Metal Contamination and Biomarkers in Cerastoderma glaucum: A Multi-level Approach.

In this study, we focused on evaluating the responses of the cockle, Cerastoderma glaucum to in situ exposures to metals at three sites in the Gulf of Gabes in the coastal zone of Tunisia differing in levels of metal contamination. Firstly, we examined the general physiological state of the organisms. Secondly, we evaluated the bioaccumulation of several metals (Cd, Cu, Zn, Ni) in the cockles. Thirdly, we focused on evaluating histologically changes in gametogenesis and sexual maturity of the organisms. Finally, we determined the expression of seven key genes encoding enzymes or proteins involved in responses to different types of environmental stressors. Results showed a decrease in the general physiological status of the cockles, including a reduced condition index, sex ratios skewed to females (70% and 80% females in the intermediate and the contaminated site, respectively) and greater mortalities in tests under anoxic conditions (i.e., stress on stress test) in cockles collected from the most contaminated site (LT50 = 2.88 days) compared to the cockles from the intermediate site (LT50 = 5 days) and the less contaminated site (LT50 = 6 days). Results for metal bioaccumulation showed that the levels of Cd, Cu, Zn and Ni in cockles were consistent with the contaminant gradient, with the highest levels in cockles from the most contaminated site (1.04; 4.92; 52.76 and 13.81 µg/g dw, respectively), followed by those from the intermediate site (0.34; 2.94; 36.94; 17.40 µg/g dw, respectively) and then the less contaminated site (0.065; 1.27; 21.62 and 5.40 µg/g dw, respectively). Results from the gametogenesis and maturity index showed few differences in the reproductive cycle of cockles collected from the three study sites. There were different patterns of gene expression that were divided into three groups in terms of responses: (1) expression of genes involved in metal detoxification, ATP Binding Cassette Subfamily B Member 1 (ABCB1) and metallothionein MT) and genes for superoxide dismutases (i.e., Mn SOD and CuZn SOD), which did not show any difference in their levels of expression; (2) heat shock protein 70 (HSP70) gene expression, which decreased in cockles according to the pollution gradient, and (3) expression of catalase (CAT) and cytochrome oxidase subunit 1 (COI) genes was threefold and 1000-fold higher in cockles from intermediate and most contaminated sites compared to the less contaminated site. Therefore, changes in overall physiological condition, sex ratios and expression of HSP70, CAT and COI genes may be appropriate biomarkers for in situ studies of the impacts of metals in cockles. However, these biomarkers should be coupled to proteomics studies.

Use of Tridacna maxima, a bivalve in the biomonitoring of the Saudi Arabian Red Sea coast.

The present study is an attempt to assess the effects of contamination of several sites in the Red Sea coasts of Saudi Arabia using bivalves as a biomonitoring tool. Oxidative stress biomarkers (including reduced glutathione level (GSH), glutathione-S-transferase activity (GST), Malondialdehyde level (MDA) and Catalase activity (CAT)), neurotoxicity acetylcholinesterase activity (ACHE), and genotoxicity micronucleus rate (MN) were measured in three distinct tissues - digestive glands, gills and mantle - of specimens of the giant clam Tridacna maxima, collected from five sites in Saudi Arabian Red Sea coast (Al-Khuraybah, Al-Wajh, Yanbu, Rabigh and Thuwal). Our results demonstrated that T. maxima showed differential biomarker responses according to the nature of pollutants and human activity that affect the coast. This study can be considered as the first one using biomarkers to assess the state of the Red Sea coast in Saudi Arabia which must be followed by periodic studies for surveillance of aquatic pollution.

Seasonal ecotoxicological monitoring of freshwater zooplankton in Bir Mcherga dam (Tunisia).

Dams represent large semi-closed reservoirs of pesticides and various organic and inorganic pollutants from agricultural and human activities, and their monitoring should receive special attention. This study evaluated the environmental health status of Bir Mcherga dam using zooplankton species. The dam has a capacity of 130 Mm3 and its waters are used for irrigation, water drinking supply, and fishery. Copepods and cladocerans (crustaceans) were collected in situ monthly between October and August 2012. Oxidative stress (CAT, MDA), neurotoxicity (AChE), and genotoxicity (micronucleus test) biomarkers were analyzed in two zooplankton species: Acanthocyclops robustus and Diaphanosoma mongolianum. High values of cells with a micronucleus were observed during summer. AChE activities were inhibited during early winter and summer. The high seasonal variability of CAT and MDA levels indicates that zooplankton is continuously exposed to different oxidative stresses. These results suggest that there is an obvious and continuous multi-faceted stress in Bir Mcherga reservoir and, consequently, an urgent monitoring of freshwater environments in Tunisia is needed, particularly those intended for human consumption and irrigation.

In situ genotoxicity assessment in freshwater zooplankton and sediments from different dams, ponds, and temporary rivers in Tunisia.

Tunisia water resources are limited. The country currently has 29 large dams, more than 1000 hill lakes, and 220 small dams which are essential for economic and social development given their contribution to irrigation, drinking water consumption, flooding protection, production of electrical energy, groundwater recharge, and industrial uses. Given the scarcity of these resources, it is crucial to be able to ensure the quality of freshwater environments, particularly those intended for human consumption. In this study, we meant to assess the health status of various freshwater ecosystems in different regions of Tunisia (north and center west) in order to detect genotoxic components in sediments and their potential effect on zooplankton (cladocerans). Sediment and cladoceran species were collected from dams, ponds, and temporary rivers in Tunisia. For each collection site, micronucleus (MN) assay was performed, in triplicates, using a pool of ten specimens of the same cladoceran species. MN occurrence in cladocerans varied from one site to another and MN frequencies varied between 0.67 and 22‰, suggesting the presence of genotoxic substances in certain sites. Sediment genotoxicity and mutagenicity were assessed using the SOS Chromotest and the Ames test. Sediment results showed that genotoxicity varies from one site to another displaying a quantitative and a qualitative variation of pollutant among the sites. These results suggest an urgent need for continuous monitoring of freshwater environments in Tunisia, particularly those intended for drinking water.

High estradiol exposure disrupts the reproductive cycle of the clam Ruditapes decussatus in a sex-specific way.

Bivalve species may be susceptible to environmental estrogenic compounds including estradiol (E2). However, they are able to biotransform the hormone quite readily and inactivate its estrogenic action. To study the long-term effects of elevated free E2 tissue levels, we transiently exceeded the biotransformation capacity of the clam Ruditapes decussatus by exposing them with high E2 concentrations (400 ng/L) and subsequently study the consequences on gametogenesis during the following reproductive cycle. Exposure to 400 ngE2/L led to a significant increase in tissue free E2 levels, which reached 10-50 ng E2Eq/gww. No deleterious effect on gonado-somatic index (GSI), condition index (CI), or ability to respond to the stress on stress test could be detected after a month of exposure, suggesting the absence of negative effects on the clam's health. However, a marked increase in gametogenesis could be observed in both sexes during the exposure. Subsequent transplantation of the clams in the field allowed the normal development of the male clams and maturation of the gonads without any detrimental effect observed after 4 months. In contrast, in early July, all female clams formerly exposed to E2 showed lower health status, and only ovaries with atretic oocytes while all control and indigenous females were normal and mature. These results show a sex-specific effect of high E2 exposure and suggest either a direct or indirect role for E2 in R. decussatus' reproduction.

Polymorphisms in XRCC1, ERCC2, and ERCC3 DNA repair genes, CYP1A1 xenobiotic metabolism gene, and tobacco are associated with bladder cancer susceptibility in Tunisian population.

Other than the established environmental risk factors associated with bladder cancer (BC), little is known about the genetic variations determining the individual susceptibility of this complex disease. This study aimed to investigate the relationship of BC with environmental agents and polymorphisms in XRCC1, ERCC2, and ERCC3 DNA repair genes and CYP1A1, CYP2D6, NAT1, and NAT2 xenobiotic metabolism genes through a hospital-based case-control study in Tunisia. The selection of the single nucleotide polymorphisms (SNPs) (rs25487, rs 13181, rs415407, rs446421, rs1058172, rs4921880, and rs1208) was performed using the dbSNP database. DNA genotyping was determined by PCR-RFLP after DNA extraction from whole blood. The risks of BC associated with every polymorphism as well as the studied environmental factors were estimated by multivariate-adjusted logistic regression using R software. In addition, gene-gene interactions were analyzed using generalized multifactor dimensionality reduction (GMDR) methods. Results showed that tobacco smoking and chewing parameters were significantly associated with BC risk. Single-gene variant analysis showed significant associations of the TT genotype of CYP1A1 and the rare GG genotype of ERCC2 with bladder cancer susceptibility (OR = 1.34, 95% CI 1.22-1.40, P < 0.0001). According to GMDR analysis, our findings indicated a significant association between BC and gene-gene interaction among the CYP1A1, ERCC3, and XRCC1. The present results suggest a potential role of XRCC1, ERCC2, ERCC3, and CYP1A1 besides tobacco intake in susceptibility to BC.

Cytotoxicity and genotoxicity effects of arsenic trioxide on SQ20B human laryngeal carcinoma cells.

This study investigates the cytotoxicity and the genotoxicity induced by arsenic trioxide As2O3in human laryngeal SQ20B carcinoma cell line. SQ20B cells were exposed to graded concentrations of arsenic trioxide (2 and 5µM) for 48h. Comet assay and γ-H2AX foci formation were used for measuring DNA damages, flow cytometry was used to identify cell cycle alterations and apoptosis, while cell morphology was visualized using transmission electron microscopy. The results show a dose-dependent induction of DNA damages and double strand breaks, alterations in cell cycle and morphologic alterations of cells. These results prove that As2O3 is highly cytotoxic and genotoxic at the micromolar range ina human laryngeal carcinoma cell line.

Responses of primary cultured haemocytes derived from the marine gastropod Haliotis tuberculata to an industrial effluent exposure.

This study assessed the responses of primary cultured haemocytes from the marine gastropod Haliotis tuberculata exposed to the increasing concentrations of industrial effluent (0, 0.5, 1, 10, 15 and 20%) discharged into the Tunisian coastal area. Analyses showed the presence of metals such as cadmium (Cd), chromium (Cr), copper (Cu), manganese (Mn), zinc (Zn), nickel (Ni) and lead (Pb) in the effluent. The effects of this mixture of pollutants on abalone haemocyte parameters were reflected by a significant decrease of cell viability, phagocytotic activity and reactive oxygen species (ROS) production as well as morphological and lysosomal membrane alterations. Thus, these results indicated that our primary culture system represents a suitable in vitro model for monitoring of anthropogenic contaminants in aquatic environments.

Gene-environment interactions between ERCC2, ERCC3, XRCC1 and cadmium exposure in nasal polyposis disease.

Gene-environment interactions have long been known to play an important role in complex disease aetiology, such as nasal polyposis (NP). The present study supports the concept that DNA repair gene polymorphisms play critical roles in modifying individual susceptibility to environmental diseases. In fact, we investigated the role of polymorphisms in DNA repair genes and cadmium as risk factors for Tunisian patients with NP. To the best of our knowledge, this is the first report on the impact of combined effects of cadmium and ERCC3 7122 A>G (rs4150407), ERCC2 Lys751Gln (rs13181) and XRCC1 Arg399Gln (rs25487) genes in the susceptibility to NP disease. Significant associations between the risk of developing NP disease and ERCC2 [odds ratio (OR) = 2.0, 95 % confidence interval (CI) = 1.1-3.7, p = 0.023] and ERCC3 (OR = 2.2, 95 % CI = 1.2-4.1, p = 0.013) genotypes polymorphisms were observed. Blood concentrations of Cd in NP patients (2.2 µg/L) were significantly higher than those of controls (0.5 µg/L). A significant interaction between ERCC3 (7122 A>G) polymorphism and blood-Cd levels (for the median of blood-Cd levels: OR = 3.8, 95 % CI = 1.3-10.8, p = 0.014 and for the 75th percentiles of blood-Cd levels: OR = 2.7, 95 % CI = 1.1-7.2, p = 0.041) was found in association with the risk of NP disease. In addition, when we stratified ERCC2, ERCC3 and XRCC1 polymorphism genotypes by the median and 75th percentiles of blood-Cd levels, we found also significant interactions between ERCC2 (Lys751Gln) and ERCC3 (7122 A>G) genotypes polymorphism and this metal in association with NP disease. However, no interaction was found between XRCC1 (Arg399Gln) polymorphism genotypes and Cd in association with NP disease.

Genotoxic effects of cadmium in human head and neck cell line SQ20B.

As cadmium may be involved in the etiology of head and neck cancers, we investigated in the present work, the cytotoxic and genotoxic effects of Cd on human larynx cells. SQ20B cells were exposed to 25 and 50 µM Cd for 48 and 72 h. Results showed a dose-dependent decrease in cell viability, especially after 48 h, associated with mitochondria alterations as showed by transmission electronic microscopy. Surprisingly, the flow cytometry shows that the cells treated with Cd have a normal proliferative cycle like the untreated cell especially in G1 or G2 phase of cell cycle. DNA damages were investigated by comet assay and immunofluorescence for gamma layer of the H2AX (g-H2AX) foci formation. Results show a strong induction of DNA double-strand breaks after Cd exposure. Overall, our results demonstrate the cytotoxicity and genotoxicity of Cd in human larynx cells and support the view that Cd could be an etiologic factor of head and neck cancers.

The cell cultures and the use of haemocytes from marine molluscs for ecotoxicology assessment.

Among aquatic organisms suitable for biological monitoring, molluscs occupy a prominent place due to their wide geographic distribution, their abundance and accessibility in the field as well as in aquaculture. Molluscs reflect the degree of environmental contamination and are the most useful bioindicator tools. The study of modulation of immune system or immunomodulation in marine molluscs has become one of the privileged ways for evaluating the physiological effects of environmental factors. Physiological responses of molluscs to environmental stresses could be mediated by haemocytes. These cells are continually exposed to the external environment due to the open circulatory system of molluscs and are affected by pollutants. In fact, several studies showed the effects of different environmental contaminants on haemocyte functions (viability, phagocytosis, ROS production) as well as on proteins involved in cytoskeletal structure maintenance using the in vitro approaches. In ecotoxicology, in vitro approach is an alternative to animal testing due to the reduced use of experimental animals, low cost and rapid performance. Although several studies showed the importance of using in vitro cell models to determine the effects of different environmental contaminants on haemocyte parameters in marine molluscs, a few reviews highlight these effects. The main purpose of this paper is to summarize the recent data on the effect of some xenobiotics on haemocyte parameters in some mollusc species and then suggest future research prospects.

The endocrine-disrupting effect and other physiological responses of municipal effluent on the clam Ruditapes decussatus.

In order to document the potential endocrine disrupting and toxic effect of the municipal wastewater effluents discharged into the Sfax coastal area (South of Tunisia), specimens of clam R. decussatus were collected from a reference site and were in vivo exposed to treated sewage effluent for 30 days. To this end, estrogenic and androgenic activities were measured in the gills to assess potential accumulation and regulation of active compounds. After effluent exposure androgenic activity in organic extracts increased up to fivefold compared to controls and remained elevated, while estrogenic activity was not significantly affected by exposure. As a consequence, remarkable disruptions in the gametogenesis activity, glycogen content, and Vitellogenin-like protein levels in male clams were observed. A parallel analysis of heavy metals in clam tissues was determined. A significant uptake of Ni, Zn, and Pb in soft tissues of exposed clams was observed. The significant increase of malondialdehyde (MDA) concentrations as a function of exposure time implies that clams have been exposed to an oxidative stress probably due to the presence of high metal concentrations in sewage effluent. Correlation analysis has revealed a statistically significant and positive relationship between MDA levels and metal concentrations in clams' tissues. The acetylcholinesterase activity was not significantly affected by exposure. Altogether, these results showed that a short-term exposure to a mixture of chemical compounds released by the Sfax wastewater treatment plant induce adverse physiological and reproductive effects in R. decussatus. Further studies are underway in order to evaluate its long-term impacts on aquatic wildlife in the gulf of Gabes area.

Association between blood arsenic levels and nasal polyposis disease risk in the Tunisian population.

Although the pathophysiology underlying nasal polyposis (NP) formation is not fully understood, systemic, local, and environmental factors appear to contribute to NP disease development. This study aimed to explore the relationship between metal blood levels and NP risk. To the best of our knowledge, the current research represents the first scientific contribution reporting levels of Cr and As in blood of NP patients. In this context, 90 NP patients and 171 controls were recruited and blood samples were analyzed to determine the concentrations of As and Cr. Metal blood levels of As in patients (2.1 µg/L) were significantly higher than those of controls (1.2 µg/L). However, no significant difference in blood Cr levels was found between cases and controls. Arsenic blood levels of cigarette smokers were significantly higher than those of non-smokers. Environmental exposure and shisha consumption presented the most significant association with NP disease (OR = 10.1 and 14.1, respectively). High levels of blood As were significantly associated with NP disease (OR = 2.1). Cr blood levels were found to be associated with the four stages of polyps in both nasal cavities. This study found a strong association between nasal polyposis disease and As blood levels. These findings merit further investigation.

The reproductive cycle, condition index, and glycogen reserves of the cockles Cerastoderma glaucum from the Gulf of Gabès (Tunisia).

The gametogenic cycle of the Cerastoderma glaucum was analyzed using both qualitative and semi-quantitative methods. The condition index and glycogen concentrations were determined in order to provide information on energy storage. The cockles were collected monthly from a Bayyadha site located 15 km south of Sfax City (Gulf of Gabès) between January 2007 and January 2008. From histological point of view, we applied two approaches: (i) the qualitative method describing the various stages of gamete development for males and females during a cycle of 13 months, and (ii) the semi-quantitative method concerning the estimation of different tissue surfaces. The results showed that there is evidence of three periods of reproduction in this population. A comparison between the surfaces occupied by the three organs showed that the foot and the gonad surfaces are higher than the surface of the adductor muscle. This could suggest that these two organs are more involved in the process of glycogen reserve storage. The results of the glycogen concentrations in the different tissues (gonad, adductor muscle, and "remainders") show that during the second and third periods of reproduction, glycogen was stored in the adductor muscle and in the remainder during sexual rest, and in the gonad during the gametogenesis phases in order to supply the reproductive effort. On the contrary, in the first period of reproduction, the low concentrations of glycogen recorded in the gonad coincided with its high degree of development. This fact could be related to environmental conditions (low temperature and food) recorded during this period.

Transcriptional response of stress-regulated genes to industrial effluent exposure in the cockle Cerastoderma glaucum.

This study assessed the responses of molecular biomarkers and heavy metal levels in Cerastoderma glaucum exposed for 1 week to two industrial effluents (1%) discharged into the Tunisian coastal area, F1 and F2, produced by different units of production of a phosphate treatment plant. A significant uptake of metals (Cd, Cu, Zn, and Ni) was observed in exposed cockles compared to controls, with an uptake higher for F1 than for F2. A decrease in LT50 (stress on stress test) was also observed after an exposure to the effluent F1. Treatments resulted in different patterns of messenger RNA (mRNA) expression of the different genes tested in this report. Gene transcription monitoring performed on seven genes potentially involved in the tolerance to metal exposure showed that for both exposures, mechanisms are rapidly and synchronically settled down to prevent damage to cellular components, by (1) handling and exporting out metal ions through the up-regulation of ATP-binding cassette xenobiotic transporter (ABCB1) and metallothionein (MT), (2) increasing the mRNA expression of antioxidant enzymes (catalase (CAT), superoxide dismutases, CuZnSOD and MnSOD), (3) protecting and/or repairing proteins through the expression of heat shock protein 70 (HSP70) mRNAs, and (4) increasing ATP production (through the up-regulation of cytochrome c oxidase 1 (CO1)) to provide energy for cells to tolerate stress exposure. The tools developed may be useful both for future control strategies and for the use of the cockle C. glaucum as a sentinel species.

Heavy metals in normal mucosa and nasal polyp tissues from Tunisian patients.

Despite growing evidence that bacteria, fungi, allergens, and superantigens play a prominent role in the pathophysiology of nasal polyps (NP), the exact cause of polyposis is still unknown. The etiology of NP is considered multifactorial. Until now, there is no information on the presence of heavy metals, such as cadmium (Cd), chromium (Cr), nickel (Ni), and arsenic (As) or of their role, in the pathogenesis of NP disease. In this study, concentrations of these four metals in tissue of NP were determined using Atomic Absorption Spectrometry. The Ni, Cr, and As levels in NP tissues were 2.1-, 3.2-, and 8.0-fold higher than those of normal mucosa (p < 0.05), respectively. A strong effect of cumulative smoking as expressed in the number of pack per year (PY), Ni, As, and Cd levels in NP tissue samples of patients ever-smokers (1-20 and >20 PY) are significantly higher than those of non-smokers (p = 0.006, 0.002, and < 0.001, respectively). The highest As concentrations among patients lived at polluted areas (1-25 and > 25 years) were observed in both nasal mucosa and NP tissues. The Ni and As in both nasal mucosa and NP tissues of patients occupationally exposed were significantly higher than non-exposed group. Cr and As levels were found to be associated with NP stage classification (p < 0.05). This is the first report to describe an association between concentrations of metals (Cr, As, and Ni) in human NP tissues and the risk of NP disease. Tissue metal levels have increased due to smoking, environmental, and occupational exposure. Therefore, heavy metal exposure may increase the risk of NP in the Tunisian population. The considerable risk in the category of highest cumulative exposure argues for an association between heavy metals exposure and nasal polyposis risk. Future investigations with larger samples should better elucidate this association.

Does a short-term exposure to cadmium chloride affects haemocyte parameters of the marine gastropod Haliotis tuberculata?

In this study, a model based on primary cultured haemocytes from the gastropod mollusc Haliotis tuberculata was established to investigate the effects of cadmium chloride in vitro. Cells were exposed for 24 h to CdCl2 concentrations of 0, 1 and 100 µg ml(-1). The effects of cadmium on haemocyte parameters were investigated using morphological, spectrophotometric and flow cytometry analysis. Results showed that cadmium has no significant effects on cell viability and phagocytotic activity under the tested conditions. However, haemocytes became more rounded after cadmium exposure, which could explain the significant decrease of cell area beginning at 1 µg ml(-1) of CdCl2.

Cadmium and nickel in blood of Tunisian population and risk of nasosinusal polyposis disease.

Nasosinusal polyposis (NSP) is a chronic inflammatory disease of the nasal mucosa. Although the pathophysiology underlying NSP formation is not fully understood, environmental factors appear to be contributed the development of this disease. A case-control study of Tunisian patients was examined to assess the levels of cadmium (Cd) and nickel (Ni) in blood and reparse the association between the exposure to these metals and the risk of nasosinusal polyposis disease. Mean blood levels of Cd in patients (2.2 ± 12.8 µg/L) were significantly higher than those of controls (0.5 ± 0.7 µg/L). Levels of blood Cd were positively correlated with tobacco smoking and chewing among controls. The Cd and Ni concentrations among control (p = 0.001) and patient (p = 0.018) tobacco consumers (smoking, chewing, and shisha) were significantly higher than those nonconsumers. Additionally, Ni blood levels of patient and control smokers were significantly higher than those of nonsmokers. Cd levels in blood samples of NSP patients occupationally exposed for more than 14 years were eight times higher than that of nonexposed. Drinking water was also found to be incriminated as exposure sources. Among risk factors, shisha consumption, environmental exposure, and occupational exposure presented the most significant association with NSP disease (odds ratio (OR) = 14.1, 10.1, and 1.7, respectively). High levels of blood Cd (OR = 3.5) were strongly associated with NSP disease (p = 0.027). Ni blood levels were shown to be associated with the four stages of polyps in both nasal cavities (right and left) (p < 0.05). This investigation suggested a potential role of toxic metals in the mechanism of NSP disease development. Exposure assessment investigations encompassing a wider population are needed.

Transcriptional response of stress-regulated genes to cadmium exposure in the cockle Cerastoderma glaucum from the gulf of Gabès area (Tunisia).

This study investigates cadmium effects on key messenger RNA (mRNA) expression (MT, MnSOD, CuZnSOD, CAT, ABCB1, HSP70, and CO1) by qPCR in the cockle Cerastoderma glaucum after chronic exposure to two high but environmentally relevant concentrations of CdCl2 (50 µg/L and 5 mg/L) for 12 h to 18 days. Cd accumulation measured in cockles' tissues is significantly higher in both treatment conditions compared to controls and in a dose-dependent manner. Stress on stress tests performed at different times of the experiment clearly demonstrated that exposure to both concentrations of Cd significantly affects cockle survival time in air. Important changes in gene transcription were also highlighted. In particular, MT, HSP70, CAT, and CuZnSOD seem to be relevant biomarkers of Cd exposure because (1) their mRNA levels increase upon exposure and (2) they are highly correlated to Cd accumulation in tissues. Results may be useful for control strategies and for the use of cockles as sentinel organisms.

Cytogenetic damage in the oral mucosa cells of bladder cancer patients exposed to tobacco in Southern Tunisia.

Bladder cancer was associated to exposure to several pollutants which can be absorbed, inhaled, or possibly ingested. We analyzed the frequency of micronuclei (MNC) and binucleated cells (BNC) in exfoliated cells of the oral mucosa of 24 bladder cancer (BC) patients and 48 controls residing in Southern Tunisia. An assessment was carried out on the incidence of MNC and BNC in 1,000 cells per individual. The data were analyzed with SPSS, using the chi-square and the Mann-Whitney U test, α = 0.05. The frequency of MN cells in BC cases was 2.5-fold higher, than in the control group (P < 0.001), while the difference for BNC between both groups was not significant. The smoking habits, age, and gender significantly influenced the MN but not the BNC alterations. The results of our study showed significantly increased frequencies of MN but not of BNC in exfoliated oral cells of BC patients associated with the smoking status, sex, and age. This study provides preliminary evidence that the frequency of MN in oral mucosa could be a predictive biomarker for cancers in parts of the body other than the upper aerodigestive tract, such as BC. Further scrupulous investigations are certainly warranted in order to implement this assay as a routine test in the planning and validation of cancer surveillance and prevention programs.