Urinary 3-OHBaP Research Articles

Prolonged exposure of environmental concentration benzo[a]pyrene promoted cancer stemness through AhR/PKA/SOX2 dependent pathway in small cell lung cancer

Benzo[a]pyrene (BaP) is commonly found in the environment as a result of incomplete combustion of organic materials and cigarette smoke. Epidemiological studies have consistently suggested that elderly smokers are at higher risk for small cell lung cancer (SCLC), with risks and clinical stages increasing with the intensity and duration of smoking. However, the underlying mechanism remains insufficiently investigated. Here, we established a positive correlation between smoking and BaP metabolite 3-hydroxybenzo[a]pyrene (3OH-BaP) in urine. The pooled standardized mean difference of urinary 3OH-BaP concentration for smokers versus nonsmokers was 5.18 (95 % CI 2.86–7.50). Clinical data suggested that smoking led to more lymph node metastasis, higher pathological N-stage, and worse overall survival in SCLC patients. We identified 75 genes that participate in BaP-associated cancer stemness of SCLC from Comparative Toxicogenomics Database and validated the expression of these candidate genes in SCLC patient samples. Protein kinase cAMP-activated catalytic subunit alpha (PRKACA) was found to be most upregulated in SCLC patients and in vitro experiments indicated that long-term exposure of SCLC cells to BaP, at the concentration equivalent to those detected in blood, increased PKA protein level. Further investigation revealed that PKA could directly interact with SOX2 and protect SOX2 from COP1-mediated ubiquitination and degradation. Upregulated SOX2 then contributed to the stemness and metastasis of SCLC cells while inhibition of aryl hydrocarbon receptor (AhR) signaling pathway abolished BaP induced PKA expression and downstream PKA/SOX2 axis. Our findings firstly pinpoint BaP exposure as a high-risk factor for SCLC and worse outcomes in patients, with the underlying mechanism being the activation of cancer stemness of SCLC via the AhR/PKA/SOX2 axis.

Consistency between air and biological monitoring for assessing polycyclic aromatic hydrocarbon exposure and cancer risk of workers

BackgroundAtmospheric levels of polycyclic aromatic hydrocarbons (PAHs) have been monitored in many companies since 1940. Because of the use of respiratory protective equipment (RPE) and cutaneous absorption, the measurement of urinary 1-hydroxypyrene (1-OHP), metabolite of pyrene (Pyr), and, more recently, 3-hydroxybenzo[a]pyrene (3-OHBaP), metabolite of benzo[a]pyrene (BaP), has been carried out to assess PAH exposure and estimate health risks. ObjectivesThis study aimed to investigate the agreement between 523 air and biological levels recorded in the Exporisq-HAP database by taking into account the effectiveness of RPE. MethodsThe agreement/consistency between 523 air and biological exposure levels was assessed by estimating and comparing the probability of exceeding French limit values (LVs) for both BaP and 3-OHBaP and ACGIH LV for 1-OHP, respectively. PAH airborne levels (wPAHs) were weighted by an assigned protection factor (APF) depending on the type of mask worn by workers, while urinary 1-OHP concentrations were adjusted with the wBaP/wPyr ratio of each industrial sector (wadj1-OHP). ResultsWithin occupational groups, there was an overall agreement between airborne PAH levels and urinary biomarker concentrations. A clear dichotomy was found between “petroleum-derived” and “coal-derived” groups, with much higher exposures in the latest group despite the use of RPEs by two-thirds of the workers. The type of RPE varied from one plant to another, which underlines the importance of taking into account their effectiveness. The analysis of urinary 3-OHBaP was not relevant for low PAH exposure levels. In addition, this biomarker underdiagnosed the exceedance of LV relative to BaP levels for 6% of “coal-derived” groups. ConclusionsThe use of urinary wadj1-OHP seemed to be more protective to assess the exceedance of LVs than those of urinary 3-OHBaP and air wBaP, but adjustment of the 1-OHP concentration by the BaP/Pyr ratio requires air sampling due to highly variable ratios observed in the studied occupational groups.

Determination of 3-Hydroxybenzo[a]pyrene Glucuronide/Sulfate Conjugates in Human Urine and Their Association with 8-Hydroxydeoxyguanosine.

While 3-hydroxybenzo[a]pyrene (3-OHBaP) is a preferable biomarker to assess human exposure to benzo[a]pyrene (BaP), a sensitive and simple method is lacking. In this study, a specific and sensitive method based on liquid chromatography coupled with electrospray tandem mass spectrometry (LC-ESI-MS/MS) was developed for direct analysis of 3-OHBaP glucuronide and sulfate conjugates in human urine samples without enzymatic hydrolysis. The limits of detection (LODs) were 0.06 ng L-1 for BaP-3-sulfate (BaP-3-S) and 0.16 ng L-1 for BaP-3-glucuronide (BaP-3-G), which showed high sensitivity. Both compounds showed excellent linearity (r2 > 0.99) in the range of 0.01-10 μg L-1 in the instrumental calibration. The absolute recoveries of the target analytes spiked in human urine for the entire analytical procedure were 68.3 ± 4.96% (mean ± SD) and 63.7 ± 5.47% for BaP-3-S and BaP-3-G, respectively. This method was applied to quantify BaP-3-G and BaP-3-S in 150 urine samples collected from healthy volunteers. The mean concentration of BaP-3-S was 0.67 ng g-1 creatinine (<LOD to 10.20 ng g-1 creatinine), about 10-fold lower than that of BaP-3-G (6.73 ng g-1 creatinine, < LOD to 52.64 ng g-1 creatinine). For comparison, we also detected the concentration of free 3-OHBaP in 15 randomly selected samples without enzymatic hydrolysis and found at least >98% of 3-OHBaP is excreted mainly in these two conjugated forms in human urine. A statistically significant positive association was observed between urinary 3-OHBaP conjugates and urinary 8-OHdG levels (p < 0.001) in the general population. This study developed a sensitive and simple method to determine urinary glucuronide/sulfate conjugated BaP metabolites and for the first time found that BaP exposure associated with 8-OHdG levels in the general population.

Maintenance of Low-Pressure Carburising Furnaces: A Source of PAH Exposure.

Low-pressure carburising is a new technology used to harden steel; the process has been shown to be a source of considerable polycyclic aromatic hydrocarbons (PAH) pollution. Some PAH are carcinogenic, and activities such as furnace maintenance may thus represent a risk to workers. Occupational exposure during these operations should therefore be assessed. In this study, the PAH-related carcinogenic risk associated with furnace maintenance was assessed by monitoring atmospheric levels of benzo[a]pyrene (BaP), a representative marker, alongside urinary levels of 3-hydroxybenzo[a]pyrene (3-OHBaP), one of its metabolites. PAH exposure levels were monitored during seven sampling campaigns in four different factories specialized in heat-treatment of mechanical workpieces for the automotive and helicopter industries. Two types of furnace were studied, and 37 individuals were monitored. Values up to 20-fold the French regulatory value of 150 ng/m3 for atmospheric BaP, and, for urinary 3-OHBaP values up to 40-fold the French biological limit value (BLV) of 0.35 nmol/mol of creatinine were detected. Very high concentrations of BaP, close to or even exceeding those found in coal-tar pitch (up to about 20 g/kg), were measured in residues (tars, dusts) deposited inside the furnace. Even when adequate and suitable personal protective equipment was used, urinary 3-OHBaP values often exceeded the BLV. We hypothesize that this exposure is linked to insidious and fortuitous dermal contamination through contact with factory equipment and staining.

Kinetics of Diol and Hydroxybenzo[a]pyrene Metabolites in Relation to DNA Adduct Formation and Gene Expression in Rats

Benzo[a]pyrene (BaP) is a human carcinogen, but there are no validated biomarkers of exposure and the relationship of carcinogenesis with early biological alterations is not fully documented. This study aimed at better documenting the toxicokinetics of diolBaP and hydroxyBaP metabolites as potential biomarkers of exposure to BaP in relation to DNA adduct formation and gene expression. Rats were intravenously (iv) injected with 40 μmol/kg BaP. BaP and several metabolites were measured in blood, tissues, and excreta collected at frequent intervals over 72 h posttreatment. BaP diol epoxide (BaPDE)–DNA adduct formation and gene expression were assessed in lungs. 3-HydroxyBaP (3-OHBaP) and 4,5-diolBaP were the most abundant measured metabolites, and differences in time courses were apparent between the two metabolites. Over the 0–72 h period, mean proportions of BaP dose recovered in urine as 3-OHBaP and 4,5-diolBaP (±SD) were 0.017 ± 0.003% and 0.1 ± 0.03%. Corresponding values in feces were 1.5 ± 0.5% and 0.42 ± 0.052%. BaPDE–DNA adducts were significantly increased in lungs and a correlation was observed with urinary 3-OHBaP and 4,5-diolBaP. Analysis of gene expression showed a modulation of expression of metabolic genes (Cyp1a1, Cyp1b1, Nqo1, Ahr) and oxidative stress and repair genes (Nrf2, Rad51). However, BaPDE adducts formation did not exhibit any significant correlation with expression of genes, except a negative correlation with Rad51 expression. Similarly, there was no significant correlation between urinary excretion of OHBaP and diolBaP and expression of genes, except for urinary 7-OHBaP excretion, which was negatively correlated with Rad51 expression. Results indicate that concomitant measurements of diolBaP and OHBaP may serve to better assess the extent of exposure as compared to single metabolite measurements, given kinetic differences between metabolites. Further, although some urinary metabolites were correlated with BaPDE adducts, links with gene expression need to be further investigated.

Development of a method to detect three monohydroxylated polycyclic aromatic hydrocarbons in human urine by liquid chromatographic tandem mass spectrometry.

A liquid chromatographic tandem mass spectrometry method (LC-MS/MS) for the simultaneous determination of 1-hydroxypyrene (1-OHP), 3-hydroxybenzo[a]pyrene (3-OHBaP), and 3-hydroxybenz[a]anthracene (3-OHBaA) in human urine has been developed. With the exception of 3-OHBaP at a low spiking level, the average recoveries were greater than 80%. The method has good accuracy (72.1–107.7%) and reproducibility (1.8–11.4%) and was successfully used to study the uptake of pyrene, benzo[a]pyrene, and benzo[a]anthracene from cigarette smoke. The results indicated that urinary 1-OHP concentration in the smoking group (66.58 ± 70.91 ng/g creatinine) was higher than that observed in the nonsmoking group (58.16 ± 49.48 ng/g creatinine). Urinary 3-OHBaA concentrations in nonsmokers and smokers with 8 mg and 10 mg tar cigarettes were 10.98 ± 4.39 ng/g creatinine, 11.01 ± 13.30 ng/g creatinine, and 9.17 ± 12.89 ng/g creatinine, respectively. Urinary 3-OHBaP concentrations in nonsmokers and smokers with 8 mg and 13 mg tar cigarettes were 1.30 ± 0.20 ng/g creatinine, 2.83 ± 1.78 ng/g creatinine, and 6.00 ± 4.44 ng/g creatinine, respectively. Urinary 1-OHP levels exhibited a significant correlation with BaP yield in cigarette smoke under the Canadian intense smoking condition (y = 3.5563x + 30.171, R 2 = 0.9916, n = 227).

Derivatization method for sensitive determination of 3-hydroxybenzo[a]pyrene in human urine by liquid chromatography–electrospray tandem mass spectrometry

While urinary 3-hydroxybenzo[a]pyrene (3-OHBaP) can indicate exposure to carcinogenic benzo[a]pyrene (BaP) in humans, most previous analytical methods could not detect it in the general population due to a lack of sufficient sensitivity. In this study, a new liquid chromatography–electrospray mass–mass spectrometry (LC–ESI-MS/MS) method was developed to analyze 3-OHBaP by employing an optimized derivatization reaction with dansyl chloride (DNS) in the aqueous phase. The instrument detection limit (IDL) of the newly developed method was 10ng/L, which is 60-fold lower than without derivatization. The method was successfully applied to analyze 3-OHBaP and 1-hydroxypyrene (1-OHPy) in human urine combined with enzymatic hydrolysis, C18 enrichment, liquid–liquid extraction and silica cartridge cleanup. The overall method recoveries were 68.7±5.47% for 3-OHBaP and 72.3±4.96% for 1-OHPy, with matrix effects of <10%. The limits of quantification (LOQs) in urine were 0.3±0.02ng/L and 0.5±0.02ng/L (n=3), respectively. The method was applied to analyze seven urine samples of volunteers, and 3-OHBaP was detected in five samples. The concentration of urinary 3-OHBaP ranged from <LOD to 2.30ng/L, much lower than those (42.0–216ng/L) of 1-OHPy. The developed method provides a new way to sensitively determine 3-OHBaP in urine of the general population.

Relevance of urinary 3-hydroxybenzo(a)pyrene and 1-hydroxypyrene to assess exposure to carcinogenic polycyclic aromatic hydrocarbon mixtures in metallurgy workers.

In metallurgy, workers are exposed to mixtures of polycyclic aromatic hydrocarbons (PAHs) in which some compounds are carcinogenic. Biomonitoring of PAH exposure has been performed by measuring urinary 1-hydroxypyrene (1-OHP), a metabolite of pyrene which is not carcinogenic. This study investigated the use of 3-hydroxybenzo(a)pyrene (3-OHBaP), a metabolite of benzo(a)pyrene (BaP) which is the main carcinogenic component in PAHs, to improve carcinogen exposure assessment. We included 129 metallurgy workers routinely exposed to PAHs during working hours. Urinary samples were collected at three sampling times at the beginning and at the end of the working week for 1-OHP and 3-OHBaP analyses. Workers in anode production showed greater exposure to both biomarkers than those in cathode or silicon production, with respectively, 71, 40, and 30% of 3-OHBaP concentrations exceeding the value of 0.4 nmol mol(-1) creatinine. No difference was observed between the 3-OHBaP levels found at the end of the penultimate workday shift and those at the beginning of the last workday shift. Within these plants, the 1-OHP/3-OHBaP ratios varied greatly according to the workers' activity and emission sources. Using linear regression between these two metabolites, the 1-OHP level corresponding to the guidance value for 3-OHBaP ranged from 0.7 to 2.4 µmol mol(-1) creatinine, depending on the industrial sector. This study emphasizes the interest of monitoring urinary 3-OHBaP at the end of the last workday shift when working week exposure is relatively steady, and the irrelevance of a single guideline value for 1-OHP when assessing occupational health risk.

Development of a sensitive method for the quantification of urinary 3-hydroxybenzo[a]pyrene by solid phase extraction, dansyl chloride derivatization and liquid chromatography-tandem mass spectrometry detection

A sensitive method for the quantification of urinary 3-OHBaP was developed by SPE, dansyl chloride derivatization and LC-MS/MS detection.

Excretion kinetics of urinary 3-hydroxybenzo[a]pyrene following dietary exposure to benzo[a]pyrene in humans

Urinary 3-hydroxy-benzo[a]pyrene (3-OHBaP) is considered as an exposure marker for assessing carcinogenic risks as it is a metabolite of benzo[a]pyrene (BaP). A controlled human study was conducted to examine the kinetics of urinary 3-OHBaP after consuming charcoal-barbecued meat. Two feeding experiments were performed, with meat doses of 15 and 30 g/kg (experiments 1 and 2, respectively). All voided urine over 7 days was collected and analyzed. The background urinary 3-OHBaP concentration was 0.002-0.085 ng/g creatinine, with large inter-individual (80-100%) and intra-individual of (90-100%) variations. The background amount of 3-OHBaP excreted by the current subjects was 40-50 pg/day. The amounts of urinary 3-OHBaP excreted within 12 h post-exposure increased significantly (P < 0.05) from background only in experiment 1. The proportion of the administered BaP dose that was excreted as urinary 3-OHBaP within 12 h post-exposure was 0.006 and 0.0012% in experiments 1 and 2, respectively. The excretion ratio declined as the dose increased. Urinary 3-OHBaP can be used to assess dietary exposure to BaP, but it may be not suitable for low-dose scenarios because of the low urinary excretion proportion and high variability of the background.

Evaluation of DNA Adducts, DNA and RNA Oxidative Lesions, and 3-Hydroxybenzo(a)pyrene as Biomarkers of DNA Damage in Lung Following Intravenous Injection of the Parent Compound in Rats

Biomarkers of exposure and effect were assessed in 40 male Sprague-Dawley rats injected intravenously with 40 micromol/kg of benzo(a)pyrene (BaP) to determine which biomarkers are more representative of BaP-induced DNA damage in lung. Lung, liver, blood, and urine were collected at t = 2, 4, 8, 16, 24, 33, 48, 72, and 360 h postdosing. Specific BaP-diol epoxide (BPDE)-DNA adducts, 8-hydroxy-7,8-dihydro-2'-deoxyguanosine (8-OHdGuo), were measured in lung, liver, and mononucleated blood cells by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Urinary 8-OHdGuo and 8-hydroxy-7,8-dihydroguanosine (8-OHGuo) were also determined by HPLC-MS/MS, and urinary 3-hydroxybenzo(a)pyrene was measured by HPLC/fluorescence. Between 2 and 72 h postdosing, BPDE-DNA adducts were significantly increased in lung, liver, and mononucleated blood cells of BaP-treated rats as compared to controls, with the highest levels found in lung. 8-OHdGuo levels also increased in lung of BaP-treated rats with values reaching statistical significance at 2, 8, and 16 h postinjection. No influence of BaP treatment was found on 8-OHdGuo and 8-OHGuo urinary excretions. BPDE-DNA adducts in lung were strongly correlated to urinary 3-OHBaP (r = 0.936 and p < 0.001) and to a lesser extent to blood BPDE-DNA adducts (r = 0.636 and p < 0.001), the latter of which were correlated to each other (r = 0.573 and p = 0.002). Urinary 3-OHBaP and BPDE-DNA adducts in mononucleated blood cells appear as relevant biomarkers of BaP genotoxic exposure and are highly promising for health risk assessment in humans.

3-Hydroxybenzo(a)pyrene as a biomarker of dermal exposure to benzo(a)pyrene

The aim of this study was to determine the percutaneous absorption flux of BaP (20 microg/cm(2) in ethanol) and the usefulness of urinary 3-OHBaP as a bio-indicator of dermal exposure to BaP. The percutaneous absorbed dose and absorption flux were estimated by comparison with intravenous administration of BaP (0.01 and 0.05 mg/kg in Cremophor) as reference way. A percutaneous absorption flux of 0.37 microg/cm(2)/h was determined by killing groups of rats, following exposure time of 4.5 and 24 h. [(14)C] skin content was 3.1 microg/cm(2), after 24 h exposure to BaP. Total urinary 3-OHBaP accounted for 0.4% of the real absorbed dose, which was fourfold higher than the percentage of an intravenous dose excreted as 3-OHBaP. This finding reveals that percutaneous absorption of BaP, based on the ratio of urinary excretion of 3-OHBaP following percutaneous exposure compared to percutaneous absorption following intravenous administration of BaP, is overestimated in the rat. In vitro, BaP was intensively metabolised by rat skin. Unchanged BaP and 3-OHBaP in receptor fluid accounted for 50 and 30% of the total radioactivity. This percutaneous first past effect of BaP in rats could, in part, explain the higher urinary excretion ratio of 3-OHBaP compared to the value based on intravenous administration of BaP. Conversely, BaP was largely lower metabolised as 3-OHBaP during percutaneous absorption by humans, so BaP absorption flux should be overestimated to a lesser extent in humans than in rats.

3-Hydroxybenzo[a]pyrene in the urine of workers with occupational exposure to polycyclic aromatic hydrocarbons in different industries

Objectives: This study was conducted to assess external and internal exposure of workers to polycyclic aromatic hydrocarbons (PAHs). In this context, the analytical and diagnostical reliability of 3-hydroxybenzo[a]pyrene (3OH-BaP) as...

3-Hydroxybenzo[a]pyrene in the urine of smokers and non-smokers

The people studied were male volunteers without occupational and dietary exposure to PAH: 27 smokers (10 cigarettes or more) and 27 non-smokers matched for age and socio-professional category. For each person, all the 24 h voided urine samples were reassembled in a single sample. 1-Hydroxypyrene (1-OHPy) and 3-hydroxybenzo[a]pyrene (3-OHBaP) were then determined by automated column-switching high-performance liquid chromatography. Urinary 1-OHPy ranged from 0.041 to 0.530 μmol/molCreatinine (arithmetic mean 0.144, median 0.115) for smokers and from 0.01 to 0.148 μmol/molCreatinine (arithmetic mean 0.044, median 0.032) for non-smokers. These values are close to those of some other studies. Urinary 3-OHBaP ranged from <0.01 to 0.084 nmol/molCreatinine (arithmetic mean 0.030, median 0.023) for smokers and from <0.01 to 0.045 nmol/molCreatinine (arithmetic mean 0.014, median 0.011) for non-smokers. Considering more particularly the urinary 3-OHBaP values, the influence of smoking could be important among workers exposed to low levels of BaP (<100 ng/m 3) and the concentrations for smokers were equivalent to most of the preshift values of exposed workers. The dietary BaP intake was slightly lower than the BaP intake for an average smoker. From the present study, temporary basic reference levels may be proposed for urinary 3-OHBaP.