NMR Untargeted and HPLC-MS/MS Targeted Metabolomic Approaches for Evaluating Styrene Exposure in the Urine of Shipyard Workers

Abstract

Due to its chemical properties, styrene is largely employed in the manufacturing of several products including rubber, polymers and resins, and it is particularly suitable for shipbuilding industry purposes. In this context, the main exposure to styrene occurs in occupational settings. Despite its widespread use, its long-term effects on human health at the occupational level are still unclear. The aim of this pilot study was to evaluate changes in styrene exposure biomarkers related to the metabolic and oxidative stress profiles in the urine of seventeen shipyard workers and seventeen non-exposed subjects. Urinary metabolites were assessed by means of NMR spectroscopy, including mandelic and phenylglyoxylic acids; four oxidative stress biomarkers, namely 8-oxo-7,8-dihydroguanine, 8-oxo-7,8-dihydroguanosine, and 8-oxo-7,8-dihydro-2'-deoxyguanosine and 3-nitrotyrosine, were evaluated via HPLC-MS/MS. The metabolic profiles of exposed workers showed both long- and short-term metabolic responses to styrene exposure compared to non-exposed subjects. From the comparison between non-exposed and before-shift workers, only 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine levels were significantly different (long term exposure response). At the same time, comparing the non-exposed group with after-shift workers, we observed lower levels of pseudouridine and 1-methylnicotinamide and higher glutamine levels in after-shift workers. The comparison between before-shift and after-shift workers showed that 8-oxo-7,8-dihydroguanine significantly increased after the shift, suggesting its involvement in the exposure to styrene (short-term exposure response). The obtained results, although preliminary, allow us to lay the basis for further human studies aimed at establishing a global understanding of styrene metabolism.

Keywords: NMR-based metabolomics; oxidative stress biomarkers; styrene exposure; urinary metabolic profiles.

Figure 1

Spectral region of urine between 1.0 and 4.5 ppm from a shipyard worker at the end of the working shift. 2-HIB: 2-hydroxyisobutyric acid; 3-H-3-MB: 3-hydroxy-3-methylbutyric acid; 3-HIB: 3-hydroxyisobutyric acid; AA: acetic acid; Ala: alanine; CA: citric acid; Crt: creatine; DMA: dimethylamine; Gln: glutamine; Gly: glycine; Ile: isoleucine; LA: lactic acid; Lys: lysine; N-AcGln: N-acetylglutamine; p-CrS: p-cresol sulfate; pyroGlu: pyroglutamylglutamic acid; Sar: sarcosine; Tau: taurine; Thr: threonine; TMAO: trimethylamine n-oxide; Trig: trigonelline; Trp: triptophan; Tyr: tyrosine; U01: unknown 01; Val: valine.

Figure 2

Spectral region of urine between 6.5 and 9.0 ppm from a shipyard worker at the end of the working shift. 1-MNA: 1-methylnicotinamide; 2PY: n-methyl-2-pyridone-5-carboxamide; 4-HPA: 4-hydroxyphenylacetic acid; FA: formic acid; Hipp: hippuric acid; Hyp; hypoxanthine; MA: mandelic acid; PAGly: phenylacetylglycine; PGA: phenylglyoxylic acid; PSI: pseudouridine.

Figure 3

Partial least squares-discriminant analysis (PLS-DA) of integrated datasets of ¹H-NMR and HPLC-MS/MS data, comparing controls (C) and workers before their shift (BS). (A) PLS-DA score plot displaying C (blue) and BS workers (green); (B) regression coefficient of the significant variables that discriminate BS (green) from C; (C) loadings plot. 1-MNA: 1-methylnicotinamide; 2-HIB: 2-hydroxyisobutyric acid; 2PY: N-methyl-2-pyridone-5-carboxamide; 3-H-3-MB: 3-hydroxy-3-methylbutyric acid; 3-HIB: 3-hydroxyisobutyric acid; 3-NO2Tyr: 3-nitrotyrosine; 4-HPA: 4-hydroxyphenylacetic acid; 8-oxodGuo: 8-oxo-7,8-dihydro-2′-deoxyguanosine; 8-oxoGua: 8-oxo-7,8-dihydroguanine; 8-oxoGuo: 8-oxo-7,8-dihydroguanosine; AA: acetic acid; Ala: alanine; CA: citric acid; Crt: crea-tine; DMA: dimethylamine; FA: formic acid; Gln: glutamine; Gly: glycine; Hipp: hippuric acid; Hyp; hypoxanthine; Ile: isoleucine; LA: lactic acid; Lys: lysine; MA: mandelic acid; N-AcGln: N-acetylglutamine; PAGly: phenylacetylglycine; p-CrS: p-cresol sulfate; PGA: phenylglyoxylic acid; PSI: pseudouridine; pyroGlu: pyroglutamylglutamic acid; Sar: sarcosine; Tau: taurine; Thr: threonine; TMAO: trimethylamine N-oxide; Trig: trigonelline; Trp: triptophan; Tyr: Tyrosine; U01: unknown 01; Val: valine.

Figure 4

Boxplot of urinary metabolites that showed statistically significant changes between C (controls, blue) and BS workers (before shift, green). Statistical significance was assessed via the Mann–Whitney rank sum test. Boxplots report p values, median, minimum and maximum values (black dots), and the 25th and 75th percentile values of metabolites concentrations.

Figure 5

Partial least squares-discriminant analysis (PLS-DA) of integrated datasets of ¹H-NMR and HPLC-MS/MS data, comparing controls (C) and workers after their shift (AS). (A) PLS-DA score plot displaying C (blue) and AS workers (magenta); (B) regression coefficient of the significant variables that discriminate BS (magenta) from C; (C) loadings plot. 1-MNA: 1-methylnicotinamide; 2-HIB: 2-hydroxyisobutyric acid; 2PY: N-methyl-2-pyridone-5-carboxamide; 3-H-3-MB: 3-hydroxy-3-methylbutyric acid; 3-hIB: 3-hydroxyisobutyric acid; 3-nO2Tyr: 3-nitrotyrosine; 4-HPA: 4-hydroxyphenylacetic acid; 8-oxodGuo: 8-oxo-7,8-dihydro-2′-deoxyguanosine; 8-oxoGua: 8-oxo-7,8-dihydroguanine; 8-oxoGuo: 8-oxo-7,8-dihydroguanosine; AA: acetic acid; Ala: alanine; CA: citric acid; Crt: creatine; DMA: dimethylamine; FA: formic acid; Gln: glutamine; Gly: glycine; Hipp: hippuric acid; Hyp; hypoxanthine; Ile: isoleucine; LA: lactic acid; Lys: lysine; MA: mandelic acid; N-AcGln: N-acetylglutamine; PAGly: phenylacetylglycine; p-CrS: p-cresol sulfate; PGA: phenylglyoxylic acid; PSI: pseudouridine; pyroGlu: pyroglutamylglutamic acid; Sar: sarcosine; Tau: taurine; Thr: threonine; TMAO: trimethylamine N-oxide; Trig: trigonelline; Trp: triptophan; Tyr: tyrosine; u01: unknown 01; Val: valine.

Figure 6

Boxplot of urinary metabolites that showed statistically significant changes between C (controls, blue) and AS workers (after shift, magenta). Statistical significance was assessed via the Mann–Whitney rank sum test. Boxplots report p values, median, minimum and maximum values (black dots), and the 25th and 75th percentile values of metabolites concentrations.

Figure 7

Boxplot plot of urinary metabolites that showed statistically significant changes between BS (before shift, green) and AS (after shift, magenta) workers. Statistical significance was assessed via the Wilcoxon signed rank Test. Boxplots report p values, median, minimum and maximum values (black dots), and the 25th and 75th percentile values of metabolites concentrations.