Pietro Apostoli

Inhalable metal-rich air particles and histone H3K4 dimethylation and H3K9 acetylation in a cross-sectional study of steel workers.

Background: Epidemiology investigations have linked exposure to ambient and occupational air particulate matter (PM) with increased risk of lung cancer. PM contains carcinogenic and toxic metals, including arsenic and nickel, which have been shown in in vitro studies to induce histone modifications that activate gene expression by inducing open-chromatin states. Whether inhalation of metal components of PM induces histone modifications in human subjects is undetermined. Objectives: We investigated whether the metal components of PM determined activating histone modifications in 63 steel workers with well-characterized exposure to metal-rich PM. Methods: We determined histone 3 lysine 4 dimethylation (H3K4me2) and histone 3 lysine 9 acetylation (H3K9ac) on histones from blood leukocytes. Exposure to inhalable metal components (aluminum, manganese, nickel, zinc, arsenic, lead, iron) and to total PM was estimated for each study subject. Results: Both H3K4me2 and H3K9ac increased in association with years of employment in the plant (p-trend = 0.04 and 0.006, respectively). H3K4me2 increased in association with air levels of nickel [β = 0.16; 95% confidence interval (CI), 0.03–0.3], arsenic (β = 0.16; 95% CI, 0.02–0.3), and iron (β = 0.14; 95% CI, 0.01–0.26). H3K9ac showed nonsignificant positive associations with air levels of nickel (β = 0.24; 95% CI, –0.02 to 0.51), arsenic (β = 0.21; 95% CI, –0.06 to 0.48), and iron (β = 0.22; 95% CI, –0.03 to 0.47). Cumulative exposures to nickel and arsenic, defined as the product of years of employment by metal air levels, were positively correlated with both H3K4me2 (nickel: β = 0.16; 95% CI, 0.01–0.3; arsenic: β = 0.16; 95% CI, 0.03–0.29) and H3K9ac (nickel: β = 0.27; 95% CI, 0.01–0.54; arsenic: β = 0.28; 95% CI, 0.04–0.51). Conclusions: Our results indicate histone modifications as a novel epigenetic mechanism induced in human subjects by long-term exposure to inhalable nickel and arsenic.

Effects of Particulate Matter on Genomic DNA Methylation Content and iNOS Promoter Methylation

Background Altered patterns of gene expression mediate the effects of particulate matter (PM) on human health, but mechanisms through which PM modifies gene expression are largely undetermined. Objectives We aimed at identifying short- and long-term effects of PM exposure on DNA methylation, a major genomic mechanism of gene expression control, in workers in an electric furnace steel plant with well-characterized exposure to PM with aerodynamic diameters < 10 μm (PM10). Methods We measured global genomic DNA methylation content estimated in Alu and long interspersed nuclear element-1 (LINE-1) repeated elements, and promoter DNA methylation of iNOS (inducible nitric oxide synthase), a gene suppressed by DNA methylation and induced by PM exposure in blood leukocytes. Quantitative DNA methylation analysis was performed through bisulfite PCR pyrosequencing on blood DNA obtained from 63 workers on the first day of a work week (baseline, after 2 days off work) and after 3 days of work (postexposure). Individual PM10 exposure was between 73.4 and 1,220 μg/m3. Results Global methylation content estimated in Alu and LINE-1 repeated elements did not show changes in postexposure measures compared with baseline. PM10 exposure levels were negatively associated with methylation in both Alu [β = −0.19 %5-methylcytosine (%5mC); p = 0.04] and LINE-1 [β = −0.34 %5mC; p = 0.04], likely reflecting long-term PM10 effects. iNOS promoter DNA methylation was significantly lower in postexposure blood samples compared with baseline (difference = −0.61 %5mC; p = 0.02). Conclusions We observed changes in global and gene specific methylation that should be further characterized in future investigations on the effects of PM.

Predictors of global methylation levels in blood DNA of healthy subjects: a combined analysis

BACKGROUND Estimates of global DNA methylation from repetitive DNA elements, such as Alu and LINE-1, have been increasingly used in epidemiological investigations because of their relative low-cost, high-throughput and quantitative results. Nevertheless, determinants of these methylation measures in healthy individuals are still largely unknown. The aim of this study was to examine whether age, gender, smoking habits, alcohol drinking and body mass index (BMI) are associated with Alu or LINE-1 methylation levels in blood leucocyte DNA of healthy individuals. METHODS Individual data from five studies including a total of 1465 healthy subjects were combined. DNA methylation was quantified by PCR-pyrosequencing. RESULTS Age [β = -0.011% of 5-methyl-cytosine (%5 mC)/year, 95% confidence interval (CI) -0.020 to -0.001%5 mC/year] and alcohol drinking (β = -0.214, 95% CI -0.415 to -0.013) were inversely associated with Alu methylation. Compared with females, males had lower Alu methylation (β = -0.385, 95% CI -0.665 to -0.104) and higher LINE-1 methylation (β = 0.796, 95% CI 0.261 to 1.330). No associations were found with smoking or BMI. Percent neutrophils and lymphocytes in blood counts exhibited a positive (β = 0.036, 95% CI 0.010 to 0.061) and negative (β = -0.038, 95% CI -0.065 to -0.012) association with LINE-1 methylation, respectively. CONCLUSIONS Global methylation measures in blood DNA vary in relation with certain host and lifestyle characteristics, including age, gender, alcohol drinking and white blood cell counts. These findings need to be considered in designing epidemiological investigations aimed at identifying associations between DNA methylation and health outcomes.

Exposure to Metal-Rich Particulate Matter Modifies the Expression of Candidate MicroRNAs in Peripheral Blood Leukocytes

Background Altered patterns of gene expression mediate the effects of particulate matter (PM) on human health, but mechanisms through which PM modifies gene expression are largely undetermined. MicroRNAs (miRNAs) are highly conserved, noncoding small RNAs that regulate the expression of broad gene networks at the posttranscriptional level. Objectives We evaluated the effects of exposure to PM and PM metal components on candidate miRNAs (miR-222, miR-21, and miR-146a) related with oxidative stress and inflammatory processes in 63 workers at an electric-furnace steel plant. Methods We measured miR-222, miR-21, and miR-146a expression in blood leukocyte RNA on the first day of a workweek (baseline) and after 3 days of work (postexposure). Relative expression of miRNAs was measured by real-time polymerase chain reaction. We measured blood oxidative stress (8-hydroxyguanine) and estimated individual exposures to PM1 (< 1 μm in aerodynamic diameter), PM10 (< 10 μm in aerodynamic diameter), coarse PM (PM10 minus PM1), and PM metal components (chromium, lead, cadmium, arsenic, nickel, manganese) between the baseline and postexposure measurements. Results Expression of miR-222 and miR-21 (using the 2−ΔΔCT method) was significantly increased in postexposure samples (miR-222: baseline = 0.68 ± 3.41, postexposure = 2.16 ± 2.25, p = 0.002; miR-21: baseline = 4.10 ± 3.04, postexposure = 4.66 ± 2.63, p = 0.05). In postexposure samples, miR-222 expression was positively correlated with lead exposure (β = 0.41, p = 0.02), whereas miR-21 expression was associated with blood 8-hydroxyguanine (β = 0.11, p = 0.03) but not with individual PM size fractions or metal components. Postexposure expression of miR-146a was not significantly different from baseline (baseline = 0.61 ± 2.42, postexposure = 1.90 ± 3.94, p = 0.19) but was negatively correlated with exposure to lead (β = −0.51, p = 0.011) and cadmium (β = −0.42, p = 0.04). Conclusions Changes in miRNA expression may represent a novel mechanism mediating responses to PM and its metal components.

Sperm count and chromatin structure in men exposed to inorganic lead: lowest adverse effect levels

Objectives: To obtain knowledge on male reproductive toxicity of inorganic lead at current European exposure levels and to establish lowest adverse effect levels, if any. Methods: A cross sectional survey of the semen of 503 men employed by 10 companies was conducted in the United Kingdom, Italy, and Belgium. The mean blood lead concentration was 31.0 μg/dl (range 4.6–64.5) in 362 workers exposed to lead and 4.4 μg/dl (range below the detection limit of 19.8) in 141 reference workers. Semen volume and sperm concentration were determined in a fresh semen sample according to an agreed protocol subject to quality assurance. The sperm chromatin structure assay (SCSA) was performed at a centralised laboratory. Extraneous determinants including centre, period of sexual abstinence, and age were taken into account in the statistical analysis. If appropriate, possible thresholds were examined by iterative threshold slope linear regression. Results: The median sperm concentration was reduced by 49% in men with blood lead concentration above 50 μg/dl. There was no indication of a linear trend of lower sperm concentration with increasing blood lead values, but threshold slope least square regression identified a blood lead concentration of 44 μg/dl (β=−0.037, F=4.35, p=0.038) as a likely threshold. Abnormal sperm chromatin structure was not related to blood lead concentration, but some indications of deterioration of sperm chromatin was found in men with the highest concentrations of lead within spermatozoa. Biological monitoring data did not indicate long term effects of lead on semen quantity or sperm chromatin. Conclusion: Adverse effects of lead on sperm concentration and susceptibility to acid induced denaturation of sperm chromatin are unlikely at blood lead concentrations below 45 μg/dl. Effects of low level exposure to lead on other measures of testicular function cannot be ruled out.

Polarization dictates iron handling by inflammatory and alternatively activated macrophages

Background Macrophages play a key role in iron homeostasis. In peripheral tissues, they are known to polarize into classically activated (or M1) macrophages and alternatively activated (or M2) macrophages. Little is known on whether the polarization program influences the ability of macrophages to store or recycle iron and the molecular machinery involved in the processes. Design and Methods Inflammatory/M1 and alternatively activated/M2 macrophages were propagated in vitro from mouse bone-marrow precursors and polarized in the presence of recombinant interferon-γ or interleukin-4. We characterized and compared their ability to handle radioactive iron, the characteristics of the intracellular iron pools and the expression of molecules involved in internalization, storage and export of the metal. Moreover we verified the influence of iron on the relative ability of polarized macrophages to activate antigen-specific T cells. Results M1 macrophages have low iron regulatory protein 1 and 2 binding activity, express high levels of ferritin H, low levels of transferrin receptor 1 and internalize – albeit with low efficiency -iron only when its extracellular concentration is high. In contrast, M2 macrophages have high iron regulatory protein binding activity, express low levels of ferritin H and high levels of transferrin receptor 1. M2 macrophages have a larger intracellular labile iron pool, effectively take up and spontaneously release iron at low concentrations and have limited storage ability. Iron export correlates with the expression of ferroportin, which is higher in M2 macrophages. M1 and M2 cells activate antigen-specific, MHC class II-restricted T cells. In the absence of the metal, only M1 macrophages are effective. Conclusions Cytokines that drive macrophage polarization ultimately control iron handling, leading to the differentiation of macrophages into a subset which has a relatively sealed intracellular iron content (M1) or into a subset endowed with the ability to recycle the metal (M2).

Male reproductive toxicity of lead in animals and humans

OBJECTIVE: To critically review the literature on male reproductive toxicity of lead in animals and humans. METHODS: A systematic literature search identified a total of 32 experimental studies in animals and 22 epidemiological studies, one case report on humans and five review articles or documents. The studies were evaluated by paying attention mainly to sample size, study design, exposure, and dose characterisation, analytical method standardisation, and quality assurance. RESULTS: Several studies on rats and other rodents indicated that blood lead concentrations > 30-40 micrograms/dl were associated with impairment of spermatogenesis and reduced concentrations of androgens. However, other animal studies, mainly about histopathological, spermatozoal, and hormonal end points, indicated that certain species and strains were quite resistant to the reproductive toxicity of lead and that different testicular lead concentrations could account for these differences. The human studies focused mainly on semen quality, endocrine function, and birth rates in occupationally exposed subjects, and showed that exposure to concentrations of inorganic lead > 40 micrograms/dl in blood impaired male reproductive function by reducing sperm count, volume, and density, or changing sperm motility and morphology. No relevant effects were detected on endocrine profile. CONCLUSION: Several factors make it difficult to extrapolate the animal data to the human situation. The difficulties are mainly due to differences between species in reproductive end points and to the level of exposure. Concentrations of blood lead > 40 micrograms/dl seemed to be associated with a decrease in sperm count, volume, motility, and morphological alterations and a possible modest effect on endocrine profile. Dose-response relation, in particular at a threshold level, is poorly understood, and site, mode, or mechanism of action are unknown. Also, the effects were not always the same or associated in the same on sperm count and concentration. Some methodological issues and indications for future studies are discussed.

The Effect of Inhaled Chromium on Different Exhaled Breath Condensate Biomarkers among Chrome-Plating Workers

Chromium is corrosive, cytotoxic, and carcinogenic for humans and can induce acute and chronic lung tissue toxicity. The aim of this study was to investigate Cr levels in exhaled breath condensate (EBC) of workers exposed to Cr(VI) and to assess their relationship with biochemical changes in the airways by analyzing EBC biomarkers of oxidative stress, namely, hydrogen peroxide (H2O2) and malondialdehyde (MDA). EBC samples were collected from 24 chrome-plating workers employed in a chrome-plating plant both before and after the Friday work shift and before the work shift on the following Monday. Cr-EBC levels increased from the beginning (5.3 μg/L) to the end of Friday (6.4 μg/L) but were considerably lower on Monday morning (2.8 μg/L). A similar trend was observed for H2O2-EBC levels (which increased from 0.36 μM to 0.59 μM on Friday and were 0.19 μM on Monday morning) and MDA-EBC levels (which increased from 8.2 nM to 9.7 nM on Friday and were 6.6 nM on Monday). Cr-EBC levels correlated with those of H2O2-EBC (r = 0.54, p < 0.01) and MDA-EBC (r = 0.59, p < 0.01), as well as with urinary Cr levels (r = 0.25, p < 0.05). The results of this study demonstrate that EBC is a suitable matrix that can be used to investigate both Cr levels and biomarkers of free radical production sampling the epithelial-lining fluid of workers exposed to Cr(VI).

Elements in environmental and occupational medicine.

Occupational and environmental medicine traditionally dealt with elements, particularly with heavy metals. The interest was justified by the wide exposure in the workplace and in the general environment and by the evidence of their specific biological and toxicological effects. During the last 2 decades of 20th century the availability of indicators of exposure or of internal dose has substantially increased thanks to improvement in AAS-ETAAS techniques and to the entrance of ICP-MS into the field of biological monitoring. There are now more and more demands for controlling pre-analytical and analytical factors, for analysing biological matrices in addition to blood and urine and for setting up methods for elements not yet extensively studied in respect to their possible biological or toxicological role. Finally, deeper knowledge has to be reached in order to evaluate the significance of elements and, possibly, of their species in biological fluids at current doses and in order to face their effects, especially those in the first portion of the dose-response curve, which is going to be the main field of interest of occupational and environmental toxicology for the next few years.

Loss of sight and sound. Could it be the hip

In September, 2007, a 58-year-old woman was admitted to our neurology department because of progressive visual and hearing loss which had started 9 months earlier. She had type 2 diabetes and hypertension, both of which were adequately controlled by medication. In 2001, she had a left hip arthroplasty which was revised in October, 2006 because of rupture of the ceramic head. 3 weeks before admission, investigations showed a mild hypothyroidism of unknown origin, which was treated with levothyroxine sodium. The neurological examination at admission showed impairment of cranial nerves II and VIII bilaterally and mild distal sensory-motor dis turbances. Laboratory investigations ruled out haema tological, infectious, neoplastic, metabolic, and immuno logical diseases. Concomitantly our patient underwent various investigations including brain MRI (showing hyperintensity of optic nerves and tracts), electromyography (showing mild lower limb nerve amplitude reduction), and acoustic and visual evoked potentials (positive for bilateral absence of brainstem acoustic responses and irregular cortical visual responses). A working diagnosis of axonal multi-neuropathy caused by a presumed immune-mediated vasculitis was made and the patient was given prednisone (50 mg/day) for 2 months with little improvement. By December, 2007, our patient became completely blind, severely deaf, and wheel-chair bound because of lower limb hyposthenia. Tests for immune-mediated process remained negative and the case was referred to toxicology for further investigation. Unexpectedly, raised concentrations of cobalt and chromium were found in diff erent biological samples (cobalt: 24 h urine collection 1187 μg/L [0·1–1·5], blood 549 μg/L [0·05–2·7], plasma 90 μg/L [0·1–0·6], CSF 11·4 μg [0·05–0·15]; chromium: 24 h urine collection 510 μg/L [0·05–2·2], blood 54 μg/L [0·1–0·5], plasma 210 μg/L [0·1–0·5], CSF 4·4 μg/L [0·01–0·2]). Cobalt-chromium poisoning due to metal wear debris from her hip prosthesis was proposed, although radiology, including CT, showed no sign of prosthesis loosening. In February, 2008, several metal ion chelating treatments were given with edetic acid. Although metal ion concentrations decreased, neurological improve ment was negligible. Therefore, in April, 2008, resection arthroplasty was done. During surgery infi ltration of the peri-prosthesic tissue by metallic debris was evident (fi gure A); analysis of peri-prosthetic fl uids showed high concentrations of cobalt and chromium, and the removed prosthesis showed wear of the head and neck (fi gure B), supporting the hypothesis of endogenous cobalt-chromium poisoning. During the following 8 months the patient showed progressive improvement, although vision only partially improved. Metal ion concentrations decreased but remained higher than reference values at the last follow-up in November, 2008. The role of cobalt or cobalt-chromium on human tissues has not been defi nitively established. Cobalt can produce various toxicological eff ects including local respiratory symptoms due to inhalation of cobalt containing dusts, and systemic eff ects (thyroiditis, cardiomyopathy, erythropoiesis). Neuro logical toxicity, mainly optic atrophy, nerve deafness, and limb paraesthesia, has been occasionally reported in association with exogenous exposure. Neuro logical toxicity as a reult of endogenous exposure (mainly associated with metal prostheses) has been described. Cobalt can induce a hypoxia-like eff ect, possibly targeting mitochondria; of note, our patient’s symptoms were similar to those observed in some mitochondrial cytopathies. Total hip replacement and hip resurfacing arthroplasty are widely used therapeutic procedures; longer-term follow-up would be necessary to evaluate adverse chronic systemic eff ects due to prolonged exposure to high serum cobalt concentrations. In addition to orthopaedic evaluation, careful neurological and toxicological examinations are recommended whenever a patient with a metallic prosthesis complains of visual loss, hearing disturbance, limb weakness, numbness, or paraesthesia, even in the absence of local osteoarticular symptoms.