Denise Doria

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.

Expression of COX-1, COX-2, and inducible nitric oxide synthase protein in human gastric antrum with Helicobacter pylori infection.

For a better understanding of the regulation of prostaglandin and nitric oxide (NO) synthesis in circumstances in which the gastric mucosa is inflamed, we have examined the ex vivo production of NO and prostaglandin E2 and the protein expression of inducible nitric oxide synthase (iNOS) and 2 cyclo-oxygenase (COX) isoforms in gastric biopsies from nine Helicobacter pylori-infected patients with active gastritis and six Helicobacter pylori (HP)-negative patients. The results indicate a significant increased of NO and PGE2 in patients with HP infection compared with uninfected samples. These findings were paralleled by marked increases in iNOS and in COX-1 and COX-2 protein expression. Expression of iNOS and COX-2 protein was absent in the mucosa of HP-negative controls. We have demonstrated that iNOS protein is expressed in the gastric mucosa of patients with HP infection. It is likely that iNOS expression and the corresponding high release of NO may play an important role in gastric inflammation associated with HP infection. However, the expression of COX-1 and COX-2 and the parallel increase of prostaglandin E2 could imply that these factors could limit the extend of mucosal damage. In previous reports NO has been shown to stimulate the COX activity, so we think that the role of NO could be both in the regulation of normal function and in the genesis of diseases.

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).

Nitric oxide enhances prostaglandin production in ethanol-induced gastric mucosal injury in rats.

The interaction between endogenous nitric oxide (NO), elicited by administration of Escherichia coli lipopolysaccharide, and cyclooxygenase system, in ethanol-induced injury in rat gastric mucosa, was investigated. Administration of graded doses of lipopolysaccharide reduced the gastric mucosal injury in response to ethanol. The ex vivo production of both nitrite and prostaglandin E2 was increased in dose-related manner by lipopolysaccharide. Pretreatment with dexamethasone, L-N6-(1-Iminoethyl)lysine(dihydrochloride) and L-NG-nitro arginine methyl ester inhibited the protection associated with lipopolysaccharide treatment and the ex vivo production of both, nitrite and prostaglandin E2. The pretreatment with L-arginine counteracted the decrease of nitrite and prostaglandin E2 production in lipopolysaccharide-treated rats in which nitric oxide synthesis was blocked by L-N6-(1-Iminoethyl)lysine(dihydrochloride). Administration of sodium nitroprusside and S-nitroso-N-acetyl-D,L-penicillamine caused a dose related enhancement in the accumulation of prostaglandin E2. Indomethacin administration and N-(2-Cyclohexyloxy-4-nitrophenyl)methanesulfonamide were ineffective in suppressing lipopolysaccharide-mediated protection against ethanol-induced damage, and in suppressing ex vivo increase of nitrite whereas the ex vivo increase of prostaglandin E2 was prevented in a dose-related fashion. These results indicate that in ethanol-induced rat gastric injury, endogenous NO elicited by lipopolysaccharide or released by NO donors is able to activate the cyclooxygenase pathway, and the protective effect of lipopolysaccharide is dependent upon NO formation.

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

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.

Comparison of hydrolysis and HPLC/MS/MS procedure with ELISA assay for the determination of S-phenylmercapturic acid as a biomarker of benzene exposure in human urine.

The present study compared three methods for the determination of S-phenylmercapturic acid (S-PMA), a metabolite of benzene, in human urine: a HPLC/MS/MS technique with two different sample treatments (strong and partial hydrolysis) and a commercial assay based on anti-S-PMA monoclonal antibodies with chemiluminescence detection. Biological monitoring was done on 126 volunteers and the results were compared for the three methods and also with benzene exposure levels (range <3.0-592.5 μg/m(3)). The correlation between environmental monitoring data and S-PMA levels in non-smokers (n=73) was highly significant (p<0.0001, Students t-test) for both HPLC/MS/MS methods (r=0.65 both for strong acidic hydrolysis of the urine and hydrolysis at pH 2) but not for the immunoassay, which overestimated the S-PMA levels by about 8 μg/g creatinine (creat.). Therefore the immunoassay is only useful as a semiquantitative screening test, but quantitative results need to be confirmed by a more accurate method like HPLC/MS/MS. The HPLC/MS/MS procedure with strong acid hydrolysis led to a recovery of S-PMA about double that using pH 2 hydrolysis, giving more accurate results. The difference between the results with the two methods makes it difficult to compare the strong acidic hydrolysis data with the ACGIH BEI value of 25 μg/g creat. since the BEI(®) documentation is based on data collected in pH conditions that were not always controlled, which may underestimate the true S-PMA concentration. Besides, as levels of benzene exposure were high, smoking was not considered a confounding factor. The BEI for S-PMA in end of shift urine samples could be reconsidered when sufficient data are available from studies where the analyses are carried out in comparable conditions of hydrolysis and monitoring only non-smoking subjects.

636 Genotoxic damage and dna repair gene polymorphisms in workers exposed to low doses of ionising radiation

Introduction DNA repair enzymes could modulate the individual susceptibility to the genotoxic effect of exposure to ionising radiation (IR). Methods The influence of polymorphisms of XRCC1, XRCC3 and XPD genes on the onset of chromosomal and DNA damage has been investigated in 43 workers exposed to low levels of IR, including 36 healthcare professionals and 7 industrial radiologists (exposed workers), and 43 subjects not occupationally exposed to IR (controls), matched for age. Chromosomal aberrations (CA) and micronuclei (MN) frequencies in peripheral blood lymphocytes were measured according to standard procedures and used as cytogenetic biomarkers, while Tail Intensity (TI) was the parameter of the Comet test used to evaluate oxidative DNA damage. Genotypic variants Arg194Trp, Arg280His and Arg399Gln for XRCC1, Thr241Met for XRCC3 and Lys751Gln for the XPD genes were analysed using the restriction fragment length polymorphism technique. Results Both total CA and chromosome breaks frequencies were significantly higher in the exposed workers than controls (p<0.05 and p<0.01 respectively), while no significant differences between the two groups were observed in terms of chromatid breaks and MN frequencies as well as the TI. In the controls only, TI was significantly higher in females than males, whereas a smoking habit did not affect the biomarkers investigated. The genetic polymorphisms of XRCC1, XRCC3 and XPD, individually analysed, did not influence any of the genotoxicity and oxidative damage biomarkers studied, either in the exposed workers or the controls. Discussion Chromosome breaks frequency resulted a valid cytogenetic biomarker for the monitoring of workers exposed to low doses of IR. The presence of single genetic variants reducing the activity of DNA repair enzymes does not seem to determine an increased risk of genotoxic effects of low doses of IR.