Maria Enrica Fracasso

Low air levels of benzene: Correlation between biomarkers of exposure and genotoxic effects

This study was aimed to identify useful biomarkers of exposure and effect in workers exposed to low levels of benzene, and to evaluate any correlations existing between these parameters. Benzene exposure was measured in 33 petrochemical industry operators (PIO), 28 service station attendants (SSA), 21 gasoline pump maintenance workers (GPMW) and 51 non-exposed controls by GC-FID analysis. Samples were collected with personal passive samplers (Radiello). End-shift urine samples were collected for t,t-muconic acid (t,t-MA) determination by HPLC and for S-phenylmercapturic acid (S-PMA) measurement by HPLC-MS/MS. The alkaline version of the comet assay and, in a subgroup of 19 SSA and 16 control subjects, chromosomal aberrations (CA) and glutathione (GSH) levels were measured in peripheral blood lymphocytes. Personal benzene exposure was significantly higher in PIO, SSA and GPMW as compared to controls. The urinary excretion of the two metabolites showed a significant increase in SSA (p=0.0258 and p=0.0001, for t,t-MA and S-PMA, respectively) and in PIO (p=0.0013 and p=0.0001, for t,t-MA and S-PMA, respectively) as compared with the control group, while no such increase was observed for GPMW, for whom occupational exposure was not continuous and occurred on specific working days only. Significant increases of DNA damage were found by the comet assay for tail moment (TM) and tail length (TL) in SSA (p<0.0001 and p=0.008, for TM and TL, respectively) and PIO (p<0.0001 and p<0.0001, for TM and TL, respectively) when compared with controls. The PIO group also displayed a significant increase in the number of cells with comet (p<0.0001). Smoking habits did not appear to interfere with these results in any of the groups. No difference was found in percentage of CA between exposed workers and controls. Significant correlations were found, in all groups, between benzene exposure and the more representative comet parameter TM (r=0.509, p=0.007; r=0.525, p=0.017 and r=0.420, p=0.046 in SSA, GPMW, and PIO, respectively). A trend of negative correlation was observed between DNA damage and either GSH or urine S-PMA for exposed workers. In summary, in present study urinary S-PMA and DNA damage by the comet assay were both sensitive to exposure to low levels of benzene, and GSH seems to play an important defence role against benzene-dependent DNA damage.

Correlation between environmental and biological monitoring of exposure to benzene in petrochemical industry operators

The present work was aimed to study in petrochemical industry operators the correlation, if any, between environmental exposure to low levels of benzene and two biological exposure indexes in end-shift urine, i.e. trans, trans-muconic acid (t,t-MA) and S-phenylmercapturic acid (SPMA). Exposure to benzene was assessed in 133 male subjects employed in outdoor operations in a petrochemical plant, using personal passive-diffusive air samplers worn at the breathing zone; adsorbed benzene was determined by GC-FID analysis. S-PMA was determined by a new HPLCMS/MS method, after (quantitative) acidic hydrolysis of the cysteine conjugate precursor. t,t-MA was measured by an HPLC-UV method. Smoking habits were assessed by means of a self-administered questionnaire. Both environmental and biological monitoring data showed that benzene exposure of petrochemical industry operators was low (mean values were 0.014ppm, 101mug/g creat, and 2.8mug/g creat, for benzene, t,t-MA, and S-PMA, respectively) if compared with the ACGIH limits. Cigarette smoking was confirmed to be a strong confounding factor for the urinary excretion of both metabolites: statistically significant increases of t,t-MA and S-PMA levels were recorded in smokers when compared to non-smokers (p<0.0001). The best correlation found was that between exposure to benzene and S-PMA levels, particularly in non-smokers. This was partly due to the hydrolysis of the S-PMA precursor N-acetyl-S-(1,2-dihydro-2-hydroxyphenyl)-l-cysteine, a crucial step of the new analytical method used, which indeed reduced the variability of the results by means of an improved standardization of this critical preanalytical factor. A weaker correlation was found between exposure to benzene and t,t-MA, possibly explained by the fact that the latter is also a metabolite of sorbic acid, a common diet component. In summary, even at such low levels of exposure, urinary metabolites proved to be a useful tool for assessing individual occupational exposure to benzene, S-PMA appearing to be a more specific biomarker than t,t-MA, particularly in non-smokers.

Synthesis of new linear guanidines and macrocyclic amidinourea derivatives endowed with high antifungal activity against Candida spp. and Aspergillus spp.

New linear and cyclic guanidines were synthesized and tested in vitro for their antifungal activity toward clinically relevant strains of Candida species, in comparison to fluconazole. Macrocyclic compounds showed a minimum inhibitory concentration in the micromolar range and a biological activity profile in some cases better than that of fluconazole. One macrocyclic derivative was also tested against Aspergillus species and showed high antifungal activity comparable to that of amphotericin B and itraconazole.

DNA single-and double-strand breaks by alkaline-and immuno-comet assay in lymphocytes of workers exposed to styrene

Occupational exposure to styrene was studied in 34 workers employed in the production of fiberglass-reinforced plastic sheets and compared to 29 unexposed healthy controls. We evaluated genotoxic effects induced by occupational styrene exposure in lymphocytes by alkaline version of the comet assay to detect single-strand breaks (SSBs), DNA oxidation products (formamido pyrimidine glycosilase (Fpg)- and endonuclease (Endo III)-sensitive sites) and DNA repair kinetics studies, as well as the neutral version of comet assay for DNA double-strand breaks (DSBs). An innovative aspect of this study was the use of immuno-comet assay, a new technique that recognizes DSBs with specific antibody by DAPI/FITC method. The battery of parameters included markers of external and internal exposure. Exposed workers showed significant high levels of SSBs (p<0.0001) and DSBs (p<0.0001) in neutral- and immuno-comet assay. A drastic decrease in DNA repair activity as compared to controls was observed (180 min vs. 35 min). Styrene workplace concentration significantly correlated with alkaline comet parameters (TM, p=0.013; TI, p=0.008), in negative with TL (p=0.022), and with DNA-base oxidation (TM Endo III, p=0.048 and TI Endo III, p=0.028). There was a significant negative correlation between urinary metabolites (MA+PGA) and TM Endo III (p=0.032) and TI Endo III (p=0.017).

Exposure to mutagenic airborne particulate in a rubber manufacturing plant.

Epidemiological studies conducted in the 1980s revealed that people working in the rubber manufacturing industry had an increased risk of cancer. Even now, workers employed in rubber processing are still at risk despite the measures adopted to improve their working conditions. The aim of the study was to evaluate the presence of a genotoxic risk in a rubber industry and to verify whether or not it was possible to locate the most dangerous position among the different rubber-working processes. The mutagenic activity of airborne particulate was evaluated in samples collected in the mixing department of a rubber manufacturing plant. Ambient air samples were taken over 3-h period in two stable positions near the mixing (Banbury mixer) and calendering areas. Personal air samples were taken over 2-h period during a normal workday from five workers employed in different rubber processing operations (mixing, weighing, calendering, compounding and extruding). The mutagenic activity of the air samples was determined by plate incorporation assay using Salmonella typhimurium strains (TA 98, TA 98NR, TA 100, YG 1021) with and without metabolic activation. Polycyclic aromatic hydrocarbon (PAH) concentrations were determined by high-performance liquid chromatography (HPLC); the presence of other presumable contaminants were carried out by gas chromatography-mass spectrometry (GC-MS). The results showed substantial direct and indirect frameshift mutagenicity in both ambient and personal samples. No mutagenic activity was present in S. typhimurium TA 100, except in the personal sample from a worker employed on the Banbury mixer. HPLC analysis revealed very low concentrations of PAHs. GC-MS analysis showed the presence of compounds such as azulene derivative, 1,2-dihydro-2,2,4-trimethylquinoline, N-methyl N-phenylbenzenamine, diphenylamine, bis(2-ethylhexyl)phthalate and bis(methyl-propyl)phthalate. We conclude that the high levels of mutagenic activity in ambiental and personal samples indicate the presence of substances with high genotoxic potency; no substantial differences were seen among the several rubber processing operations. PAHs were not involved in indoor pollution. GC-MS analysis revealed the presence of compounds which may be produced by high temperatures to which the raw materials are subjected during rubber manufacturing processes. These substances are potential carcinogen though their mutagen properties have not been clearly determined.

Increases in polycyclic aromatic hydrocarbon content and mutagenicity in a cutting fluid as a consequence of its use

SummaryThe aim of this study was to evaluate the relationships between the length of time a cutting fluid was used, its content in polycyclic aromatic hydrocarbons (PAHs) and its mutagenic potential. The PAH concentrations were determined by means of a high-resolution gas chromatograph-mass spectrometer in samples of new cutting fluid and in samples used for 3, 6 and 9 months. The following PAHs were measured: phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene + triphenylene, benzo [e] pyrene, benzo [a]-pyrene and perylene. Mutagenicitiy assays were carried out on the aforementioned samples using the Ames test. Salmonella typhimurium TA98 was used as an indicator to show up mutagens capable of inducing frame-shift genetic changes, and Escherichia coli WP2 uvrA was used as an indicator to detect mutagens capable of inducing base pair genetic changes. The mutagenic tests were carried out with and without microsomial activation, using 1:1, 1:10, 1:20 and 1: 50 dilutions of cutting fluid samples. An increase in the concentrations of total PAHs over time was observed in the samples of cutting fluid used for 3, 6 and 9 months. The highest percentage increase in PAH concentrations was observed in the 6-month-old sample (10 times the initial concentration, from 45 to 411.8 μg of oil). None of the samples were mutagenic to S. typhimurium without metabolic activation or to E. coli with and without metabolic activation. All samples except for the 1:1 diluted sample showed moderate but significant mutagenic activity in the S. typhimurium test with metabolic activation. Mutagenicity rose to its highest levels (similar to those observed with the mutagenic compound 2-acetylaminofluorene, which was used as a positive control) in the 9-month-old sample and in the 6-month-old 1:20 and 1:50 diluted sample. This study shows an increase in the PAH concentrations and mutagenicity of cutting fluids following prolonged use. In order to minimize the risk caused by such an increase in PAH content, it is recommended that the cutting fluid be changed at least every 6 months.

In vivo CYP2E1 phenotyping as a new potential biomarker of occupational and experimental exposure to benzene.

Assessing CYP2E1 phenotype in vivo may be important to predict individual susceptibility to those chemicals, including benzene, which are metabolically activated by this isoenzyme. Chlorzoxazone (CHZ), a specific CYP2E1 substrate, is readily hydroxylated to 6-OH-chlorzoxazone (6-OH-CHZ) by liver CYP2E1 and the metabolic ratio 6-OH-CHZ/CHZ in serum (MR) is a specific and sensitive biomarker of CYP2E1 activity in vivo in humans. We used this MR as a potential biomarker of effect in benzene-treated rats and, also, in humans occupationally exposed to low levels of benzene. Male Sprague-Dawley rats (375-400g b.w.) were treated i.p. for 3 days with either a 0.5ml solution of benzene (5mmol/kg b.w.) in corn oil, or 0.5ml corn oil alone. Twenty-four hours after the last injection, a polyethylene glycol (PEG) solution of CHZ (20mg/kg b.w.) was injected i.p. in both treated and control animals. After 2, 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, and 240min from injection, 0.2ml blood was taken from the tip tail and stored at -20 degrees C until analysis. A modified reverse phase HPLC method using a 5microm Ultrasphere C18 column equipped with a direct-connection ODS guard column, was used to measure CHZ and its metabolite 6-OH-CHZ in serum. No statistically significant difference in the MR was observed, at any sampling time, between benzene-treated and control rats. The concentration-versus-time area under the curve (AUC), however, was lower (p<0.05, Mann-Whitney test), whereas the systemic clearance was higher (p<0.05) in treated than in control rats. Eleven petrochemical workers occupationally exposed to low levels of airborne benzene (mean+/-SD, 25.0+/-24.4microg/m(3)) and 13 non-exposed controls from the same factory (mean+/-SD, 6.7+/-4.0microg/m(3)) signed an informed consent form and were administered 500mg CHZ p.o. Two hours later a venous blood sample was taken for CHZ and 6-OH-CHZ measurements. Despite exposed subjects showed significantly higher levels of t,t-MA and S-PMA, two biomarkers of exposure to benzene, than non-exposed workers, no difference in the MR mean values+/-SD was found between exposed (0.59+/-0.29) and non-exposed (0.57+/-0.23) subjects. So, benzene was found to modify CHZ disposition, but not CYP2E1 phenotype in benzene-treated rats, nor in workers exposed to benzene, probably due to the levels of exposure being too low.

Outdoor formaldehyde and NO2 exposures and markers of genotoxicity in children living near chipboard industries.

Background: Industrial air pollution is a public health hazard. Previous evidence documented increased respiratory symptoms and hospitalizations in children who live near the factories in the largest chipboard manufacturing district in Italy (Viadana). Objectives: We evaluated the association of outdoor exposure to formaldehyde and nitrogen dioxide (NO2) with markers of early genotoxic damage in oral mucosa cells of randomly selected children (6–12 years of age) living in Viadana. Methods: In 2010–2011, DNA strand breaks and nuclear abnormalities were evaluated in exfoliated buccal cells by the comet and micronucleus assays, respectively, and formaldehyde and NO2 were monitored by passive sampling. Annual exposure estimates to pollutants were assigned to children’s houses by spatial interpolation. Results: Of 656 children, 413 (63%) participated. Children living near (< 2 km) the chipboard industries had the highest average exposure to formaldehyde and NO2 (p < 0.001). A 1-SD increase in formaldehyde (0.20 μg/m3) was associated with a 0.13% (95% CI: 0.03, 0.22%) higher comet tail intensity, a 0.007 (95% CI: 0.001, 0.012) higher tail moment, and a 12% relative increase [relative risk (RR) = 1.12; 95% CI: 1.02, 1.23] in nuclear buds. A 1-SD NO2 increase (2.13 μg/m3) was associated with a 0.13% (95% CI: 0.07, 0.19%) increase in binucleated cells and a 16% relative increase (RR = 1.16; 95% CI: 1.06, 1.26) in nuclear buds. Conclusions: Exposure to pollutants was associated with markers of genotoxicity in exfoliated buccal cells of children living in a region with chipboard industries. These findings, combined with previously reported associations between chipboard industrial activities and respiratory outcomes in children, add to concerns about potential adverse effects of industry-related exposures in the Viadana district. Citation: Marcon A, Fracasso ME, Marchetti P, Doria D, Girardi P, Guarda L, Pesce G, Pironi V, Ricci P, de Marco R. 2014. Outdoor formaldehyde and NO2 exposures and markers of genotoxicity in children living near chipboard industries. Environ Health Perspect 122:639–645; http://dx.doi.org/10.1289/ehp.1307259

Urinary mutagenic activity after different immunosuppressive protocols in renal transplant patients

Cyclosporin (CsA) and azathioprine (AZA) are useful immunosuppressive drugs in the management of kidney and liver transplant recipients. We investigated urinary mutagenicity in three groups of kidney transplant recipients after different immunosuppressive protocols. Urinary mutagenicity was detected in a base-pair strain, E. coli WP2uvrA, in a liquid incubation assay. No mutagenic activity was detected in the urines of patients treated with CsA (4.5 mg/kg); 85% of the urines in the second group treated with AZA (1.26 mg/kg) showed high mutagenic activity, whereas mutagenic activity was found in 40% of the urines of subjects treated with CsA and AZA (3.89 mg/kg + 1.15 mg/kg). These data suggest that immunosuppressive therapy with AZA carriers a high risk of urinary mutagenicity, while immunosuppressive combined treatment with CsA and AZA significantly reduces this risk.

DNA damage and repair capacity in workers exposed to low concentrations of benzene.

DNA damage and cellular repair capacity were studied in 18 male fuel tanker drivers and 13 male filling‐station attendants exposed to low and very low concentrations of benzene, respectively, and compared to 20 males with no occupational exposure (controls). Exposure to airborne benzene was measured using passive personal samplers, and internal doses were assayed through the biomarkers t,t‐muconic acid, S‐phenylmercapturic acid and urinary benzene. DNA damage was evaluated using tail intensity (TI) determined by the comet assay in peripheral lymphocytes. Urinary 7‐hydro‐8‐oxo‐2’‐deoxyguanosine (8‐oxodG) was measured as a biomarker of oxidative damage. DNA repair kinetics were assessed using the comet assay in lymphocytes sampled 20 and 60 min post H2O2 exposure. Benzene exposure differed significantly between the drivers (median 246.3 µg/m3), attendants (median 13.8 µg/m3), and controls (median 4.1 µg/m3). There were no differences in TI and 8‐oxodG among the three groups, or between smokers and non‐smokers. DNA repair kinetics were similar among the drivers, attendants and controls, although the comet assay on H2O2‐damaged lymphocytes after 60 min revealed significantly lower levels of TI only in drivers. The DNA repair process in smokers was similar to that observed in drivers. In conclusion, this study found no relationship between low levels of benzene exposure and DNA damage, although there was evidence that exposure interferes with DNA repair kinetics. The biological impact of this finding on the onset of genotoxic effects in exposed workers has still to be ascertained. Environ. Mol. Mutagen. 57:151–158, 2016.