Towards an alternative testing strategy for nanomaterials used in nanomedicine: lessons from NanoTEST.

In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it still remains unclear how exactly NPs interact with their cellular targets. Size, surface, mass, geometry, and composition may all play a beneficial role as well as causing toxicity. Concerns of scientists, politicians and the public about potential health hazards associated with NPs need to be answered. With the variety of exposure routes available, there is potential for NPs to reach every organ in the body but we know little about the impact this might have. The main objective of the FP7 NanoTEST project ( www.nanotest-fp7.eu ) was a better understanding of mechanisms of interactions of NPs employed in nanomedicine with cells, tissues and organs and to address critical issues relating to toxicity testing especially with respect to alternatives to tests on animals. Here we describe an approach towards alternative testing strategies for hazard and risk assessment of nanomaterials, highlighting the adaptation of standard methods demanded by the special physicochemical features of nanomaterials and bioavailability studies. The work has assessed a broad range of toxicity tests, cell models and NP types and concentrations taking into account the inherent impact of NP properties and the effects of changes in experimental conditions using well-characterized NPs. The results of the studies have been used to generate recommendations for a suitable and robust testing strategy which can be applied to new medical NPs as they are developed.

A pilot study on the feasibility of European harmonized human biomonitoring: Strategies towards a common approach, challenges and opportunities.

In 2004 the European Commission and Member States initiated activities towards a harmonized approach for Human Biomonitoring surveys throughout Europe. The main objective was to sustain environmental health policy by building a coherent and sustainable framework and by increasing the comparability of data across countries. A pilot study to test common guidelines for setting up surveys was considered a key step in this process. Through a bottom-up approach that included all stakeholders, a joint study protocol was elaborated. From September 2011 till February 2012, 17 European countries collected data from 1844 mother-child pairs in the frame of DEMOnstration of a study to COordinate and Perform Human Biomonitoring on a European Scale (DEMOCOPHES).(1) Mercury in hair and urinary cadmium and cotinine were selected as biomarkers of exposure covered by sufficient analytical experience. Phthalate metabolites and Bisphenol A in urine were added to take into account increasing public and political awareness for emerging types of contaminants and to test less advanced markers/markers covered by less analytical experience. Extensive efforts towards chemo-analytical comparability were included. The pilot study showed that common approaches can be found in a context of considerable differences with respect to experience and expertize, socio-cultural background, economic situation and national priorities. It also evidenced that comparable Human Biomonitoring results can be obtained in such context. A European network was built, exchanging information, expertize and experiences, and providing training on all aspects of a survey. A key challenge was finding the right balance between a rigid structure allowing maximal comparability and a flexible approach increasing feasibility and capacity building. Next steps in European harmonization in Human Biomonitoring surveys include the establishment of a joint process for prioritization of substances to cover and biomarkers to develop, linking biomonitoring surveys with health examination surveys and with research, and coping with the diverse implementations of EU regulations and international guidelines with respect to ethics and privacy.

Gender differences in cadmium and cotinine levels in prepubertal children.

Susceptibility to environmental stressors has been described for fetal and early childhood development. However, the possible susceptibility of the prepubertal period, characterized by the orchestration of the organism towards sexual maturation and adulthood has been poorly investigated and exposure data are scarce. In the current study levels of cadmium (Cd), cotinine and creatinine in urine were analyzed in a subsample 216 children from 12 European countries within the DEMOCOPHES project. The children were divided into six age-sex groups: boys (6-8 years, 9-10 years and 11 years old), and girls (6-7 years, 8-9 years, 10-11 years). The number of subjects per group was between 23 and 53. The cut off values were set at 0.1 µg/L for Cd, and 0.8 µg/L for cotinine defined according to the highest limit of quantification. The levels of Cd and cotinine were adjusted for creatinine level. In the total subsample group, the median level of Cd was 0.180 µg/L (range 0.10-0.69 µg/L), and for cotinine the median wet weight value was 1.50 µg/L (range 0.80-39.91 µg/L). There was no significant difference in creatinine and cotinine levels between genders and age groups. There was a significant correlation between levels of cadmium and creatinine in all children of both genders. This shows that even at such low levels the possible effect of cadmium on kidney function was present and measurable. An increase in Cd levels was evident with age. Cadmium levels were significantly different between 6-7 year old girls, 11 year old boys and 10-11 year old girls. As there was a balanced distribution in the number of subjects from countries included in the study, bias due to data clustering was not probable. The impact of low Cd levels on kidney function and gender differences in Cd levels needs further investigation.

Interpreting biomarker data from the COPHES/DEMOCOPHES twin projects: Using external exposure data to understand biomarker differences among countries.

In 2011 and 2012, the COPHES/DEMOCOPHES twin projects performed the first ever harmonized human biomonitoring survey in 17 European countries. In more than 1800 mother-child pairs, individual lifestyle data were collected and cadmium, cotinine and certain phthalate metabolites were measured in urine. Total mercury was determined in hair samples. While the main goal of the COPHES/DEMOCOPHES twin projects was to develop and test harmonized protocols and procedures, the goal of the current paper is to investigate whether the observed differences in biomarker values among the countries implementing DEMOCOPHES can be interpreted using information from external databases on environmental quality and lifestyle. In general, 13 countries having implemented DEMOCOPHES provided high-quality data from external sources that were relevant for interpretation purposes. However, some data were not available for reporting or were not in line with predefined specifications. Therefore, only part of the external information could be included in the statistical analyses. Nonetheless, there was a highly significant correlation between national levels of fish consumption and mercury in hair, the strength of antismoking legislation was significantly related to urinary cotinine levels, and we were able to show indications that also urinary cadmium levels were associated with environmental quality and food quality. These results again show the potential of biomonitoring data to provide added value for (the evaluation of) evidence-informed policy making.

Meta-analysis of data from human ex vivo placental perfusion studies on genotoxic and immunotoxic agents within the integrated European project NewGeneris.

In the E.U. integrated project NewGeneris, we studied placental transport of thirteen immunotoxic and genotoxic agents in three ex vivo placental perfusion laboratories. In the present publication, all placental perfusion data have been re-analyzed and normalized to make them directly comparable and rankable. Antipyrine transfer data differed significantly between the studies and laboratories, and therefore normalization of data was necessary. An antipyrine normalization factor was introduced making the variance significantly smaller within and between the studies using the same compound but performed in different laboratories. Non-normalized (regular) and normalized data showed a good correlation. The compounds were ranked according to their transplacental transfer rate using either antipyrine normalized AUC120 or transfer index (TI120(%)). Normalization generated a division of compounds in slow, medium and high transfer rate groups. The transfer rate differed slightly depending on the parameter used. However, compounds with passage similar to antipyrine which goes through the placenta by passive diffusion, and good recovery in media (no accumulation in the tissue or adherence to equipment) were highly ranked no matter which parameter was used. Antipyrine normalization resulted in the following ranking order of compounds according to AUC(120NORM) values: NDMA ≥ EtOH ≥ BPA ≥ IQ ≥AA ≥ GA ≥ PCB180 ≥ PhIP ≥ AFB1 > DON ≥ BP ≥ PCB52 ≥ TCDD. As the variance in all parameters within a study decreased after antipyrine normalization, we conclude that this normalization approach at least partially corrects the bias caused by the small methodological differences between studies.

Studying placental transfer of highly purified non-dioxin-like PCBs in two models of the placental barrier.

Currently, toxicology and toxicokinetics of purified non-dioxin-like polychlorinated biphenyls (NDL-PCBs) are poorly characterised. Transplacental kinetics of NDL-PCBs can be studied in a variety of models, but careful validation of each model is crucial. We aimed to develop a standard operating procedure for establishing an in vitro model of the human placental barrier. Using this model, we sought to investigate placental transport kinetics of two NDL-PCB congeners. Firstly, we compared the BeWo cell line of the American Type Culture Collection with the BeWo b30 clone and determined parameters for monolayer formation. Secondly, we performed placental perfusions to validate the in vitro model. To that end, the transport of radiolabelled PCB52 and 180 was investigated in both models. We were not able to grow the ATCC cell line to confluency, but determined monolayer formation using BeWo b30. A confluent monolayer is present by day 4 post-seeding, transepithelial electrical resistance being 44.65 ± 11.06 Ω cm(2) and sodium fluorescein transport being 4.1% ± 0.18. Both measures can be used as indicators for monolayer formation. Results from kinetic studies in vitro and ex vivo were in excellent agreement. Both NDL-PCBs crossed the placental barrier within 2.5 h. We found PCB180 to transfer more rapidly and PCB52 to associate more with placental tissue. Since transport and association patterns were similar in vitro and ex vivo, we conclude that the protocol provided here forms the basis for a good model of the placental barrier using BeWo b30. We hypothesise that the observed differences in transport and association patterns of NDL-PCBs may indicate that toxic effects of PCB52 play a more important role regarding placental function, whereas PCB180 may be of greater importance for fetal toxicity.

Transcriptomic network analysis of micronuclei-related genes: a case study.

Mechanistically relevant information on responses of humans to xenobiotic exposure in relation to chemically induced biological effects, such as micronuclei (MN) formation can be obtained through large-scale transcriptomics studies. Network analysis may enhance the analysis and visualisation of such data. Therefore, this study aimed to develop a 'MN formation' network based on a priori knowledge, by using the pathway tool MetaCore. The gene network contained 27 genes and three gene complexes that are related to processes involved in MN formation, e.g. spindle assembly checkpoint, cell cycle checkpoint and aneuploidy. The MN-related gene network was tested against a transcriptomics case study associated with MN measurements. In this case study, transcriptomic data from children and adults differentially exposed to ambient air pollution in the Czech Republic were analysed and visualised on the network. Six genes from the network, i.e. BAX, DMNT1, PCNA, HIC1, p21 and CDC20, were retrieved. Based on these six genes and in combination with p53 and IL-6, a dedicated network was created. This dedicated network is possibly suited for the development of a reporter gene assay that could be used to screen populations complementary to the current MN test assay. In conclusion, we have shown that network analysis of transcriptomics data in relation to the formation of MN is possible and provides a novel mechanistic hypothesis by indicating which genes are regulated and influence others.

Toxicokinetics of the food-toxin IQ in human placental perfusion is not affected by ABCG2 or xenobiotic metabolism.

Metabolizing enzymes and transporters affect toxicokinetics of foreign compounds (e.g. drugs and carcinogens) in human placenta. The heterocyclic amine, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is a food-borne carcinogen being metabolically activated by cytochrome P450 (CYP) enzymes, especially by CYP1A1/2. IQ is also a substrate for ABCG2 transporter. Placental transfer of (14)C-IQ was evaluated in 4-6 h ex vivo human placental perfusions in Finland and Denmark. In Finland placentas were perfused with (14)C-IQ alone (0.5 microM, n = 6) or in combination with GF120918 (inhibitor of ABCG2, 1 microM, n = 6) or Ko143 (specific inhibitor of ABCG2, 2 microM, n = 4) to study the role of ABCG2 inhibition in transfer while in Denmark perfusions were performed with (14)C-IQ alone. Critical parameters (leak from fetal to maternal circulation, pH values, blood gases, glucose consumption, the production of hCG hormone and transport of antipyrine) were analyzed during the perfusions. (14)C-IQ on maternal and fetal sides was determined by liquid scintillation counting. In Finland IQ and its metabolites in final perfusates were determined also by LC/TOF-MS. ABCG2 expression and EROD activity (CYP1A1/2) were analyzed from perfused tissues. (14)C-IQ was easily transferred through the placenta from maternal to fetal side in both laboratories. Neither significant EROD activity nor IQ metabolites were found in placentas from non-smoking mothers. Inhibition of ABCG2 by GF120918 (FM-ratio of IQ 0.95) or Ko143 (FM-ratio of IQ 0.94) did not affect (14)C-IQ transfer (FM-ratio of IQ in IQ only perfusions 0.97), which indicates that placental ABCG2 does not have a significant role in protecting fetus from IQ.

Optimization of chemically defined cell culture media--replacing fetal bovine serum in mammalian in vitro methods.

Quality assurance is becoming increasingly important. Good laboratory practice (GLP) and good manufacturing practice (GMP) are now established standards. The biomedical field aims at an increasing reliance on the use of in vitro methods. Cell and tissue culture methods are generally fast, cheap, reproducible and reduce the use of experimental animals. Good cell culture practice (GCCP) is an attempt to develop a common standard for in vitro methods. The implementation of the use of chemically defined media is part of the GCCP. This will decrease the dependence on animal serum, a supplement with an undefined and variable composition. Defined media supplements are commercially available for some cell types. However, information on the formulation by the companies is often limited and such supplements can therefore not be regarded as completely defined. The development of defined media is difficult and often takes place in isolation. A workshop was organised in 2009 in Copenhagen to discuss strategies to improve the development and use of serum-free defined media. In this report, the results from the meeting are discussed and the formulation of a basic serum-free medium is suggested. Furthermore, recommendations are provided to improve information exchange on newly developed serum-free media.

Increased micronuclei and bulky DNA adducts in cord blood after maternal exposures to traffic-related air pollution.

Exposure to traffic-related air pollution in urban environment is common and has been associated with adverse human health effects. In utero exposures that result in DNA damage may affect health later in life. Early effects of maternal and in utero exposures to traffic-related air pollution were assessed through the use of validated biomarkers in blood cells from mother-newborn pairs. A cross-sectional biomonitoring study with healthy pregnant women living in the Greater Copenhagen area, Denmark, was conducted. Bulky DNA adducts and micronuclei (MN) were measured in blood from 75 women and 69 umbilical cords, concurrently collected at the time of planned Caesarean section. Modeled residential traffic density, a proxy measure of traffic-related air pollution exposures, was validated by indoor levels of nitrogen dioxide and polycyclic aromatic hydrocarbons in 42 non-smoking homes. DNA adduct levels were similar and positively correlated in maternal and cord blood (1.40 vs. 1.37 n/10(8) nucleotides; r=0.99; p<0.01). Maternal MN frequencies were significantly associated with age (p<0.01), and higher than those of the newborns (7.0 vs. 3.2 MN per 1000 binucleated cells). Adduct levels were highest among mother-newborn pairs who lived near medium-traffic-density (>400-2500 vehicle km/24h; p<0.01) places. MN frequencies among newborns from women who lived at high-traffic-density homes (>2500 vehicle km/24h) were significantly increased (p=0.02). This trend remained after adjusting for potential confounders and effect modifiers. For the first time increased bulky DNA adducts and MN in cord blood after maternal exposures to traffic-related air pollution are found, demonstrating that these transplacental environmental exposures induce DNA damage in newborns. Given that increased DNA damage early in life indicate an increased risk for adverse health effects later in life, these findings justify intervention of pregnant women.

Typical signature of DNA damage in white blood cells: a pilot study on etheno adducts in Danish mother-newborn child pairs.

The impact of DNA damage commonly thought to be involved in chronic degenerative disease causation is particularly detrimental during fetal development. Within a multicenter study, we analyzed 77 white blood cell (WBC) samples from mother-newborn child pairs to see if imprinting of DNA damage in mother and newborn shows a similar pattern. Two adducts 1,N(6)-ethenodeoxyadenosine (epsilondA) and 3,N(4)-ethenodeoxycytidine (epsilondC) were measured by our ultrasensitive immunoaffinity (32)P-post-labeling method. These miscoding etheno-DNA adducts are generated by the reaction of lipid peroxidation (LPO) end products such as 4-hydroxy-2-nonenal with DNA bases. Mean epsilondA and epsilondC levels when expressed per 10(9) parent nucleotides in WBC-DNA from cord blood were 138 and 354, respectively; in maternal WBC-DNA, the respective values were 317 and 916. Thus, the DNA-etheno adduct levels were reliably detectable and about two times lower in child cord blood, the difference being significant at P < 0.0004. Analysis of epsilondA and epsilondC levels in cord versus maternal blood WBC showed strong positive correlations (R(2) approximately 0.9, P < 0.00001). In conclusion, LPO-induced DNA damage arising from endogenous reactive aldehydes in WBC of both mother and newborn can be reliably assessed by epsilondA and epsilondC as biomarkers. The high correlation of etheno adduct levels in mother and child WBC suggests that a typical signature of DNA damage is induced similarly in fetus and mother. Prospective cohort studies have to reveal whether these two WBC-DNA adducts could serve as risk indicator for developing hematopoietic cancers and other disorders later in life.

Genomic damage in children accidentally exposed to ionizing radiation: a review of the literature.

During the last decade, our knowledge of the mechanisms by which children respond to exposures to physical and chemical agents present in the environment, has significantly increased. Results of recent projects and programmes focused on children's health underline a specific vulnerability of children to environmental genotoxicants. Environmental research on children predominantly investigates the health effects of air pollution while effects from radiation exposure deserve more attention. The main sources of knowledge on genome damage of children exposed to radiation are studies performed after the Chernobyl nuclear plant accident in 1986. The present review presents and discusses data collected from papers analyzing genome damage in children environmentally exposed to ionizing radiation. Overall, the evidence from the studies conducted following the Chernobyl accident, nuclear tests, environmental radiation pollution and indoor accidental contamination reveals consistently increased chromosome aberration and micronuclei frequency in exposed than in referent children. Future research in this area should be focused on studies providing information on: (a) effects on children caused by low doses of radiation; (b) effects on children from combined exposure to low doses of radiation and chemical agents from food, water and air; and (c) specific effects from exposure during early childhood (radioisotopes from water, radon in homes). Special consideration should also be given to a possible impact of a radiochemical environment to the development of an adaptive response for genomic damage. Interactive databases should be developed to provide integration of cytogenetic data, childhood cancer registry data and information on environmental contamination. The overall aim is to introduce timely and efficient preventive measures, by means of a better knowledge of the early and delayed health effects in children resulting from radiation exposure.

Ethics in studies on children and environmental health.

Children, because of age-related reasons, are a vulnerable population, and protecting their health is a social, scientific and emotional priority. The increased susceptibility of children and fetuses to environmental (including genotoxic) agents has been widely discussed by the scientific community. Children may experience different levels of chemical exposure than adults, and their sensitivity to chemical toxicities may be increased or decreased in comparison with adults. Such considerations also apply to unborn (fetal exposure) and newborn (neonatal exposure) children. Therefore, research on children is necessary in both clinical and environmental fields, to provide age-specific relevant data regarding the efficacy and safety of medical treatments, and regarding the assessment of risk from unintended environmental exposure. In this context, the stakeholders are many, including children and their parents, physicians and public health researchers, and the society as a whole, with its ethical, regulatory, administrative and political components. The important ethical issues are information of participants and consent to participate. Follow-up and protection of data (samples and information derived from samples) should be discussed in the context of biobanks, where children obtain individual rights when they become adults. It is important to realise that there are highly variable practices within European countries, which may have, in the past, led to differences in practical aspects of research in children. A number of recommendations are provided for research with children and environmental health. Environmental research with children should be scientifically justified, with sound research questions and valid study protocols of sufficient statistical power, ensuring the autonomy of the child and his/her family at the time of the study and later in life, if data and samples are used for follow-up studies. When children are enrolled, we recommend a consent dyad, including (1) parental (or legal guardian) informed consent and (2) the child's assent and/or informed consent from older minors. For evaluation of the studies including children, a paediatrician should always be involved in the research ethics committee.

In vitro susceptibilities in lymphocytes from mothers and cord blood to the monofunctional alkylating agent EMS.

It has been reported that children may experience different levels of chemical exposures than adults and that their sensitivities to chemical toxins may be increased or decreased when compared to adults. The perinatal period is one period in which these susceptibilities may be examined. Midwives at the Bradford Royal Infirmary collected venous blood samples from mothers at the time of birth and venous cord blood post-delivery. Lymphocytes were isolated from both blood types and examined in the alkaline comet assay using the monofunctional alkylating agent ethyl methanesulphonate (EMS). There were no biologically significant differences when subjects were categorized into subgroups based on lifestyle habits and physical characteristics, and overall there were no statistically significant differences in levels of DNA damage in mothers (n=22) and babies (n=22), except at the basal level (P<0.05), but mean values in babies were always lower over the EMS dose range. Whole blood was used in the micronucleus (MN) assay, and there was a significantly (P<0.05) higher rate of MN in mothers (n=17), per 1000 binucleates, as compared with lymphocytes from their offspring (n=17) at the basal level. This may be accounted for by age and endogenous factors. Overall, this current study cannot provide statistically significant evidence that children have either increased or decreased levels of susceptibility to a chemical toxin in comparison to adults when EMS is examined in vitro.

Chromosomal aberrations and SCEs as biomarkers of cancer risk.

Previous studies have suggested that the frequency of chromosomal aberrations (CAs), but not of sister chromatid exchanges (SCEs), predicts cancer risk. We have further examined this relationship in European cohorts comprising altogether almost 22,000 subjects, in the framework of a European collaborative project (CancerRiskBiomarkers). The present paper gives an overview of some of the results of the project, especially as regards CAs and SCEs. The results confirm that a high level of CAs is associated with an increased risk of cancer and indicate that this association does not depend on the time between CA analysis and cancer detection, i.e., is obviously not explained by undetected cancer. The present evidence indicates that both chromatid-type and chromosome-type CAs predict cancer, even though some data suggest that chromosome-type CAs may have a more pronounced predictive value than chromatid-type CAs. CA frequency appears to predict cancers at various sites, although there seems to be a particular association with gastrointestinal cancers. SCE frequency does not appear to have cancer predictive value, at least partly due to uncontrollable technical variation. A number of genetic polymorphisms of xenobiotic metabolism, DNA repair, and folate metabolism affect the level of CAs and might collectively contribute to the cancer predictivity of CAs. Other factors that may influence the association between CAs and cancer include, e.g., exposure to genotoxic carcinogens and internal generation of genotoxic species. Although the association between CA level and cancer is seen at the group level, an association probably also exists for the individual, although it is not known if an individual approach could be feasible. However, group level evidence should be enough to support the use of CA analysis as a tool in screening programs and prevention policies in occupational and environmental health.

Genome-wide differential gene expression in children exposed to air pollution in the Czech Republic.

The Teplice area in the Czech Republic is a mining district where elevated levels of air pollution including airborne carcinogens, have been demonstrated, especially during winter time. This environmental exposure can impact human health; in particular children may be more vulnerable. To study the impact of air pollution in children at the transcriptional level, peripheral blood cells were subjected to whole genome response analysis, in order to identify significantly modulated biological pathways and processes as a result of exposure. Using genome-wide oligonucleotide microarrays, we investigated differential gene expression in children from the Teplice area (n=23) and compared them with children from the rural control area of Prachatice (n=24). In an additional approach, individual gene expressions were correlated with individual peripheral blood lymphocyte micronuclei frequencies, in order to evaluate the linkage of individual gene expressions with an established biomarker of effect that is representative for increased genotoxic risk. Children from the Teplice area showed a significantly higher average micronuclei frequency than Prachatice children (p=0.023). For considerable numbers of genes, the expression differed significantly between the children from the two areas. Amongst these genes, considerable numbers of genes were observed to correlate significantly with the frequencies of micronuclei. The main biological process that appeared significantly affected overall was nucleosome assembly. This suggests an effect of air pollution on the primary structural unit of the condensed DNA. In addition, several other pathways were modulated. Based on the results of this study, we suggest that transcriptomic analysis represents a promising biomarker for environmental carcinogenesis.

Perceptions of parents on the participation of their infants in clinical research.

OBJECTIVE: To analyse the motivations and perceptions of parents on the participation of their infants and young children in a comprehensive and invasive clinical research study. METHODS: Semistructured qualitative interviews were conducted with 23 mothers with asthma whose infants and young children were participating in the Copenhagen Prospective Study on Asthma in Childhood. The interviews were audiotaped, transcribed and analysed using the template analysis method. RESULTS: Parents were motivated by altruism and by the opportunity to get their child checked regularly by medical experts to prevent the possible development of asthma. Parents found it very important that their children enjoyed their visits to the research clinic, and that they could withdraw from the study if their child started responding negatively to those visits. No apparent difference was seen in the attitude between the parents of children with lung or skin symptoms and those of healthy children. CONCLUSIONS: It is possible to design and accomplish invasive clinical research on infants and young children in a manner that parents find ethically sound.

Laboratory work and pregnancy outcomes: a study within the National Birth Cohort in Denmark.

AIMS: To examine pregnancy outcomes in women doing laboratory work. METHODS: Using data from the Danish National Birth Cohort (1997-2003), the authors conducted a prospective cohort study of 1025 female laboratory technicians and 8037 female teachers (as reference). The laboratory technicians were asked about laboratory work tasks during pregnancy in an interview (at around 16 weeks of gestation). Pregnancy outcomes were obtained by linking the cohort to the national registers. Hazard ratios (HRs) of late fetal loss and diagnosing of congenital malformations were calculated by using Cox regression, and odds ratios (ORs) of preterm birth and small for gestational age were calculated by using logistic regression. RESULTS: Overall, there were no significant differences in pregnancy outcomes between laboratory technicians and teachers. However, we found that laboratory technicians working with radioimmunoassay or radiolabelling had an increased risk of preterm birth (OR = 2.2, 95% CI 0.8 to 6.2 for radioimmunoassay, and OR = 1.9, 95% CI 0.8 to 4.6 for radiolabelling) and "major" malformations (HR = 2.1, 95% CI 1.0 to 4.7 for radioimmunoassay, and HR = 1.8, 95% CI 0.9 to 3.7 for radiolabelling). The ORs of preterm birth doubled for women working with these tasks every day or several times a week. When an exposure matrix was applied, an increased risk of "major" malformations for exposure to organic solvents was seen. CONCLUSIONS: The results did not indicate any high risk of reproductive failures in laboratory technicians in general. Exposure to radioisotopes may carry a high risk of preterm birth and congenital malformations. This finding deserves further investigation.

Genotoxic damage in mine workers exposed to diesel exhaust, and the effects of glutathione transferase genotypes.

This study was performed in an Estonian shale-oil mine with the purpose to develop and apply a number of biomarkers for occupational diesel-exhaust exposure monitoring. Increased breathing-zone exposures to exhaust from operators of diesel-powered trucks in the mine was confirmed in the environmental monitoring part of the study, showing a 7.5-fold higher exposure to particle-associated 1-nitropyrene (1-NP) in 50 underground workers compared with 42 surface workers [P.T.J. Scheepers, D. Coggon, L.E. Knudsen, R. Anzion, H. Autrup, S. Bogovski, R.P. Bos, D. Dahmann, P. Farmer, E.A. Martin, V. Micka, V. Muzyka, H.-G. Neumann, J. Poole, A. Schmidt-Ott, F. Seiler, J. Volf, I. Zwirner-Baier, Biomarkers for occupational diesel exhaust exposure monitoring (BIOMODEM)-a study in underground mining, Toxicol. Lett. 134 (2002) 305-317; P.T.J. Scheepers, V. Micka, V. Muzyka, R. Anzion, D. Dahmann, J. Poole, R.P. Bos, Exposure to dust and particle-associated 1-nitropyrene of drivers of diesel-powered equipment in underground mining, Ann. Occp. Hyg. 47 (2003) 379-388]. Analysis of DNA damage by the Comet assay on frozen blood samples was performed on the total study group and showed significantly higher levels (p=0.003) in underground workers (smokers) driving diesel-powered excavation machines (median 155 on a scale from 0 to 400, among 47 persons), compared with surface workers who smoked (median of 90, among 46 persons). The level of DNA damage in underground smokers was significantly higher (p=0.04) than in non-smokers. Samples from 2 of the 3 sampling weeks had significantly lower DNA damage compared with the third week, probably due to timely processing and freezing. These samples also showed significant differences (p<0.001) between underground workers (median 145, among 41 persons) and surface workers (median 60, among 30 persons). An HPLC method was developed for the analysis of (32)P-postlabelled 1-NP-DNA-adducts, and was applied to a sub-sample of 20 workers. No significant differences between surface and underground workers were found in this sub-sample with respect to the minor, unidentified adducts that had similar chromatographic properties to 1-NP adducts, and smoking did not have any effect on adduct levels. No significant effects of the genotypes of GSTM1, GSTP1 and GSTT1 on DNA-adducts and on DNA damage as measured by the Comet assay were found in the total study group. The study confirms an increased level of DNA damage in workers exposed to exhaust from truck-driving in the mine. However, the results of the environmental and biological monitoring of 1-NP did not correlate, suggesting that inhalation exposure to diesel exhaust is not reflected by an increase in 1-NP-DNA-adduct levels and/or that factors other than occupational exposure to diesel exhaust are primary determinants of these DNA-adduct levels.