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Mitochondrial ability of shaping Ca(2+) signals has been demonstrated in a large number of cell types, but it is still debated in heart cells. Here, we take advantage of the molecular identification of the mitochondrial Ca(2+) uniporter (MCU) and of unique targeted Ca(2+) probes to directly address this issue. We demonstrate that, during spontaneous Ca(2+) pacing, Ca(2+) peaks on the outer mitochondrial membrane (OMM) are much greater than in the cytoplasm because of a large number of Ca(2+) hot spots generated on the OMM surface. Cytoplasmic Ca(2+) peaks are reduced or enhanced by MCU overexpression and siRNA silencing, respectively; the opposite occurs within the mitochondrial matrix. Accordingly, the extent of contraction is reduced by overexpression of MCU and augmented by its down-regulation. Modulation of MCU levels does not affect the ATP content of the cardiomyocytes. Thus, in neonatal cardiac myocytes, mitochondria significantly contribute to buffering the amplitude of systolic Ca(2+) rises.

Mitochondria from every tissue are quite similar in their capability to accumulate Ca²⁺ in a process that depends on the electrical potential across the inner membrane; it is catalyzed by a gated channel (named mitochondrial Ca²⁺ uniporter), the molecular identity of which has only recently been unraveled. The release of accumulated Ca²⁺ in mitochondria from different tissues is, on the contrary, quite variable, both in terms of speed and mechanism: a Na⁺-dependent efflux in excitable cells (catalyzed by NCLX) and a H⁺/Ca²⁺ exchanger in other cells. The efficacy of mitochondrial Ca²⁺ uptake in living cells is strictly dependent on the topological arrangement of the organelles with respect to the source of Ca²⁺ flowing into the cytoplasm, i.e., plasma membrane or intracellular channels. In turn, the structural and functional relationships between mitochondria and other cellular membranes are dictated by the specific architecture of different cells. Mitochondria not only modulate the amplitude and the kinetics of local and bulk cytoplasmic Ca²⁺ changes but also depend on the Ca²⁺ signal for their own functionality, in particular for their capacity to produce ATP. In this review, we summarize the processes involved in mitochondrial Ca²⁺ handling and its integration in cell physiology, highlighting the main common characteristics as well as key differences, in different tissues.

Here we describe use of a mitochondrial targeted Cameleon to produce stably transformed Arabidopsis plants that enable analyses of mitochondrial Ca²⁺ dynamics in planta and allow monitoring of the intra-mitochondrial Ca²⁺ concentration in response to physiological or environmental stimuli. Transgenic plants co-expressing nuclear and mitochondrial targeted Cameleons were also generated and analyzed. Here we show that mitochondrial Ca²⁺ accumulation is strictly related to the intensity of the cytoplasmic Ca²⁺ increase, demonstrating a tight association between mitochondrial and cytoplasmic Ca²⁺ dynamics. However, under all experimental conditions, mitochondrial Ca²⁺ dynamics were substantially different from those monitored in the cytoplasm, demonstrating that mitochondria do not passively sense cytosolic Ca²⁺, but actively modulate the intra-mitochondrial level of the cation. In particular, our analyses show that the kinetics of Ca²⁺ release from mitochondria are much slower than in the cytoplasm and nucleus. The mechanisms and functional implications of these differences are discussed., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

To study the effect of eating foods with a high arsenic (As) content on the intra and inter-individual variability of urinary concentrations of the As species, daily urine samples were collected for 10 consecutive days from 12 healthy male subjects. A daily food diary was kept throughout the study period. Personal exposure to airborne As was measured once during the study. As(3), As(5), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and arsenobetaine were determined in all urine samples by inductive coupled plasma mass spectrometry, and the sum of As(3)+As(5)+MMA+DMA (iAs) by hydride generation-atomic absorption spectrophotometry. Exposure to airborne As was below the limit of detection in all samplings. As(3) was found in only 19.2% and As(5) in only 3.3% of the urine samples, whereas high urinary concentrations of arsenobetaine were observed. With the exception of arsenobetaine, expressed as a percentage, a significant inter-individual variability was observed for all species of As, for iAs and for the MMA/DMA ratio (p<0.001). Instead, the intra-individual variability was significant only for the MMA/DMA ratio (p<0.001). Among foods with a high As content, only a heavy consumption of seafood was shown to influence inter-individual variability of DMA%, arsenobetaine expressed as μg g(-1) creatinine and iAs. In conclusion, even in populations with a high intake of organic As through foods, the finding of a significant inter-individual but no significant intra-individual variability of urinary species confirms the usefulness of urinary As speciation for biological monitoring of exposure to As., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The urinary excretion of t,t-muconic acid (t,t-MA), S-phenylmercapturic acid (SPMA) and urinary benzene and the influence of a smoking habit and of exposure to urban traffic on the urinary excretion of these biomarkers were investigated in 137 male adults from the general population. All subjects were not occupationally exposed to benzene and resident in two cities in Puglia (Southern-Italy). Environmental exposure to benzene was measured using passive personal samplers. The biomarkers t,t-MA, SPMA and urinary benzene were determined in urine samples collected from each subject at the end of the environmental sampling. The percentage of cases above the limit of detection was higher for SPMA and urinary benzene in smokers than in non-smokers, and for airborne benzene and urinary benzene in subjects exposed to urban traffic. Airborne benzene was correlated with the time spent in urban traffic during the environmental sampling. Among the biomarkers, urinary benzene was found to be correlated with airborne benzene only in non-smokers, and with the time spent in urban traffic, both in smokers and non-smokers considered together, and in non-smokers only. Finally, multiple regression analysis showed that the urinary excretion of all the biomarkers was dependent on the number of cigarettes smoked per day and, for urinary benzene, also on the time spent in urban traffic. In conclusion, urinary benzene seems to be a more valid biomarker than t,t-MA and SPMA to assess environmental exposure to extremely low concentrations of benzene. Cigarette smoking prevailed over traffic exhaust fumes in determining the internal dose of benzene.

Mitochondrial Ca(2+) uptake and release play a fundamental role in the control of different physiological processes, such as cytoplasmic Ca(2+) signalling, ATP production and hormone metabolism, while dysregulation of mitochondrial Ca(2+) handling triggers the cascade of events that lead to cell death. The basic mechanisms of mitochondrial Ca(2+) homeostasis have been firmly established for decades, but the molecular identities of the channels and transporters responsible for Ca(2+) uptake and release have remained mysterious until very recently. Here, we briefly review the main findings that have led to our present understanding of mitochondrial Ca(2+) homeostasis and its integration in cell physiology. We will then discuss the recent work that has unravelled the biochemical identity of three key molecules: NCLX, the mitochondrial Na(+)/Ca(2+) antiporter, MCU, the pore-forming subunit of the mitochondrial Ca(2+) uptake channel, and MICU1, one of its regulatory subunits.

Aim: To study the validity of urinary benzene as a biomarker of low and very low exposure to this toxicant, as compared with t,t-muconic acid (t,t-MA) and S-phenylmercapturic acid (SPMA), also taking into account the influence of cigarette smoking and co-exposure to toluene on the urinary excretion of benzene., Materials and Methods: The results obtained in two different studies were compared: in the first, workers occupationally exposed to low concentrations of benzene (18 fuel tanker drivers and 23 filling station attendants) were compared with 31 controls and in the second, workers exposed to very low concentrations of benzene (the same 23 filling station attendants) were compared with the 31 controls. Exposure to airborne benzene and toluene was monitored with passive personal samplers (Radiello). Then the urine collected at the end of the work shift was analyzed for t,t-MA, SPMA and urinary benzene. All participants also filled out a questionnaire about their lifestyle habits., Results: There were no differences among the three groups in terms of age and smoking habit. Occupational exposure to benzene and toluene and the urinary concentrations of t,t-MA, SPMA and urinary benzene were higher in the fuel tanker drivers than the filling station attendants and higher in the latter than in the controls. Cigarette smoking was found to be associated with urinary excretion of t,t-MA, SPMA and urinary benzene at both low and very low exposure to benzene. The biomarkers t,t-MA, SPMA and urinary benzene were almost always correlated, for both low and very low exposure to benzene. Notably, for low exposure to benzene a dependency relation was found with the levels of t,t-MA, SPMA and urinary benzene on both cigarette smoking and airborne benzene, whereas for very low exposure to benzene there was a dependency relation of SPMA on cigarette smoking and airborne benzene, of urinary benzene only on cigarette smoking and of t,t-MA on none of the variables considered., Conclusions: For occupational exposure to low concentrations of benzene, urinary benzene and SPMA showed a comparable validity, while for exposure to very low concentrations of this toxicant the validity of SPMA was confirmed while urinary benzene was found to be less useful. Cigarette smoking was the main factor conditioning the excretion of all the biomarkers of benzene in conditions of both low and very low exposure to the toxicant, so for the analysis of occupational exposure to benzene it is best to recommend abstention from smoking at least in the hours immediately before urine collection.

Objective: To verify whether urinary benzene is an applicable biomarker of occupational exposure to very low concentrations of benzene, considering the influence of cigarette smoke and benzene-toluene co-exposure., Materials and Methods: 23 filling station attendants with occupational exposure to benzene and 31 controls were analyzed. Occupational and environmental exposure was monitored and t,t-muconic acid (t,t-MA), S-phenylmercapturic acid (SPMA), urinary benzene and creatinine in the urine samples were tested., Results: Occupational exposure to benzene and toluene was significantly higher in the filling station attendants than in the controls, whereas t,t-MA, SPMA and urinary benzene were not different in the two groups. Instead, the smoker group showed significantly higher values for the above biomarkers than the non-smoker group, each of which included both exposed workers and controls. SPMA was dependent on airborne benzene and cigarette smoking, and urinary benzene only on cigarette smoking, while t,t-MA was not dependent on either of these variables., Conclusions: At very low concentrations of occupational exposure to benzene, urinary benzene is less valid than SPMA as a biomarker, even if both are strongly influenced by smoking habit. Abstention from smoking should therefore be recommended for at least two hours before urine collection.

Calcium handling by mitochondria is a key feature in cell life. It is involved in energy production for cell activity, in buffering and shaping cytosolic calcium rises and also in determining cell fate by triggering or preventing apoptosis. Both mitochondria and the mechanisms involved in the control of calcium homeostasis have been extensively studied, but they still provide researchers with long-standing or even new challenges. Technical improvements in the tools employed for the investigation of calcium dynamics have been-and are still-opening new perspectives in this field, and more prominently for mitochondria. In this review we present a state-of-the-art toolkit for calcium measurements, with major emphasis on the advantages of genetically encoded indicators. These indicators can be efficiently and selectively targeted to specific cellular sub-compartments, allowing previously unavailable high-definition calcium dynamic studies. We also summarize the main features of cellular and, in more detail, mitochondrial calcium handling, especially focusing on the latest breakthroughs in the field, such as the recent direct characterization of the calcium microdomains that occur on the mitochondrial surface upon cellular stimulation. Additionally, we provide a major example of the key role played by calcium in patho-physiology by briefly describing the extensively reported-albeit highly controversial-alterations of calcium homeostasis in Alzheimer's disease, casting lights on the possible alterations in mitochondrial calcium handling in this pathology., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Oxidative stress is a major challenge for all cells living in an oxygen-based world. Among reactive oxygen species, H2O2, is a well known toxic molecule and, nowadays, considered a specific component of several signalling pathways. In order to gain insight into the roles played by H2O2 in plant cells, it is necessary to have a reliable, specific and non-invasive methodology for its in vivo detection. Hence, the genetically encoded H2O2 sensor HyPer was expressed in plant cells in different subcellular compartments such as cytoplasm and peroxisomes. Moreover, with the use of the new green fluorescent protein (GFP)-based Cameleon Ca2+ indicator, D3cpv-KVK-SKL, targeted to peroxisomes, we demonstrated that the induction of cytoplasmic Ca2+ increase is followed by Ca2+ rise in the peroxisomal lumen. The analyses of HyPer fluorescence ratios were performed in leaf peroxisomes of tobacco and pre- and post-bolting Arabidopsis plants. These analyses allowed us to demonstrate that an intraperoxisomal Ca2+ rise in vivo stimulates catalase activity, increasing peroxisomal H2O2 scavenging efficiency.

Although it is widely accepted that mitochondria in living cells can efficiently uptake Ca(2+) during stimulation because of their vicinity to microdomains of high [Ca(2+)], the direct proof of Ca(2+) hot spots' existence is still lacking. Thanks to a GFP-based Ca(2+) probe localized on the cytosolic surface of the outer mitochondrial membrane, we demonstrate that, upon Ca(2+) mobilization, the [Ca(2+)] in small regions of the mitochondrial surface reaches levels 5- to 10-fold higher than in the bulk cytosol. We also show that the [Ca(2+)] to which mitochondria are exposed during capacitative Ca(2+) influx is similar between near plasma membrane mitochondria and organelles deeply located in the cytoplasm, whereas it is 2- to 3-fold higher in subplasma membrane mitochondria upon activation of voltage-gated Ca(2+) channels. These results demonstrate that mitochondria are exposed to Ca(2+) hot spots close to the ER but are excluded from the regions where capacitative Ca(2+) influx occurs., (Copyright 2010 Elsevier Inc. All rights reserved.)

Objectives: This study analyzes the validity of new, more sensitive and specific urinary biomarkers of internal dose, namely, urinary benzene for benzene and urinary toluene and S-benzylmercapturic acid (SBMA) for toluene, to assess their efficacy when compared to traditional biomarkers for biological monitoring of occupational exposure to low concentrations of these two toxic substances., Methods: Assessment was made of 41 workers occupationally exposed to benzene and toluene, 18 fuel tanker drivers and 23 filling-station attendants, as well as 31 subjects with no occupational exposure to these toxic substances (controls). Exposure to airborne benzene and toluene was measured using passive Radiello personal samplers worn throughout the work shift. In urine samples collected from all subjects at the end of the workday, both the traditional and the new internal dose biomarkers of benzene and toluene were assessed, as well as creatinine so as to apply suitable adjustments., Results: Occupational exposure to benzene and toluene resulted significantly higher in the fuel tanker drivers than the filling-station attendants, and higher in the latter than in controls. Significantly higher concentrations of t,t-muconic acid (t,t-MA), S-phenylmercapturic acid (SPMA), urinary benzene, SBMA and urinary toluene were found in the drivers than the filling-station attendants or the controls. Instead, urinary phenol and hippuric acid were not different in the three groups. In the entire sample, airborne benzene and toluene values were significantly correlated, as were the respective urinary biomarkers, showing coefficients ranging from 0.36 to 0.98. Subdividing the subjects by smoking habit, higher coefficients were evident in non-smokers than in smokers; at multiple regression analysis t,t-MA, SPMA and urinary benzene and toluene were dependent on the number of cigarettes smoked daily and on airborne benzene and toluene, respectively. Instead, SBMA was dependent only on airborne toluene., Conclusions: Our research confirmed the validity of t,t-MA and SPMA for use in the biological monitoring of exposure to low concentrations of benzene. Urinary benzene showed comparable validity to SPMA; both parameters are affected by smoking cigarettes in the hours before urine collection, so it is best to ask subjects to refrain from smoking for 2 h before urine collection. Urinary toluene was found to be a more specific biomarker than SBMA.

Indoor and outdoor formaldehyde and acetaldehyde levels were assessed to characterize pollution in dwellings in the city and the Province of Bari, also taking into account seasonal variability, and to investigate health effects of aldehyde exposure on the general population. In 2007, passive environmental monitoring was performed, for 24 hours, in the kitchen of 59 dwellings, as well as outdoors for 27 of them. A questionnaire probing personal and home characteristics was administered to all 182 subjects habitually resident in the homes. During the period January-June 2008, formaldehyde and acetaldehyde levels were monitored monthly in 20 of the investigated dwellings inhabited only by non smokers. Indoor formaldehyde and acetaldehyde concentrations were significantly higher (16.0 +/- 8.0 and 10.7 +/- 8.8 microg m(-3)) than outdoor concentrations (4.4 +/- 1.7 and 3.4 +/- 2.0 microg m(-3)), showing a correlation between indoor levels of the two aldehydes (r = 0.41; p = 0.001). In dwellings inhabited only by non smokers, formaldehyde concentrations were higher in the presence of furniture bought new or restored less than one year before (p = 0.03). Formaldehyde and acetaldehyde levels were significantly higher in winter months than in spring-summer months (F = 2.86, p = 0.02; F = 5.39, p < 0.001) and seemed to be influenced by the time that kitchen windows were kept open. As regards the effects on human health, a low prevalence of allergic disease and no association between any irritant or allergic complaints and indoor levels of the two aldehydes was observed. In conclusion, the results showed low indoor and outdoor concentrations of formaldehyde and acetaldehyde, not generally posing a risk for human health.

In order to characterize the different sources of exposure to arsenic (As), urinary excretion of total As, the sum of inorganic As+MMA+DMA determined by the hydride generation-atomic absorption spectrophotometry technique, and the species As3, As5, monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and arsenobetaine were determined in 49 workers at a steel foundry, with presumed occupational exposure to As, and 50 subjects from the general population, all males. No evidence of occupational exposure to As resulted from environmental monitoring performed in the foundry, although the analysis of minerals used as raw materials showed the presence of As, particularly in fossils and fine ores. The urinary concentrations of As3, MMA, DMA, the sum of inorganic As+MMA+DMA and total As were not different in the two groups, while arsenobetaine appeared significantly higher in the controls than in the workers. The different species of urinary As were all significantly correlated. Urinary excretion of As3 was associated with the consumption of mineral water and with residence in an industrial zone, while MMA, DMA, arsenobetaine, the sum of inorganic As+MMA+DMA and total As urinary excretion were associated with the consumption of crustaceans and/or shellfish 3 days or less before urine collection. Multiple regression analysis confirmed these results. In conclusion, in populations with a high consumption of seafood, living in areas characterized by coastal/marine As pollution, only speciation of As can identify a prevalent role of environmental sources, like the consumption of seafood contaminated by As, in determining urinary As excretion, and exclude an occupational origin of the exposure.

Obstructive Sleep Apnea Syndrome (OSAS) and Excessive Daytime Sleepiness (EDS) are sleep disorders which can increase cardiovascular risk. An health survey was performed on the cement workers to estimate the prevalence of sleep disorders and to investigate occupational, personal and health risk factors that could influence it. A total of 761 male workers, employed at 10 different cement plants of South Italy and Sicily, were examined. All subjects gave informed consent to take part in the survey. The following questionnaires were administered: Berlin Questionnaire to estimate the high risk of OSAS, Epworth Sleepiness Scale for EDS, a questionnaire posing questions about working conditions, personal characteristic, lifestyle, past history of disease and present illness. Statistical analysis was performed with the statistical package SPSS. The prevalence of high risk of OSAS and of EDS resulted respectively in 24.2% and 3.4% of workers. Sleep disorders detected with the two questionnaires were significantly associated. A positive and significant association between OSAS and respectively age, time of employment, BMI, ex-smoker status, neck, waist or hip circumferences, chronic fatigue and arterial hypertension was observed. Subjective variables regarding working conditions (job interest, evaluation oforganization of work and job satisfaction) and alcohol consumption were not associated with the high risk of OSAS. Shift work (2 and 3 shifts) was not associated with the high risk of OSAS. An healthy worker effect was observed for workers who changed from shift work (2 or 3 shifts) to fixed daytime work. For them, this change to fixed daytime work was conditioned by chronic disease like hypertension and obesity. EDS was not dependent, associated or correlated with any of the occupational, personal or pathologic variables investigated in the study. In conclusion the research showed no relationship between working conditions, particularly shift work, and the high risk of OSAS, and the influence of obesity in determining the high risk of OSAS, itself a potential cardiovascular risk factor. The interest of occupational physician has been focused on introducing in health surveillance also measures of health promotion regarding sleep disorders with the aim of preserving health condition in workers.

We here describe the generation of novel, green fluorescent protein-based Ca(2+) indicators targeted to the peroxisome lumen. We show that (i) the Ca(2+) concentration of peroxisomes in living cells at rest is similar to that of the cytosol; (ii) increases in cytosolic Ca(2+) concentration (elicited by either Ca(2+) mobilization from stores or Ca(2+) influx through plasma membrane Ca(2+) channels) are followed by a slow rise in intraperoxisomal [Ca(2+)]; (iii) Ca(2+) influx into peroxisomes is driven neither by an ATP-dependent pump nor by membrane potential nor by a H(+)(Na(+)) gradient. The peroxisomal membrane appears to play a low pass filter role, preventing the organelle from taking up shortlasting cytosolic Ca(2+) transients but allowing equilibration of the peroxisomal luminal [Ca(2+)] with that of the cytosol during prolonged Ca(2+) increases. Thus, peroxisomes appear to be an additional cytosolic Ca(2+) buffer, but their influx and efflux mechanisms are unlike those of any other cellular organelle.

Creatinine adjustment has been used to remove the influence of the effect of urine dilution on exposure biomarkers measured in spot samples. This research aimed to determine the reliability of creatinine adjustment for urinary chromium and arsenic in subjects from general population considering interferences able to influence creatinine excretion. 444 male subjects were examined and each participant was administered an anamnestic questionnaire. Chromium and arsenic were determined on second morning void urine samples by atomic absorption spectrophotometry and creatinine by Jaffè method. Urinary creatinine showed a significant negative decrease with age increasing (p < 0.001). Chromium concentrations resulted higher in subjects with renal disease, particularly after creatinine adjustment (p = 0.014). Age was negatively correlated with urinary creatinine (rho = -0.23; p < 0.001), and positively with chromium (rho = 0.13; p = 0.007) and arsenic (rho = 0.17; p = 0.004), only after creatinine adjustment. A positive correlation was also found between unadjusted chromium and arsenic (rho = 0.10; p = 0.039) and between urinary creatinine and both unadjusted chromium (rho = 0.32; p < 0.001) and arsenic (rho = 0.18; p < 0.001). An high coefficient of correlation was observed between unadjusted chromium (rho = 0.88; p < 0.001) and arsenic (rho = 0.90; p < 0.001) and the respective adjusted values. Multiple regression showed a dependence of urinary creatinine and adjusted chromium and arsenic concentrations on age. In conclusion, although chromium and arsenic seem to have a renal kinetics of excretion comparable to the creatinine one, the influence of age on creatinine elimination suggests that the expression of urinary values of these metallic elements as microg/L is more reliable.

Mitochondrial Ca(2+) homeostasis is today at the center of wide interest in the scientific community because of its role both in the modulation of numerous physiological responses and because of its involvement in cell death. In this review, we briefly summarize a few basic features of mitochondrial Ca(2+) handling in vitro and within living cells, and its involvement in the modulation of Ca(2+)-dependent signaling. We then discuss the role of mitochondrial Ca(2+) in the control of apoptotic death, focusing in particular on the effects of pro- and anti-apoptotic proteins of the Bcl-2 family. Finally, the potential involvement of Ca(2+) and mitochondria in the development of two diseases, Ullrich muscular dystrophy and familial Alzheimer's disease, is briefly discussed.

The influence of cigarette smoking on concentrations of urinary benzene, a sensitive and specific biomarker proposed for biological monitoring of exposure to very low doses of benzene, was investigated in 24 filling-station attendants and 31 workers non occupationally exposed to benzene. Environmental monitoring was performed by personal passive samplers "Radiello", and a spot urine sample was collected at the end of the work shift, from all subjects, for the determination of urinary benzene. Exposure to benzene resulted significantly higher in filling-station attendants (mean 23.3 +/- 17.0 microg/m3; range 4.5-66.3 microg/m3) than in controls (mean 4.6 +/- 2.6 microg/m3; range < 3-11.5 microg/m3), while concentrations of urinary benzene did not show any significant difference between the two groups. Considering all subjects as a single group, it was observed that urinary benzene concentrations were positively correlated with the number of cigarettes smoked during the sampling time (rho = 0.38; p = 0.047) and with airborne benzene levels (rho = 0.32; p = 0.019), and negatively correlated with the time elapsed between the last smoked cigarette and urine collecting (rho = -0.40; p = 0.045). Multiple regression analysis confirmed the influence of cigarette smoking on urinary benzene concentrations. In conclusion, our study showed the validity of urinary benzene as a biomarker for biological monitoring of exposure to very low doses of benzene, although cigarette smoking determined a prevalent etiological role at the low environmental benzene concentrations observed.

Introduction: Urinary inorganic arsenic is an expression of occupational exposure to the metal, provided that there is no history of ingestion of foods containing high concentrations of inorganic and/or organic arsenic. The present study was conducted to assess the contribution of professional and environmental exposure to inorganic arsenic on urinary excretion of the metal., Materials and Methods: We examined 195 workers at a steelfoundry in Taranto, exposed to very low concentrations of inorganic arsenic and two control groups consisting of 105 subjects resident near the factory and 144 subjects resident approximately 20 Km away. All participants were administered a questionnaire enquiring about general characteristics, lifestyle, occupational and extra occupational exposure to arsenic. Urinary arsenic was determined by atomic-absorption spectrophotometry., Results: Exposed and non exposed subjects were similar as regards general characteristics and lifestyle. The environmental concentration of arsenic for the foundryworkers was invariably lower than 0.1 microg/m3. Urinary excretion of arsenic was higher in the subjects in all three groups, examined singly and together, if they had eaten crustaceans and/or shellfish in the three days before urine collection. There was a significant positive correlation with the consumption of shellfish and a significant negative correlation with the number of days since the last crustacean/shellfish meal. Multiple regression analysis showed a dependence of urinary elimination of arsenic on the days since the last crustacean/shellfish meal., Discussion: The absence of occupational exposure to arsenic allowed us to attribute the higher urinary elimination of arsenic to ingestion of crustaceans and/or shellfish in the three days before collection of the urine, both in subjects exposed to inorganic arsenic and in the two groups belonging to the general population. Our results support the hypothesis that inorganic arsenic, determined by atomic-absorption spectrophotometry, may derive from the catabolism of organic arsenic compounds contained in crustaceans and/or shellfish included in the diet.

The results of a polycentric study to assess the reference values of urinary mercury (U-Hg) in four Italian cities are presented. A total of 383 subjects were selected on the basis of standardised criteria by a questionnaire on personal habits, lifestyle, occupational or non-occupational exposure to Hg, medical history, number and area of dental amalgams. U-Hg was determined by hydride generation atomic absorption method (HG-AAS), with a detection limit of 0.5 microg/l and by flow injection (FI) inductively coupled plasma mass spectrometry (ICP-MS), with a detection limit of 0.03 microg/l. The median value of U-Hg, determined by HG-AAS, was 0.78 microg/g creatinine (0.75 for males and 0.83 for females), with 5 degrees and 95 degrees percentiles, respectively, of 0.17 and 3.66 microg/g creatinine. When determined by FI ICP-MS, the median value was 0.79 microg/g creatinine (0.77 for males and 0.79 for females) with 5 degrees and 95 degrees percentiles of, respectively, 0.12 and 5.02 microg/g creatinine. Among the independent variables, city of origin, area of dental amalgams, fish intake and tobacco smoking significantly influenced the U-Hg levels. The U-Hg reference values from this survey are lower than those from other recent investigations, probably due to characteristics and selection of the examined individuals and to the strict control of pre-analytical and analytical factors of variability.

Our previous research (Soleo L, Vacca A, Vimercati L et al. Occup Environ Med 1997;54:437-442) showed a reduction in tumor necrosis factor-alpha (TNF-alpha) serum levels in workers with prolonged exposure to low doses of inorganic mercury, suggesting an in vivo functional defect of the monocyte-macrophage system. On this basis, here we wondered whether workers exposed to lower doses of metallic mercury displayed possible changes in the monocyte-macrophage system. In this particular cohort of workers, we also sought for the effects of the exposure on the polymorphonuclear leukocytes (PMNL) chemotaxis. The monocyte-macrophage system and the natural killer (NK) cells were examined in 19 exposed workers and in 25 unexposed workers, as the control group (controls). Specifically, the circulating monocyte-macrophage cells and their CD13, CD15 and CD33 subsets, serum cytokines (IL-8, GM-CSF and TNF-alpha) and the NK cells were analyzed. In seven exposed and seven controls randomly chosen workers the PMNL chemotaxis was also assessed. The selected indicator of mercury exposure were the levels of mercury in the urine (U-Hg), that was significantly higher in exposed workers than the controls (9.7 +/- 5.5 microg/l and 2.4 +/- 1.2 microg/l, respectively). None of the exposed workers had shown signs of either acute or chronic inorganic mercury toxicity or any form of hypersensitivity. Several immunological variables tested, monocyte-macrophage cells and their subsets, NK cells and serum cytokines overlapped between the exposed and the control workers. When the workers were considered as a whole (exposed plus controls), no correlation was found between current U-Hg and all immunological parameters. However, when exposed workers were studied separately, an inverse correlation was disclosed between cumulative U-Hg and cells (as percentage) expressing the CD13 (r = -0.599; P = 0.007) and CD15 (r = -0.614; P = 0.005) molecules, and NK cells (r = -0.455; P = 0.05). Moreover, a significant impairment in the PMNL chemotaxis (t = 3.70; P = 0.003) was observed in the exposed workers. The results of our study suggest that the exposure to very low levels of metallic mercury led to subtle impairment of circulating monocyte and NK cells (as percentages) according to the increase in U-Hg levels, as well as of the PMNL chemotactic function in this particular group of workers, even though they remain clinically asymptomatic. Therefore, we suggest that impairment of these parameters provide a sensitive indicator of metallic mercury and other chemical contaminants present in the environment.

The aim of the research was to assess the contribution of dental amalgams and other non-occupational factors of exposure to inorganic mercury (diet, etc.) to the quantity of mercury excreted with urine in workers exposed to low level concentrations of inorganic mercury. Two groups of workers (Groups I and II) were studied who were exposed to low and different environmental concentrations of inorganic mercury. These two groups were compared with a group of subjects not occupationally exposed to mercury in the same geographical area (Group III). All subjects were administered a questionnaire concerning personal data, lifestyle, recent removal and/or insertion of dental amalgam fillings, presence of nasal obstruction or bruxism and consumption of fish. The number of amalgam-filled teeth was established for each subject. Mean environmental exposure to inorganic mercury was 0.0087 mg/m3 for Group I and 0.0030 mg/m3 for Group II. Urinary excretion in the 3 groups was 4.2 +/- 2.8 micrograms/l for Group I, 3.0 +/- 2.1 micrograms/l for Group II and 1.6 +/- 1.2 micrograms/l for Group III. The results showed that of the factors of exposure to inorganic mercury, only occupational exposure (T = 9.18; p = 0.000) and the number of amalgam-filled teeth (T = 2.03; p = 0.043) were able to influence significantly urinary excretion of mercury; the sources of non-occupational exposure did not appear to play any role. The contribution of each amalgam filling to urinary mercury excretion was calculated to be 0.08 microgram/l. Occupational exposure therefore, even at low level doses, is still the main cause of urinary mercury excretion in workers exposed to inorganic mercury; of the non-occupational exposure factors, a significant role is played by amalgam dental fillings, whose contribution needs to be taken into consideration in order to make a correct interpretation of the results of biological monitoring of exposed workers.

The purpose of this study was to assess the role of dental amalgams and diet upon urinary mercury (U-Hg) excretion. 98 subjects (50 men and 48 women) not exposed to inorganic mercury, for either occupational or environmental reasons, and living in coastal and inland districts of Apulia (Southern Italy) were considered. All the subjects were administered a questionnaire with questions concerning life style, medical history, and occupational activity. Dental amalgams were evaluated with respect to their number and their surface areas. Urinary mercury was measured by the cold vapour atomic absorption technique. Expressed in terms of arithmetic mean, U-Hg excretion was found to amount to 1.03 micrograms/g creatinine (5th and 95th percentile: 0.31 and 2.40; range 0.30-3.25). Multiple linear regression analysis showed that, of the several tested independent variables (dental amalgams, age, body mass index, consumption of tuna, bass, swordfish, etc.), only the number of amalgam fillings (T = 5.25; p = 0.025) and the number of restored surfaces (T = 2.33; p = 0.020) were found liable to affect urinary mercury excretion in a significant manner. In conclusion, the results of this study confirm the primary role of amalgam fillings in affecting urinary mercury excretion in those subjects who are not occupationally exposed to inorganic mercury, The resulting urinary mercury levels can no doubt be taken as the reference values for the population of Apulia.