An ecotoxicological view on neurotoxicity assessment.

The numbers of potential neurotoxicants in the environment are raising and pose a great risk for humans and the environment. Currently neurotoxicity assessment is mostly performed to predict and prevent harm to human populations. Despite all the efforts invested in the last years in developing novel in vitro or in silico test systems, in vivo tests with rodents are still the only accepted test for neurotoxicity risk assessment in Europe. Despite an increasing number of reports of species showing altered behaviour, neurotoxicity assessment for species in the environment is not required and therefore mostly not performed. Considering the increasing numbers of environmental contaminants with potential neurotoxic potential, eco-neurotoxicity should be also considered in risk assessment. In order to do so novel test systems are needed that can cope with species differences within ecosystems. In the field, online-biomonitoring systems using behavioural information could be used to detect neurotoxic effects and effect-directed analyses could be applied to identify the neurotoxicants causing the effect. Additionally, toxic pressure calculations in combination with mixture modelling could use environmental chemical monitoring data to predict adverse effects and prioritize pollutants for laboratory testing. Cheminformatics based on computational toxicological data from in vitro and in vivo studies could help to identify potential neurotoxicants. An array of in vitro assays covering different modes of action could be applied to screen compounds for neurotoxicity. The selection of in vitro assays could be guided by AOPs relevant for eco-neurotoxicity. In order to be able to perform risk assessment for eco-neurotoxicity, methods need to focus on the most sensitive species in an ecosystem. A test battery using species from different trophic levels might be the best approach. To implement eco-neurotoxicity assessment into European risk assessment, cheminformatics and in vitro screening tests could be used as first approach to identify eco-neurotoxic pollutants. In a second step, a small species test battery could be applied to assess the risks of ecosystems.

Application of an optimized system for the well-defined exposure of human lung cells to trichloramine and indoor pool air.

In this study an in vitro exposure test to investigate toxicological effects of the volatile disinfection by-product trichloramine and of real indoor pool air was established. For this purpose a set-up to generate a well-defined, clean gas stream of trichloramine was combined with biotests. Human alveolar epithelial lung cells of the cell line A-549 were exposed in a CULTEX(®) device with trichloramine concentrations between 0.1 and 40 mg/m(3) for 1 h. As toxicological endpoints the cell viability and the inflammatory response by the cytokines IL-6 and IL-8 were investigated. A decreasing cell viability could be observed with increasing trichloramine concentration. An increase of IL-8 release could be determined at trichloramine concentrations higher than 10 mg/m(3) and an increase of IL-6 release at concentrations of 20 mg/m(3). Investigations of indoor swimming pool air showed similar inflammatory effects to the lung cells although the air concentrations of trichloramine of 0.17 and 0.19 mg/m(3) were much lower compared with the laboratory experiments with trichloramine as the only contaminant. Therefore it is assumed that a mixture of trichloramine and other disinfection by-products in the air of indoor pool settings contribute to that effect.

Benzalkonium chloride (BAC) and dimethyldioctadecyl-ammonium bromide (DDAB), two common quaternary ammonium compounds, cause genotoxic effects in mammalian and plant cells at environmentally relevant concentrations.

Quaternary ammonium compounds (QACs) are cationic surfactants that are widely used as disinfectants. In the present study, we tested two important representatives, namely, benzalkonium chloride (BAC) and dimethyldioctadecyl-ammonium bromide (DDAB) in four genotoxicity tests, namely, in the Salmonella/microsome assay with strains TA 98, TA 100 and TA 102, in the single-cell gel electrophoresis (SCGE) assay with primary rat hepatocytes and in micronucleus (MN) assays with peripheral human lymphocytes and with root tip cells of Vicia faba. In the bacterial experiments, consistently negative results were obtained in the dose range between 0.001 and 110 microg per plate in the presence and absence of metabolic activation while significant induction of DNA migration was detected in the liver cells. With BAC, a moderate but significant effect was found with an exposure concentration of 1.0 mg/l while DDAB caused damage at lower doses (0.3 mg/l). The effects were not altered when the nuclei were treated with formamidopyridine glycosylase, indicating that they are not due to formation of oxidized purines. The MN assays with blood cells were carried out under identical conditions to the SCGE experiments and a significant increase was seen at the highest dose levels (BAC: 1.0 and 3.0 mg/l; DDAB: 1 mg/l). Both compounds also caused significant induction of MN as well as inhibition of cell division in plant cells, the lowest effective levels were 1.0 and 10 mg/l for DDAB and BAC, respectively. Our findings show that both chemicals induce moderate but significant genotoxic effects in eukaryotic cells at concentrations which are found in wastewaters and indicate that their release into the environment may cause genetic damage in exposed organisms. Furthermore, the direct contact of humans to QAC-containing detergents and pharmaceuticals that contain substantially higher concentrations than those which were required to cause effects in eukaryotic cells in the present study should be studied further in regard to potential DNA-damaging effects in man.

[In vitro determination of the cytotoxic activity of saliva samples of smokers]. / In-vitro-Bestimmung zytotoxischer Aktivität in Speichelproben von Rauchern.

BACKGROUND: The question arised whether saliva can be included in populations monitoring of high risk groups for the development of head and neck squamous cell carcinomas. Cytotoxic mechanisms strongly influence the carcinogenesis of squamous cell carcinomas. PATIENTS AND METHODS: Saliva specimen of 131 abusing and non-abusing probands were tested on their biological (cytotoxic) effects to draw conclusions on the individual cancer risk. To determine the cytotoxic activity of saliva, we used the "plating efficiency index" of lungfibroblasts of the chinese hamster. RESULTS: We found significantly increased cytotoxic effects in the saliva of smoking probands (p < 0.002). Regularly combined smoking and drinking of alcohol led to a highly significant increased risk of cytotoxic saliva in the tested persons (odds ratio: 17.4; p < 0.005). CONCLUSIONS: Including patients with head and neck squamous cell carcinomas, ongoing studies must prove the practical relevance of this biomarker for estimating the relative cancer risk in the upper aerodigestive tract.

[Ames test as biomarker]. / Der Ames-Test als Biomarker.

BACKGROUND AND OBJECTIVE: The incidence of squamous cell carcinomas in the upper aerodigestive tract has increased worldwide. The main risk factors are chronic tobacco and alcohol consumption. The detection of high-risk persons is important because early diagnosis of these tumors provides a good chance for permanent healing. Biomonitoring programs may help to give precise information about the individual cancer risk among smoking and drinking persons. The aim of this study was to evaluate the Ames test as a biomarker to detect the genotoxicity of saliva. PATIENTS AND METHODS: Saliva specimens of 131 probands were investigated for their genotoxic effects using the Ames test. RESULTS: Our results showed an increased trend of genotoxic activity in the saliva of smokers. A highly significant additional increase of genotoxicity was measured in smoking and drinking individuals. CONCLUSIONS: Our study shows that the Ames test could be used to show genotoxic effects in saliva specimens. In combination with other biomarkers, this test may help to develop a valid concept for detecting cancer-endangered people.

Exfoliative cytology of normal buccal mucosa to predict the relative risk of cancer in the upper aerodigestive tract using the MN-assay.

The high frequency of second or third primary tumors was first explained by Slaughter et al. with the concept of field cancerisation. Another theory postulates micrometastatic lesions as a reason for this phenomenon. The micronuclei (MN)-assay was evaluated to provide evidence for the concept of field cancerisation and to quantify the premalignant field change of normal mucosa in order to predict the individual cancer risk. MN-assay was carried out in 55 patients with squamous cell carcinoma of the head and neck, in 16 patients with a leucoplakia and in 99 healthy controls. A detailed questionnaire for population monitoring was completed. Buccal cytosmears of healthy mucosa of the study participants were examined for the MN count per 1000 cells. There was a direct correlation between tobacco abuse and increasing MN count as a sign of a cytogenetic damage of buccal mucosa cells. Alcohol did not influence the formation of MN. Both buccal sites were damaged in the same degree as proof of field cancerisation. The relative cancer risk (odds ratio) for smoking healthy controls with a definite MN frequency was estimated. Our study underscores the importance of the MN-assay as a biomarker to predict the relative cancer risk in the upper aerodigestive tract under suspicion of the individual susceptibility and the exposition to known carcinogenic agents such as tobacco and alcohol. The concept of field cancerisation was confirmed.

[Micronuclei as biological markers for the detection of local cancer transformation in the upper aerodigestive tract]. / Mikrokerne als Biomarker zum Nachweis der Feldkanzerisierung im oberen Aerodigestivtrakt.

A lot of different endogenous and exogenous factors are accused to promote squamous cell carcinomas in the upper aerodigestive tract. Main risk factors are the chronic tobacco- and alcohol consumption. The fact, that many patients develop syn- or metachronic carcinomas in this area was first described by Slaughter et al. 1953 and explained with the phenomenon of field cancerisation. Concerning to this hypothesis the whole mucosa of the upper aerodigestive-tract is premalignant damaged. In our study the micronucleus-frequency was determined as a biomarker for the genetic injury to prove the fieldcancerisation on the cellular level at 159 people (control-group, abuser, patients with HNSCC). Our results confirm the hypothesis of fieldcancerisation of the mucosa of the upper aerodigestive-tract at strong tobacco- and alcohol consumers and patients with head and neck squamous cell carcinoma. Furthermore there is a highly statistically significant correlation between increasing micronucleus frequency and increasing tobacco abuse. As a final result of our study the micronucleus assay seems to be of good value to show a genotoxic damage in healthy mucosa at people with a high risk to develop HNSCC, but it's not usable to give any answer if and when such carcinomas arise.

Monitoring of occupational exposure to epichlorohydrin by genetic effects and hemoglobin adducts.

The present work is focused on the determination of in vivo doses and studies of genetic effects in workers exposed to epichlorohydrin (ECH). The studied endpoints were hemoglobin (Hb) adducts, frequencies of hprt mutants, micronuclei in cytochalasin B blocked binucleated lymphocytes, sister chromatid exchanges (SCE) and high frequency cells (HFC). Blood samples were collected from office clerks and ECH exposed factory workers at an industrial plant in Germany. The workers were exposed to 0.11-0.23 ppm ECH in the air 45 h per week and to 0.2-2.6 ppm for 3 h per week. Some Swedish non-exposed subjects were also used for Hb adduct measurements. The genetic data, HFC and SCE, showed a significant difference between exposed and unexposed donors. In contrast to earlier studies on SCE, no impact of smoking was observed. Effects on micronuclei were on the borderline of significance, whereas there was no effect for HPRT mutants. The average Hb adduct level was higher in exposed than in non-exposed donors, although the difference was only significant when the exposed group was compared to Swedish controls. Smoking gave significantly increased adduct levels. The absence of significant correlations between individual data for Hb adducts and genetic effects, may be explained by the different periods of time covered by the responses in these endpoints. Whereas Hb adducts reflect the exposure during up to 4 months (i.e. the life span of human erythrocytes), the SCE, and particularly the HFC, seem to accumulate for years in a long-lived fraction of T-lymphocytes without DNA repair. Thus, the adduct data does not reflect the exposure backwards in time unless it can be shown that exposure conditions have remained unchanged. The origin of the background adduct levels in non-smoking control persons is at present not known.

Measurement of frequencies of HPRT mutants, chromosomal aberrations, micronuclei, sister-chromatid exchanges and cells with high frequencies of SCEs in styrene/dichloromethane-exposed workers.

Frequencies of HPRT mutants (MFs), chromosomal aberrations with or without gaps (CA+; CA-), aberrant cells (AC), micronuclei (MN), sister-chromatid exchanges (SCEs) and cells with high frequencies of SCEs (HFCs) were measured in lymphocytes collected from 46 workers occupationally exposed to styrene and dichloromethane (DCM = methylene chloride). These parameters were also determined in 23 controls. Time-weighted average (TWA) values for styrene and DCM exposure during an 8-h working day were respectively 70 mg/m3 (range: 0-598) and 108 mg/m3 (range: 0-742). These values correspond to TWA values of 17 ppm styrene and 31 ppm DCM. In exposed workers, all cytogenetic parameters were significantly enhanced (P < 0.0001; one-sided), but, due to the lack of appropriate control data, no definite conclusions could be drawn concerning the mutagenicity of styrene/DCM exposure. Duration of exposure was not correlated with genetic effects analyzed. The TWA value for styrene was not correlated with the extent of genetic damage detected, but the TWA value for DCM was positively correlated with the frequencies of chromosome aberrations (with gaps) and aberrant cells. These observations make it difficult to decide whether styrene or DCM, or both chemicals, induced the cytogenetic effects observed in exposed workers. Using the present styrene/DCM data, earlier ethylene oxide data and unpublished epichlorohydrin data, the relative sensitivity of the genetic endpoints to detect genotoxic exposure was: HFC > CA- > CA+ > SCE > MN > HPRT.

Chromosomal aberrations in peripheral lymphocytes of nurses and physicians handling antineoplastic drugs.

The frequencies of structural chromosome aberrations of persons occupationally exposed to antineoplastic drugs without adequate protection were measured in peripheral blood lymphocytes of 106 persons from five oncological units and in an adequate control population. The observed chromosomal aberration frequencies were 3.3 +/- 0.1 aberrations per 100 cells in the exposed group and 0.6 +/- 0.1 aberrations per 100 cells in the controls. Chromosomal aberration frequencies were not correlated with age, duration of exposure and smoking habits. The results stress the necessity to protect hospital staff against the potential risk related to the handling of antineoplastic drugs.

Biological and chemical monitoring of occupational exposure to ethylene oxide.

Studies were carried out on two populations occupationally exposed to ethylene oxide (EtO) using different physical and biological parameters. Blood samples were collected from 9 hospital workers (EI) and 15 factory workers (EII) engaged in sterilization of medical equipment with EtO and from matched controls (CI and CII). Average exposure levels during 4 months (the lifespan of erythrocytes) prior to blood sampling were estimated from levels of N-(2-hydroxyethyl)valine adducts in hemoglobin. They were significantly enhanced in EI and EII and corresponded to a 40-h time-weighted average of 0.025 ppm in EI and 5 ppm in EII. Exposures were usually received in bursts with EtO concentrations in air ranging from 22 to 72 ppm in EI and 14 to 400 ppm in EII. All samples were analyzed for HPRT mutants (MFs), chromosomal aberrations (CAs), micronuclei (MN) and SCEs. MFs were significantly enhanced by 60% in EII but not in EI. These results are the first demonstration of mutation induction in man by ethylene oxide. CAs were significantly enhanced in EI and EII by 130% and 260% respectively. MN were not enhanced in EI but significantly in EII(217%). The mean frequency of SCEs was significantly elevated by 20% in EI and by almost 100% in EII. SCE was the only parameter that allowed distinction between daily and occasionally exposed workers in EII. An interesting finding in exposed workers was the large increase of the percentage of cells with high frequencies of SCE (3-4 times in EI and 17-fold in EII). The relative sensitivity of endpoints for detection of EtO exposure in the present investigation was in the following order: HOEtVal adducts greater than SCEs greater than chromosomal aberrations greater than micronuclei greater than HPRT mutants.