Multi-endpoint biological monitoring in combined, carcinogenic occupational exposures.

We aimed to develop a relevant multi-endpoint biomonitoring system by studying different genotoxicity biomarkers in complex carcinogenic exposures under occupational situations. Altogether 109 workers were followed in five different workplaces. The combined carcinogenic exposures were monitored in the urine and peripheral blood samples using Ames mutagenicity test and cytogenetic analyzes. The different genotoxicity endpoints studied showed different results in the same carcinogenic exposure situations. The urinary mutagenicity tests provided more information and proved to be more sensitive compared to the cytogenetic tests in the majority of cases. In complex exposures multistep biomonitoring panel should be applied, because the exact mechanisms of the combination of single exposing agents are not known. Such a panel should involve monitoring different endpoints, e.g. point mutations, chromosomal mutations. A relatively affordable and rapid testing panel was developed using validated tests as Ames and cytogenetic assays, but its practical use should be confirmed by further investigations.

Use of low density polyethylene membranes for assessment of genotoxicity of PAHs in the Seine River.

The genotoxicity of river water dissolved contaminants is usually estimated after grab sampling of river water. Water contamination can now be obtained with passive samplers that allow a time-integrated sampling of contaminants. Since it was verified that low density polyethylene membranes (LDPE) accumulate labile hydrophobic compounds, their use was proposed as a passive sampler. This study was designed to test the applicability of passive sampling for combined chemical and genotoxicity measurements. The LDPE extracts were tested with the umu test (TA1535/pSK1002 ± S9) and the Ames assay (TA98, TA100 and YG1041 ± S9). We describe here this new protocol and its application in two field studies on four sites of the Seine River. Field LDPE extracts were negative with the YG1041 and TA100 and weakly positive with the TA98 + S9 and Umu test. Concentrations of labile mutagenic PAHs were higher upstream of Paris than downstream of Paris. Improvement of the method is needed to determine the genotoxicity of low concentrations of labile dissolved organic contaminants.

Reporter Gene Assays in Ecotoxicology.

The need for simple and rapid means for evaluating the potential toxic effects of environmental samples has prompted the development of reporter gene assays, based on tester cells (bioreporters) genetically engineered to report on sample toxicity by producing a readily quantifiable signal. Bacteria are especially suitable to serve as bioreporters owing to their fast responses, low cost, convenient preservation, ease of handling, and amenability to genetic manipulations. Various bacterial bioreporters have been introduced for general toxicity and genotoxicity assessment, and the monitoring of endocrine disrupting and dioxin-like compounds has been mostly covered by similarly engineered eukaryotic cells. Some reporter gene assays have been validated, standardized, and accredited, and many others are under constant development. Efforts are aimed at broadening detection spectra, lowering detection thresholds, and combining toxicity identification capabilities with characterization of the toxic effects. Taking advantage of bacterial robustness, attempts are also being made to incorporate bacterial bioreporters into field instrumentation for online continuous monitoring or on-site spot checks. However, key hurdles concerning test validation, cell preservation, and regulatory issues related to the use of genetically modified organisms still remain to be overcome.

Automation of the in vitro micronucleus and chromosome aberration assay for the assessment of the genotoxicity of the particulate and gas-vapor phase of cigarette smoke.

Total particulate matter (TPM) and the gas-vapor phase (GVP) of mainstream smoke from the Reference Cigarette 3R4F were assayed in the cytokinesis-block in vitro micronucleus (MN) assay and the in vitro chromosome aberration (CA) assay, both using V79-4 Chinese hamster lung fibroblasts exposed for up to 24 h. The Metafer image analysis platform was adapted resulting in a fully automated evaluation system of the MN assay for the detection, identification and reporting of cells with micronuclei together with the determination of the cytokinesis-block proliferation index (CBPI) to quantify the treatment-related cytotoxicity. In the CA assay, the same platform was used to identify, map and retrieve metaphases for a subsequent CA evaluation by a trained evaluator. In both the assays, TPM and GVP provoked a significant genotoxic effect: up to 6-fold more micronucleated target cells than in the negative control and up to 10-fold increases in aberrant metaphases. Data variability was lower in the automated version of the MN assay than in the non-automated. It can be estimated that two test substances that differ in their genotoxicity by approximately 30% can statistically be distinguished in the automated MN and CA assays. Time savings, based on man hours, due to the automation were approximately 70% in the MN and 25% in the CA assays. The turn-around time of the evaluation phase could be shortened by 35 and 50%, respectively. Although only cigarette smoke-derived test material has been applied, the technical improvements should be of value for other test substances.

Application of a modified gaseous exposure system to the in vitro toxicological assessment of tobacco smoke toxicants.

Tobacco smoke is a complex mixture of over 6,000 individual chemical constituents. Approximately 150 of these have been identified as 'tobacco smoke toxicants' due to their known toxicological effects. A number of these toxicants are present in the gaseous phase of tobacco smoke. This presents a technical challenge when assessing the toxicological effects of these chemicals in vitro. We have adapted a commercially available tobacco smoke exposure system to enable the assessment of the contribution of individual smoke toxicants to the overall toxicological effects of whole mainstream cigarette smoke (WS). Here we present a description of the exposure system and the methodology used. We use the example of a gaseous tobacco smoke toxicant, ethylene oxide (EtO), a Group 1 IARC carcinogen and known mutagen, to illustrate how this methodology can be applied to the assessment of genotoxicity of gaseous chemicals in the context of WS. In the present study we found that EtO was positive in Salmonella typhimurium strain YG1042, a strain that is sensitive to tobacco smoke. However, EtO did not increase the mutagenicity of the WS mixture when it was added at greatly higher concentrations than those found typically in WS. The findings presented here demonstrate the suitability of this exposure system for the assessment of the mutagenic potential of gases in vitro. Whilst we have focused on tobacco smoke toxicants, this system has broad application potential in studying the biological effects of exposure to a wide range of gaseous compounds that are present within complex aerosol mixtures.

Evaluación de riesgo genotóxico: biomonitorización de trabajadores agrícolas de Caranavi, Guanay, Palca y Mecapaca, expuestos a plaguicidas / Assessment of genoptoxic risk biomonitoring of farm workers exposed to pesticides in Caranavi, Guanay, Palca and Mecapaca

OBJETIVO: detectar los efectos citotóxicos y genotóxicos en trabajadores agrícolas, mediante estudios de biomonitoreo genético. DISEÑO: casos y controles Participantes Trabajadores agrícolas de Caranavi, Guanay, Mecapaca y Palca del Departamento de La Paz Lugar Localidades de Caranavi, Guanay, Palca y Mecapaca. Unidad de Genética, toxicológica Instituto de Genética MATERIAL Y MÉTODOS: se aplicó cuestionario a 259 trabajadores agrícolas. Se evaluó el efecto genotóxico en linfocitos de sangre heparinizada, a través de la frecuencia de Intercambios entre Cromátides Hermanas (ICH), el Índice de Proliferación Celular (PRI), el % de células con alta frecuencia de intercambios (%HFC), frecuencia de micronúcleos en células binucleadas (MNBN), el índice de división nuclear (IDN), la presencia de aberraciones cromosómicas estructurales (AC), y parámetros de la prueba del cometa, como DNA de la cola, DNA de la cabeza, longitud de la cola, longitud del cometa, el momento de la cola y momento Olive. RESULTADOS: Los casos presentaron un aumento estadísticamente significativo (p<0.05) en la frecuencia de ICH, MN/BN y aberraciones cromosómicas, en relación a los controles. Así mismo, los parámetros de DNA de la cola, DNA de la cabeza, longitud de la cola, longitud del cometa, el momento de la cola y momento Olive, mostraron un aumento en relación a los controles, (p<0.05). Los valores promedio (± ES) de los parámetros del ensayo del cometa, fueron mayores y estadísticamente significativos en los expuestos y RPP's en relación a los no expuestos. En el grupo de RPP´s se observó daño genotóxico en menor proporción pero no significativo en relación a los expuestos, posiblemente por su capacitación en medidas de protección. El análisis divariado entre exposición a plaguicidas y daño genotóxico mostró que las personas expuestas a plaguicidas tienen 1.49 veces más probabilidad de sufrir daño genotóxico con un OR de 2.49 (IC 95% 1.48 - 4.20). CONCLUSIÓN: los resultados indican que los trabajadores agrícolas expuestos sin protección ni medidas de seguridad a mezclas de plaguicidas, han experimentado riesgo genotóxico, que fue manifestado con elevada frecuencia de intercambios entre cromátides hermanas, micronúcleos, aberraciones cromosómicas y parámetros del cometa, en linfocitos de sangre periférica. Así mismo, la presencia de aberraciones cromosómicas, que son las que determinan la asociación con efecto carcinogénico, muestra que los trabajadores agrícolas expuestos a plaguicidas tienen mayor probabilidad de que las mutaciones encontradas al momento del estudio, puedan volverse irreversibles por la saturación de los sistemas de reparación del DNA y en el futuro desarrollar diversos tipos de cáncer. Estos hallazgos son indicativos de la necesidad de realizar biomonitorización permanente de los agricultores ocupacionalmente expuestos a varias mezclas de plaguicidas, utilizando una batería de pruebas de genotoxicidad. Por otra parte, ilustra la necesidad de implementar pautas generales para minimizar o prevenir la exposición.

OBJECTIVE: to detect the cytotoxic and genotoxic effects in farm workers, by means of genetic biomonitoring studies. Design Cases and controls Participants Farm workers from Caranavi, Guanay, Palca and Mecapaca Place Towns of Caranavi, Guanay, Palca and Mecapaca, Genetic Toxicology unit. Genetic Institute. MATERIAL AND METHODS: Questionnaires to 257 agricultural workers were applied genotoxic effect was evaluated in lymphocytes from heparinized blood, through analysis of sister chromatid Exchange (SCE), cells with a high frecuency of SCE (HFC), proliferation rate index (PRI) the micronucleus (MN) assay, nuclear division index (NDI), chromosomal aberrations (CA) and comet assay parameters like DNA tail, DNA head, tail length comet length, tail moment and Olive moment. RESULTS: the frequency of SCE, MN/BN and CA was significantly increased (p<0.05) in cases vs. control group. Likewise, the parameters of Tail DNA, DNA head , tail length, comet length, tail moment and Olive moment, showed increased values in relation to controls (p<0.05). Averages of comet parameters were significantly higher in exposed and RPP's group than in un exposed group. RPP`s groups showed minor DNA damage but not as significant as exposed group, possibly due to their training in protective measures. The bivariated analysis between pesticides exposure and genotoxic damage showed that the people exposed to pesticides have 1.49 times more probability of suffering genotoxic damage with OR 2.49 (IC 95% 1.48 - 4.20). CONCLUSIONS: the results indicate that the farm workers exposed to mixture of pesticides without protection and safety measures, are at genotoxic risk hazard , with high frequency of sister chromatid exchange, micronuclei, chromosomal aberrations and parameters of the comet assay in lymphocytes of peripheral blood. Also, the presence of chromosomal aberrations, which are those that determine the association with carcinogenic effect, shows that the farm workers exposed to pesticides have greater probability that the mutations found at the time of the study, can become irreversible by saturation of the DNA repair systems and in the future develop diverse types of cancer. These findings are indicative of the necessity to do permanent biomonitoring of the farmers occupationally exposed to several mixtures of pesticides, using a battery of genotoxicity tests. On the other hand, it illustrates the necessity to implement general guidelines to diminish or to prevent the exposure.

Special considerations for conducting genotoxicity tests with protein materials.

Standard genotoxicity tests are often inappropriate for testing new biological entities, in particular for recombinant proteins which are nature-identical. Arguments that these may contain mutagenic impurities are not substantiated; however, we have produced evidence that such impurities would be detected amidst a vast excess of protein. Concerns that human patients receiving therapy may be at risk from higher-than-physiological levels of proteins are also somewhat theoretical. However, it is apparent that genotoxicity testing will be required for these products for the time being, even if pragmatic approaches reduce the battery of in vitro tests to Ames and chromosomal aberrations only, and reduce the top dose in vivo to 1000x the human therapeutic dose. There is a number of physical and chemical properties of proteins that demand special approaches to methodology if the tests are to produce accurate results. The potential for adsorption to certain forms of glass and plastic means special care must be taken in dissolving and diluting test solutions; adherence to filters means special low protein binding, non-pyrogenic filters should be used for sterilisation of test solutions, where this is necessary; freeze-dried powders aliquotted in multiple vials should be dissolved in minimal solvent and cascaded from vial to vial rather than trying to empty the solid contents for bulk weighing. As proteins are often supplied in solution, in order to achieve sufficiently high test concentrations, it may be necessary to resuspend test bacteria/cells in the test solutions for short periods of time before centrifuging and resuspending in selective or growth media.(ABSTRACT TRUNCATED AT 250 WORDS)