, the new European Chemicals Legislation (REACH) favours alternative methods to conventional in vivo testing, if validated and appropriate. Even alternative methods in the status of prevalidation or validation, but without scientific or regulatory acceptance may be used under certain conditions. Considerable progress in the establishment of alternative methods has been made in some fields, in particular with respect to methods predicting local toxic effects and genotoxicity. In more complex important fields of safety and risk assessment such as systemic single and repeated dose toxicity, toxicokinetics, sensitisation, reproductive toxicity and carcinogenicity, it is expected that the development and validation of in silico methods, testing batteries (in vitro and in silico) and tiered testing systems will have to overcome many scientific and regulatory obstacles which makes it extremely difficult to predict the outcome and the time needed. The main reasons are the complexity and limited knowledge of the biological processes involved on one hand and the long time frame until validation and regulatory acceptance of an alternative method on the other. New approaches in safety testing and evaluation using "Integrated Testing Strategies" (ITS) (including combinations of existing data, the use of chemical categories/grouping, in vitro tests and QSAR) that have not been validated or not gained wide acceptance in the scientific community and by regulatory authorities will need a thorough justification of their appropriateness for a given purpose. This requires the availability of knowledge and experience of experts in toxicology. The challenging deadlines for phasing out of in vivo tests in the Cosmetics Amending Directive 2003/15/EC appear unrealistic. Likewise, we expect that the application of validated alternative methods will only have a small or moderate impact on the reduction of in vivo tests under the regimen of REACH, provided that at least the same level of protection of human health as in the past is envisaged. As a consequence, under safety aspects, it appears wise to consider established in vivo tests to be indispensable as basic tools for hazard and risk assessment with respect to systemic single and repeated dose toxicity, sensitisation, carcinogenicity and reproductive toxicity, especially regarding quantitative aspects of risk assessment such as NOAELs, LOAELs and health-related limit values derived from them. Based on the overall evaluation in this review, the authors are of the opinion that in the short- and mid-term, the strategy of the development of alternative methods should be more directed towards the refinement or reduction of in vivo tests. The lessons learnt during these efforts will provide a substantial contribution towards the replacement initiatives in the long-term.

Autofluorescence detection of tumors in the human lung--spectroscopical measurements in situ, in an in vivo model and in vitro.

To detect bronchial carcinoma by autofluorescence, we measured the spectra of tumor and normal tissue in situ, in an in vivo model and in vitro by fiber optic spectrometer and two-dimensional resolved microspectroscopy. The in situ measurements were performed in bronchi of nine patients with squamous cell carcinoma during regular bronchoscopy with autofluorescence assistance. The fluorescence was monitored with a fiber optical spectrometer under blue light excitation (lambda=405nm). In an in vivo model, the resected lobe of a lung was perfused under physiological conditions. Tumorous and normal tissues were examined spectroscopically during perfusion and after blood removal and substitution with formol. In another setup the wavelength dependency of autofluorescence was examined on resected parts of physiological bronchi and central bronchial carcinomas. Under the variation of the excitation from 385 to 465nm the autofluorescence response was monitored with a fiber optic spectrometer. For investigation of the origin of autofluorescence, two-dimensional resolved spectroscopy was performed with the SpectraCube system on several sections of tumor and normal tissues All measurements, performed in vivo, in the in vivo model and in vitro agreed, that the main difference of the autofluorescence between tumor and normal bronchus tissue is the intensity of the fluorescences' main peak at 505nm. The signal on tumor tissue is in all cases significantly lower than that of normal tissue. The shape of the autofluorescence peaks is in healthy and carcinoma tissue approximately the same with two characteristic minima at 540 and 580nm. After the preparation with formaldehyde those minima disappeared from the spectra. A comparison with the absorption spectra of hemoglobin showed, that the variation of the spectra may be due to the blood content in the tissue. Two-dimensional spatially resolved spectroscopy showed, that the lower intensity of fluorescence in tumor tissue is due to the irregular and low-concentrated formation of fluorescent structures, which seen to be the elastic structures of bronchial tissue.

Does the anaerobic formation of hydroxyl radicals by paraquat monocation radicals and hydrogen peroxide require the presence of transition metals?

This in vitro study investigated the formation of hydroxyl radicals (*OH) under anaerobic conditions through the direct reaction between paraquat radicals (PQ(+)*) and hydrogen peroxide (H(2)O(2)) by quantitative UV-VIS and electron spin resonance (ESR) spectroscopy. PQ(+)* was formed by paraquat reduction using either sodium dithionite or the xanthine/xanthine oxidase reaction as electron donors. The anaerobic formation of PQ(+)* was quantified both by measuring light absorption at 605 nm or by ESR techniques respectively, using either the absorption coefficient or ultramarine as a stable spin standard. Detection of *OH took place with aid of the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline- N-oxide (DEPMPO). Generation or addition of H(2)O(2) to PQ(+)* eliminates the 35-line ESR signal of PQ(+)* and subsequently generates the 8-line ESR signal of the DEPMPO-OH adduct. The elimination of PQ(+)* as well as the formation of OH-DEPMPO adduct was not influenced by 1.0 mM deferoxamine, indicating that iron or other transition metals are, at least under anoxic conditions, not necessarily involved in the generation of the most aggressive reactive oxygen species *OH.

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).

Expression of cytochrome P450 2E1 in normal human bronchial epithelial cells and activation by ethanol in culture.

Serum-free primary cultures of human bronchial epithelial cells and freshly isolated samples of human bronchial epithelium were used to investigate basal expression of the cytochrome P450 enzyme CYP2E1 and its activation or induction by ethanol in bronchial epithelial cells. The cultures consisted of > or =95% cells of epithelial characteristics as determined by transmission electron microscopy and immunohistochemical staining. Monolayers were obtained from explants over a period of several months via transfer of tissue into new dishes ('generations'1-5). Using RT-PCR analysis, basal expression of mRNAs coding for CYP2B7, CYP2F1 and CYP2E1 were detected in cultures from several donors. The basal expression of CYP2E1 protein and mRNA showed differences between the donors. The mRNA was detected even in cultures from higher generations and increased in some cultures over time. The CYP2E1 protein content was low and in most cultures of generations 2-5 could not be detected by immunoblot analysis of native protein extracts. Nevertheless, in some cases immunoreactive CYP2E1 protein was present in monolayers obtained from the fourth and fifth transfer (18-week 'generation'). CYP2E1 activity was measured via 6-hydroxylation of chlorzoxazone either by a destructive assay using cell lysate or by a non-invasive assay using the medium of cell cultures. In short-term cultured isolated bronchial epithelium, ethanol treatment increased CYP2E1 activity by up to 5-fold within 4 days but with inter-individual differences. In cells up to 4 weeks in culture, CYP2E1 activity remained inducible by a single dose of ethanol. Differentiated primary human cells in culture may be useful tools as model systems for the evaluation of CYP2E1-driven processes in man.

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.

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.

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.

Metabolism of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in primary cultures of rat alveolar type II cells.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces primarily lung tumors, which are assumed to derive from malignant transformation of alveolar type II (AII) cells within the lung. To elicit its carcinogenic effects, NNK requires metabolic activation by cytochrome P-450 (CYP)-mediated alpha-hydroxylation. Therefore, in this study the metabolism of NNK and expression of the NNK-activating CYP isoform CYP2B1 were investigated in primary cultures of rat AII cells. Although basal expression of CYP2B1 decreased in a time-dependent manner during culture of AII cells, substantial CYP2B1 protein expression was observed in AII cell cultures after the first 24 h. When AII cells were incubated with 0. 05 microM [5-(3)H]NNK, N-oxidation of NNK, which is thought to represent a detoxification pathway, was predominant (42%). alpha-Hydroxylated metabolites resulting from metabolic activation of NNK amounted to 35% of all detected metabolites. However, the proportion of alpha-hydroxylated metabolites decreased to 17% of all detected metabolites when AII cells were incubated with a 100-fold higher concentration of NNK (5 microM). In summary, this study indicates a remarkable activity of cultured AII cells to metabolize NNK, leading to substantial metabolic activation of NNK, which was more pronounced in incubations at low NNK concentration. Because exposure to NNK via cigarette smoking is thought to lead to very low plasma NNK concentrations (1-15 pM), these data suggest that metabolic activation of NNK in cigarette smokers might occur to a larger extent than would be expected according to previous metabolic studies performed with high (micromolar) NNK concentrations.