Urinary caffeine metabolites and assessment of CYP1A2 activity

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Biological and physical hazards

In addition to chemicals, sewage workers are also exposed to biological and physical agents. Various pathogenic bacteria and viruses as well as protozoa and helminthes have been reported in sewage system [1]. Accordingly, infections and associated health effects have been the focus of the majority of research in sewage exposed workers in the past [1,2]. A summary of a brief review of literature among sewage workers showed that enteric viruses, bacteria, parasites and pathogenic fungi have been found in sewage and aerosols from wastewater treatment plants, suggesting that sewage workers are at increased risk of infection [19-22]. In France, hepatitis A virus seroprevalence in Parisian sewage workers was found increased with age, reaching 80% for workers older than 40 years [23]. Giardiasis and entamoeba histiolytica have been also reported among sewage workers in France and other countries [24,25].
Sewage workers are also exposed to a variety of physical hazards putting them at risk for significant injuries. Physical contact with the effluent is possible through cleaning up work, contact with spray during decontamination processes, and while touching contaminated surfaces, clothes, shoes, etc [7]. A study found that in 255 sewage workers, the 12 month prevalence symptoms rates were 52.4% neck symptoms, 54.8% upper back and 72.8% low back symptoms [26].

Sewage drains indoor air pollution

Urban surfaces receive deposits of PAHs and volatile organic compounds (VOCs) from different sources such as car traffic, industries, waste incinerators, and domestic heating, via both atmospheric transport and local activity [39]. Industrial wastewater treatment plants might as also be a source for these hazardous substances with known health effects [41]. No study has measured the PAHs or VOCs in the air of the sewage system workplace. However, many have measured these substances in the wastewater treatment plants, in municipal solid waste, and in the sewage sludge, although rarely in the air of these plants. In Paris, a study assessed concentrations of certain pollutants in wastewater during dry and wet periods across different sampling sites and sewer networks. For each sample, a total of 66 elements, including PAHs, VOCs were analysed. A broad range of pollutants was observed during dry as well as wet weather periods. Of the 66 elements investigated, 33 and 40 substances were observed in raw sewage and wet weather effluent, respectively. The majority of organic pollutants were identified within the µg.L-1 range [42]. In the sewage sludge and wastewater samples, PAHs and VOCs have also been reported in other studies [43,44].

Polycyclic aromatic hydrocarbons (PAHs)

We found no study that assessed the PAHs in the air of the under-ground sewage workplaces. However, in Italy, possible and probable carcinogens PAHs, such as benzo(a)anthracene, benzo(a)pyrene, benzo(a)fluoranthene, and benzo(k)fluoranthene, were found in the aerosol collected from the aeration tanks of the sewage treatment plant in Prato, Italy [45].
In Korea, a study reviewed the characteristics of PAH found in sewage sludge. The study was performed on 16 PAHs. It showed that concentrations of the PAHs on the inlet and on the outlet of the air measurement devices to be ranged from 3.926-925.748 µg/m3 and from 1.153-189.449 µg/m3, respectively [46]. In Greece, higher concentrations of benzo[a]pyrene (B[a]P), and total PAHs were found in the urban sewage sludge than in the industrial sludge
[47]. Pham et al. reported an increase of PAHs concentration in Montreal wastewaters during the winter period [48]. Blanchard et al. [6] found the concentration of PAHs in raw wastewater entering the Seine Aval treatment plant in Paris to be (as ng L-1) 0.2 to 400 for fluoranthene (mean, 77.4), 2 to 104 (mean, 21.4) for benzo(a)pyrene, and 0.3 to 63 (mean, 12.2) for benzo(b)fluoranthene. These results are consistent with that of Pham and Proulx (1997) in the Montreal wastewater treatment plant (83 to 216, 20 to 77, and 42 to 168 ngL-1 for fluoranthene, benzo(a)pyrene, and benzo(b)fluoranthene; respectively) [49]. Similar toxicants were found by other authors who analyzed the wastewater of municipal sewage treatment plants [28,29,50]. Over a period of two years (2000-2001), sediment samples were extracted from 40 silt traps spread through the combined sewer system of Paris. All samples were analysed for 16 PAHs. The results show that there are some important (between- and within-site) variations in hydrocarbon contents. PAHs contamination levels (50th percentile) in the Parisian sewer sediment were estimated at 18 µg g-1 [51]. In USA, study in the wastewater, some PAHs, like fluoranthrene, pyrene, chrysene, phenanthrene, benzo[a]anthracene, and naphthalene have been revealed [52].

Volatile organic compounds (VOCs)

We found no study that evaluated VOCs concentrations in the confined air of the under-ground sewage workplaces. However, VOCs have been identified in the air of the municipal solid waste treatment plants [53]. Benzene, toluene and organic solvents have been reported in the air of the sewage treatment plants receiving industrial sewage [9,27]. In a recent study on sewage management plants, concentrations of seven volatile organic sulfur compounds samples were determined. A wide range of concentrations was observed.
Dimethyl sulfide, carbon disulfide and dimethyl disulfide were the most abundant compounds, the highest concentrations being 608.5µg m-3, 658.5µg m-3 and 857.8µg m-3, respectively. The results were strongly influenced by the characteristics of the sampling point, e.g. whether the sample was taken at a confined site or in the open air [54]. The emissions of different VOCs from a wastewater treatment plant in Turkey has been studied; 1,3-dichlorobenzene, tetrachloroethylene were the most abundant with a total hourly emission rate of 0.041 kg h-1 [55]. VOCs were monitored in the different sections of a wastewater treatment plant in Taiwan samples over several air sampling points. In the drainage and effluent system in each season, acetone, isopropanol and dimethyl sulfide were the major species and maximum concentrations were 400.4, 22.8 and 641.2 ppbv (part per billion by volume), respectively [56]. In Greece, 41 VOCs was analysed in wastewater samples collected seasonally in four wastewater treatment plants: 31 VOCs was observed [57]. The total organic vapour concentrations were measured in the influents of two wastewater treatment plants in a Midwestern city in USA and identified chlorobenzene, tetrachloroethylene, trichlorobenzene, 1, 1, 1- trichloroethane, trichloroethylene, toluene, xylene, benzene, methylethylketone, methylisobutylketone, and aliphatic naphtha [58,59].

Other exposures in sewage system (skin contact and ingestion)

Sewage workers might also come in contact with industrial waste from accidental or illegal release, putting them at risk from extremely irritants to the mucous membrane, eyes, and skin [38,29]. An outreak of airborne irritant contact dermatitis developed among incinerator workers employed in a sewage treatment facility. Contamination of the workplace and workers’ clothing by sludge from the interstices of an incinerator exhaust fan proved to be the cause of the problem [60]. Exposure to wastewater in Mexico revealed no significant risk from ingestion or dermal contact except from nitrate exposure [61]. Dermal contact to acrylamide in sewage and wastewater treatment plants have been suggested [62].


Comet and micronucleus assays in vitro human occupational studies

The comet and MN assays have been suggested suitable for monitoring occupational exposure [96,98,129139]. To our knowledge, no study has specifically investigated the use of comet and micronucleus tests among the urine extracts of sewage workers or other occupationally or environmentally exposed workers. Only one study investigated the level of DNA damage among sewage workers using the comet test but on blood lymphocytes and it did not find significant differences between the sewage workers and the control group [76]. In this study, the sewage workers were classified into three different groups of exposure, based on self-reported levels of exposure, and the group with the highest exposure was defined as those workers who had worked in sewage-contaminated environments for at least 8 h in the preceding 2 weeks. This may indicate that the sewage workers were not exposed to an extent that causes DNA damage in lymphocytes. However, the assessment of exposure may not accurately discriminate between different exposure circumstances. It would have been of great value if some sort of definition of the DNA damaging agents and measurement of exposure had been included in the study. However, these tests have been evaluated in various occupational studies in using other body or tissue samples, mainly blood lymphocytes or oral mucosa. Analysis of leukocytes from workers exposed to environmental pollutants at a waste disposal site exhibited significant DNA damage as presented by comet assay in exposed group [135].
Regarding other settings of occupational exposures to complex mixture of chemicals and low doses of carcinogens, comet and micronucleus tests have been widely applied in ex vivo epidemiological biomonitoring studies to evaluate genotoxic effects and DNA damage.
In Italy, paving workers exposed to complex mixture including several PAHs contained in asphalt fumes during road paving, were evaluated for early genotoxic and oxidative anomalies on lymphocytes. Using the comet assay, they found a significant increase in DNA damage in exposed group than the control [136]. In Turkey, a significant increase in MN frequency among asphalt workers compared to control on peripheral lymphocytes was reported [137], while a lack of MN induction was reported for road pavers in Sweden [138]. Another work evaluated the occupational genotoxicity risk through the comet and MN test applied to exfoliated cells of the buccal mucous among storage battery renovation workers and car painters in Brazil. Highly significant effects of occupational exposure were found in both tests [139].

Comet assay “Single Cell Gel Electrophoresis” (SCGE)

The urine extracts kept at -20°C will be thawed and warmed to room temperature shortly before the assay. Comet assay will be performed basically according to Sing et al. 1988 [65], with modifications according to Muller et al. 2000 [66]. Briefly, the cells will be incubated with the organic extract of urine (200 μl) during 24h (typical division duration of these cells). Viability of cells will be determined by trypan blue test. Microscopic slides will be precoated with 100 μl of agarose (1%). The slides will be gently immersed in ice-cold freshly lysis solution and will be covered with fresh electrophoresis buffer for 20 min and placed in a horizontal electrophoresis unit tank filled with new fresh electrophoresis buffer. After electrophoresis, they will be washed with a freshly made neutralizing buffer and stained with 50 μl ethidium bromide solution. They will then be examined for analysis of DNA migration under a fluorescence microscope (Olympus BX-40, Olympus, Japan) using a computerized image analysis system (Komet 5, Kinetic Imaging). Two slides will be analyzed for each sample with fifty cells scored in each slide. Olive tail moment will be used for analysis of results [67].

Table of contents :

1. Introduction
1.1. Background
1.2. Health hazards in the workplace of sewage workers
1.2.1. Biological and physical hazards
1.2.2. Chemical hazard
1.3. Characterization of exposure among sewage workers
1.3.1. Sewage drains indoor air pollution Polycyclic aromatic hydrocarbons (PAHs) Volatile organic compounds (VOCs)
1.3.2. Other exposures in sewage system (skin contact and ingestion)
1.4. Health effects among sewage workers
1.4.1. Morbidity studies
1.4.2. Mortality and cancer studies
1.5. Biomarkers of exposure to genotoxicants
1.5.1. In vitro comet assay History and definition General principle of the assay Fields of application
1.5.2. In vitro micronucleus assay History and definition General principle of the assay Fields of application
1.5.3. Comet and micronucleus assays in vitro human occupational studies
1.6. Biomarkers of early effects of genotoxicants
1.6.1. The 24h urinary 8-oxodG
1.6.2. DNA-adducts
1.6.3. Metabolism of caffeine and CYP1A2 activity
1.7. Description of the parisian sewage system
1.8. Rationale and justification of the study
1.9. Aim of the study
1.10. Study hypothesis
1.11. Thesis overview
2. Materials and methods
2.1. Protocol development
2.1.1. Study protocol article (1st article)
2.1.2. Comet article (2nd article)
2.1.3. Validation of urine extraction protocol on comet assay
2.1.4. Validation of micronucleus assay on B[a]P using Hep G2 cells
2.1.5. Urinary caffeine metabolites and assessment of CYP1A2 activity
2.2. Epidemiological study
2.2.1. Details out of protocol article Study setting, administrative and ethical consideration Sample size Recruitment procedure and data collection
2.2.2. Details on the tests out of the article Blood samples Treatment of urine samples Extraction of organic fraction from urine samples Cellular line used (Hep G2) Chemicals and media for cell culture
2.2.3. Comet assay The solutions used Culture and treatment of cellular line used (Hep G2) Methodology Selection of comets and image analysis parameter used
2.2.4. Micronucleus assay The solutions used Methodology Selection and calculation of the parameters
2.2.5. Sewage system‟s indoor air sampling protocol Sampling and collection of pahs and VOCs Extraction and analysis of PAHs Extraction and analysis of VOCs
2.2.6. Measurement of 24h urinary 8-oxodG
2.2.7. Statistical analysis
3. Results
3.1. Exposure and genotoxicity article (3rd article)
3.2. Multivariate analysis of in vitro assays with the study groups
3.3. Population characteristics factors and 8-oxodG associations
3.4. Multivariate analysis of 8-oxodG with the study groups
3.5. Multivariate analysis of 8-oxodG with exposure duration
3.6. The associations between 8-oxodG and workplace air pollutants
3.7. The association between the two biomarkers of exposure
4. General discussion
4.1. The study‟s main results
4.2. The results of urinary caffeine metabolites and CYP1A2 activity
4.3. Cancer risk estimates and workplace sampling
4.4. The urinary biomarkers of exposure associations
4.5. The early effect biomarker (8-oxodG) associations
4.6. Choice of the cellular line (Hep G2)
4.7. The study sample size
4.8. Study questionnaires
4.9. Bias of the study
5. Conclusion and research perspectives
Sommaire (Français)
General references
Partnerships of the study
Funding of the study
Résumé :


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