Frans J. Jongeneelen

Determination of hydroxylated metabolites of polycyclic aromatic hydrocarbons in urine

9,101, show excess rates of lung cancers. Estimates of the health risks due to environmental or occupational exposure to these compounds strongly depend on the quality of analytical methods used to assess the intake or uptake of PAHs. In this respect, an important role is played by biological monitoring, i.e. the systematic registration of the exposure level of individual workers and groups of workers, measured in biological specimens. PAHs are metabolized extensively and the enzymes involved are classified in two broad categories: phase 1 enzymes, which catalyse oxidative reactions, and phase 2 enzymes, which catalyse conju- gative reactions of oxidized PAHs with endogenous compounds such as sulphuric acid, glucuronic acid and glutathione. PAH metabolites may be excreted either as free or as conjugated compounds. The known carcinogenic PAHs are of a large molecular type

1-Hydroxypyrene in human urine after exposure to coal tar and a coal tar derived product

SummaryA method for isolating 1-hydroxypyrene from urine is described. The presence of 1-hydroxypyrene in urine was identified by fluorescence excitation and emission scanning after HPLC-separation. 1-Hydroxypyrene could be detected in the urine of rats following oral administration of as little as 0.5 μg pyrene. The dose-dependence of 1-hydroxypyrene in urine was evident after a wide range of pyrene dosing. After therapeutical coal tar treatment of dermatological patients the enhanced excretion of 1-hydroxypyrene was highly significant. Employees of a creosote impregnating plant showed an excretion pattern of 1-hydroxypyrene which could be related to their work. 1-Hydroxypyrene in urine of non-exposed people was very low, but detectable. It is suggested that the method reported is suitable for the assessment of uptake of man to pyrene, a compound that is commonly present in work environments which are associated with pollution of polycyclic aromatic hydrocarbons.

Smoking and dietary intake of polycyclic aromatic hydrocarbons as sources of interindividual variability in the baseline excretion of 1-hydroxypyrene in urine.

SummarySeventy-six male volunteers, who were not occupationally exposed to polycyclic aromatic hydrocarbons (PAHs), participated in a study on the effect of tobacco smoking, alcohol consumption, dietary PAH intake, age, and body fat content on the baseline excretion of 1-hydroxypyrene in urine. Major determinants of urinary 1-hydroxypyrene excretion were smoking, dietary PAH intake, and age. The mean 1-hydroxypyrene concentrations in the urine of the volunteers in this study ranged between 0.05 and 0.79 μmol/mol creatinine. Smokers excreted on average 0.25 μmol/mol creatinine (range: 0.10–0.79 μmol/mol creatinine), and nonsmokers on average 0.12 μmol/mol creatinine (range: 0.04–0.29 μmol/mol creatinine). The average number of cigarettes smoked per day correlated well with urinary 1-hydroxypyrene concentrations (rs = 0.67, P < 0.001). The consumption of PAH-containing food products and active smoking account for 99% of total pyrene intake. The effect of age on 1-hydroxypyrene excretion is probably caused by a lower creatinine excretion in the elderly. Passive smoking and fat content had a statistically significant, but negligible effect on urinary 1-hydroxypyrene excretion. Passive smoking and the inhalation of ambient air are relatively inimportant for total pyrene intake (both account for less than 1%). Neither the consumption of alcohol nor the inhalation of ambient air significantly affected urinary 1-hydroxypyrene excretion. It is concluded that when urinary 1-OH-pyrene excretion is used in the assessment of PAH exposure, one should particularly be aware of the interindividual variability of the baseline excretion of PAH metabolites due to tobacco smoking and dietary PAH intake.

Biological exposure limit for occupational exposure to coal tar pitch volatiles at cokeovens.

SummaryBiological monitoring is an efficient tool in the evaluation of exposure to chemical agents. However, the dose-response of adverse health effects using biological exposure indices and biological limit values are rarely available. This paper presents an estimation of the occupational exposure limit value of 1-hydroxypyrene in urine, a biological exposure indicator of polycyclic aromatic hydrocarbons (PAH). A large-scale study of the exposure of cokeoven workers to PAH, in wich both air sampling (benzene soluble matter and individual PAH including benzo(α)pyrene) and biological monitoring (1-hydroxypyrene in urine) were applied, made it possible to establish an empirical mathematical relationship between the air sampling data and biological monitoring data. It was calculated that cokeoven workers with a urinary concentration of 1-hydroxypyrene of 2.3 μmol/mol creatinine after a 3-day working period equals the airborne threshold limit value (TLV) of coal tar pitch volatiles (CTPV). Epidemiological studies have quantified the relative risk of lung cancer for topside and non-topside cokeoven workers. The published environmental exposure data of topside and non-topside cokeoven workers were used to determine the time-average exposure. The data of 1-hydroxypyrene in the urine of cokeoven workers and data of epidemiological studies from different coke plants were combined according to the concentrations of PAH in the air. Thus, it was possible to establish an indirect relationship between lung cancer mortality risk and the biological exposure indicator for cokeoven workers. Exposure at the level of the suggested tentative biological exposure limit (BEL) of 2.3 μmol/ mol creatinine is estimated to be equal to a relative risk of lung cancer of approximately 1.3.

The relationship between polycyclic aromatic hydrocarbons in air and in urine of workers in a Söderberg potroom.

The relationships between increase of urinary 1-hydroxypyrene over the workweek and the airborne concentrations of benzo(a)pyrene and coal tar pitch volatiles (CTPVs) were studied among groups of workers in a vertical-stud Söderberg potroom of an aluminum smelter. There was a strong correlation between the natural logarithm of the pyrene concentration and the natural logarithm of the total polycyclic aromatic hydrocarbon (PAHs) concentration in personal air samples (r = 0.94). 1-Hydroxypyrene as the major metabolite of pyrene, a polycyclic aromatic hydrocarbon, was used as a marker for exposure to PAHs. A strong positive correlation was found between the natural logarithm of increase of urinary 1-hydroxypyrene and the natural logarithm of the estimated airborne PAH exposure (R2 = 0.84 with CTPV) when the use of facial protective clothing was taken into account. The relationship between increase of urinary 1-hydroxypyrene and PAHs differed for workers who used facial protective clothing under their respirators compared to those who did not. A contradictory fact was found: the use of facial protection seemed to lead to an elevated increase of 1-hydroxypyrene over the workweek. The regression model for the relationship between increase of urinary 1-hydroxypyrene over the workweek and airborne CTPV was: LN(change in 1-hydroxypyrene) = -4.7 + 1.2 LN(CTPV) -0.44 LN(CTPV)*(use of facial protection) + 3.5 (use of facial protection).

Evaluation of urinary excretion of l-hydroxypyrene and thioethers in workers exposed to bitumen fumes

SummaryBiological monitoring of exposure to bitumen fumes during road-paving operations was carried out. In order to evaluate the biological uptake of the workers, the nonselective urinary thioether assay and a selective method for the determination of urinary l-hydroxypyrene were used. Urinary thioether data of exposed workers were higher than those of nonexposed subjects. The effect of smoking, however, was stronger than the effect of occupational exposure. Levels of l-hydroxypyrene in road-paving workers were significantly higher than those in control subjects. The 1-hydroxypyrene level was also influenced by smoking habits, but the effect of occupational exposure was stronger. Our present data suggest that enhanced urine levels of both thioethers and 1-hydroxypyrene in bitumen workers may indicate an increased genotoxic risk. Furthermore, our results demonstrate the applicability of the 1-hydroxypyrene assay after occupational exposure to petroleum-based products.

Determination of four metabolites of the plasticizer di (2-ethylhexyl) phthalate in human urine samples

SummaryA method for biological monitoring of exposure to the plasticizer di(2-ethylhexyl)phthalate (DEHP) is described. In this method the four main metabolites of DEHP [i.e., mono (2-ethylhexyl) phthalate (MEHP), mono (5-carboxy-2-ethylpentyl)phthalate, mono(2-ethyl-5-oxohexyl)phthalate, and mono(2-ethyl-5-hydroxyhexyl)-phthalate] are determined in urine samples. The procedure includes enzymatic hydrolysis, ether extraction, and derivatization with triethyloxonium tetrafluoroborate. Analysis is performed by gas chromatography electron impact mass spectrometry. The detection limit for all four metabolites is less than 25 μg/l urine. The coefficient of variation based on duplicate determinations of urine samples of workers occupationally exposed to DEHP was 16% for MEHP (mean concentration 0.157 mg/l) and 6% -9% for the other three metabolites (mean concentrations 0.130-0.175 mg/1). The method described here was used to study DEHP metabolism in man. Most persons excrete mono(2-ethyl-5-oxohexyl)-phthalate and mono (2-ethyl-5-hydroxyhexyl)phthalate as a (glucuronide) conjugate. Mono (5-carboxy-2-ethyl-pentyl)phthalate is mainly excreted in free form, while for MEHP a large interindividual variation in conjugation status was observed. Of the four metabolites quantified, 52% are products of a ((ω-l)-hydroxylation reaction of MEHP [i.e., mono (2-ethyl-5-oxohexyl)phthalate and mono (2-ethyl-5-hydroxyhexyl)phthalate], 22% is the product of a ω-hydroxylation reaction of MEHP [i.e., mono (5-carboxy-2-ethylpentyl)phthalate], and 26% is not oxidized further (i.e., MEHP). A good correlation is obtained when the amount of MEHP ω-hydroxylation products is compared with the amount of MEHP (ω-1)hydroxylation products in urine samples. When the internal dose of DEHP has to be established we recommend that the levels of all four metabolites of DEHP be studied in urine samples.

Absorption of polycyclic aromatic hydrocarbons through human skin: differences between anatomical sites and individuals.

In order to determine differences in absorption of polycyclic aromatic hydrocarbons (PAH) between anatomical sites and individuals, coal-tar ointment was applied to skin of volunteers at various sites. The surface disappearance of PAH and the excretion of urinary 1-OH-pyrene after skin application of coal-tar ointment were used as parameters for dermal PAH absorption. The surface disappearance was determined by the measurement of the fluorescence of PAH on skin. Surface disappearance measurements show low but significant differences in dermal PAH absorption between anatomical sites: shoulder > forehead, forearm, groin, > ankle, hand (palmar site). The average PAH absorption rate constant at different skin sites ranges from 0.036/h to 0.135/h (overall mean: 0.066/h). This indicates that after 6 h of exposure, 20-56% of a low dermal dose of PAH (e.g., about 1.0 ng pyrene/cm2) will be absorbed. The interindividual differences in PAH absorption are small (7%) in comparison with differences between anatomical sites (69%). Results based on the urinary excretion of 1-OH-pyrene are less clear. The site of application of the coal-tar ointment (dose: 2.5 mg/cm2 during 6 h) has no significant effect on the excreted amount of 1-OH-pyrene in urine. It is estimated that after coal-tar ointment application on skin, 0.3-1.4% of the pyrene dose (about 2 micrograms pyrene/cm2) becomes systemically available. For the accurate estimation of PAH uptake through skin of workers, it seems relevant to distinguish different body regions, not only because of the regional variation in percutaneous PAH absorption, but also because of the high dispersal of PAH contamination on skin of workers.

Metabolites of the plasticizer di(2-ethylhexyl)phthalate in urine samples of workers in polyvinylchloride processing industries

SummaryLittle is known about occupational exposure to the plasticizer di(2-ethylhexyl)phthalate (CAS number 117-81-7), a compound widely used in polyvinylchloride (PVC) plastics. We have studied the uptake of DEHP in workers by determining the concentrations of four metabolites of DEHP in urine samples, i.e., mono(2-ethylhexyl)phthalate (MEHP), mono (5-carboxy-2-ethylpentyl)phthalate, mono(2-ethyl-5-oxohexyl)phthalate, and mono(2-ethyl-5-hydroxyhexyl)phthalate. In addition DEHP concentrations in the air were determined by personal air sampling. Nine workers in a PVC boot factory exposed to a maximum of 1.2 mg/m3 DEHP showed an increase in the urinary concentrations of all four metabolites over the workshift. These results were obtained on both the first and the last day of the workweek. With the exception of MEHP, the increases in the concentrations of the metabolites during a workday were statistically significant. Six workers from a PVC cable factory exposed to a maximum of 1.2 mg/m3 DEHP showed a one-to fourfold increase in the concentrations of the four metabolites over the workshift, but these increases were not statistically significant. These results indicate that measurement of DEHP metabolites in urine samples may be of use for monitoring the occupational exposure to DEHP.

Biological monitoring of human exposure to coal tar. Urinary excretion of total polycyclic aromatic hydrocarbons, 1-hydroxypyrene and mutagens in psoriatic patients.

SummaryThree methods for the biological monitoring of human exposure to coal tar were compared. Levels of 1-hydroxypyrene(1-OH PYR), polycyclic aromatic hydrocarbons (PAH) and mutagens (Ames plate incorporation assay using Salmonella typhimurium strain TA98 in the presence of S9 and β-glucuronidase) were determined in urinary samples from psoriatic patients undergoing topical treatment with mineral coal tar. A single sample of urine with a high content of PAH was diluted with urine of non-exposed, non-smoking subjects in order to obtain nine samples with a decreasing content of PAH metabolites. Mutagenicity of the extracts was detectable down to the dilution corresponding to a content in 1-OH PYR of about 50 μg/g creatinine and total PAH of 7 μg/g creatinine. In a second phase the three indicators of exposure to PAH were compared in 16 urinary samples from four psoriatic patients. The total PAH levels determined by the acidic deconjugation/reduction method were confirmed to be nearly always lower than the corresponding levels of 1-OH PYR alone. Most of the extracts were mutagenic, however, some of the samples with a high content in PAH metabolites were not mutagenic. In all the urinary samples analyzed the excretion of 1-OH PYR was markedly greater than in control subjects. 1-OH PYR and urinary mutagenicity levels were well correlated. The present data suggest that both the determination of mutagenicity and 1-OH PYR in urine may be used to monitor occupational exposure to PAH, the latter method being cheaper and of greater specificity and sensitivity.