Jytte Molin Christensen

Human exposure to toxic metals: factors influencing interpretation of biomonitoring results

An important approach to assessment of risk from environmental and occupational exposures is biomonitoring which provides an estimate of the total dose absorbed and gives indirect access to determination of target site concentrations. It is also a tool for assessing residual exposure, when respiratory protection is used. The interest in biological effects from toxic metals has increased during the last decades, as large amounts of metals have been released into industrial areas, and it is estimated that approximately 150,000 Danish workers are exposed to various metals. Since biomonitoring results play an important role in decision-making regarding great health and economic impact, understanding the factors influencing validity of such data is essential. In the present survey, the toxic elements arsenic, cadmium, chromium, cobalt, lead and nickel are used as examples to illustrate the disturbing factors in the interpretation of biomonitoring results. The aim of collecting samples is to obtain a small and representative sample of subjects or of a subpopulation being investigated for specific purposes, e.g. the pollution at Mundelstrup, where arsenic exposure of inhabitants and workers engaged in removing contaminated soil was monitored. As exposures vary over time and between subjects, it should be recognized that sampling as well as analytical variations contribute both to bias and random errors. Also biomonitoring data are a function of demographic, lifestyle and geographic factors. Therefore, stratified sampling designs are of the utmost importance. Half-lives play an important role. For short half-lives below 10 h, no decision can be made on long-term exposure if only one urine sample has been taken. For arsenic, cobalt, chromium and nickel in urine measured at the end of a workweek (t1/2: 20-100 h), 2-3 samples should be taken to monitor a single worker, and the results should be interpreted from the average of the results. For groups of workers, it is recommended that results from at least 5-10 workers be used to obtain a useful group mean value. In general, pharmacokinetics modelling contributes to information on sampling time and sampling size. Intake of cobalt in mineral tablets containing soluble cobalt compounds was a factor with a large influence on blood and urinary levels. Age and gender influence the blood and urine concentrations of arsenic, cadmium, cobalt and lead, e.g. B-Pb in females is 20-30% lower than B-Pb in males.(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of a radioimmunoassay and establishment of a reference interval for salivary cortisol in healthy subjects in Denmark

A commercial radioimmunoassay (RA) for salivary cortisol was evaluated using certified reference material in water and spiked to pooled saliva in the range 2.1–89.1 nmol/L. A variance component model for describing the effects of age, body mass index (BMI), diurnal variation, gender, days of sick leave during the past year, and smoking habits was established. Reference intervals for salivary cortisol in 120 healthy individuals performing their routine work were established according to the International Union of Pure and Applied Chemistry (IUPAC) and the International Federation of Clinical Chemistry (IFCC). The method evaluation of the certified reference material in water did not show any bias of the method, i.e. recovery was 97% [CI: 94%; 100.9%]. LOD (detection limit) was 1.59 nmol/L. The ratio between analytical and within‐subject variation (CVa/CVi) was 0.14, indicating that the method was adequate for measurement in healthy subjects. Reference intervals were estimated to be from 3.6 to 35.1 nmol/L for samples at the time of awakening (05.27–07.27), 7.6–39.4 nmol/L for peak level in saliva samples collected 20 min after awakening (05.47–07.47), and LOD 10.3 nmol/L for late afternoon samples (17.00–19.00). Reactivity (increase from awakening to 20 min after awakening) was estimated to be 82% [CI: −179; 345%] and recovery (decrease from 20 min after awakening to 18.00) to be 80% [CI: 51; 109%]. Eighteen percent of the subjects showed a decrease in cortisol in saliva from awakening to 20 min after awakening. Salivary cortisol was not affected by age, body mass index, gender, smoking habits or days of sick leave during the past year.

Seasonal and biological variation of urinary epinephrine, norepinephrine, and cortisol in healthy women.

BACKGROUND There is a significant circadian and seasonal periodicity in various endocrine functions. The present study describes the within-day and seasonal fluctuation for urinary catecholamines and cortisol and estimates the within- (CV(i)) and between-subject (CV(g)) coefficients of variation for healthy women undertaking their routine work. In addition, index of individuality (I(i)) and power calculations were derived. METHODS Eleven healthy females undertaking their routine life-style at work participated in the study. Each subject collected six samples during 24 h 15 days over a year, giving a total number of 990 samples. Using a random effect analysis of variance, we estimated CV(g) and total within-subject variation (CV(ti)), i.e. combined within-subject and analytical variation, from logarithmically transformed data. Analytical variation was subtracted from CV(ti) to give CV(i). CV(i) was estimated from samples collected monthly during 1 year (CV(iy)), weekly during 1 month (CV(im)), and six to eight times/day (CV(id)). RESULTS A seasonal variation was demonstrated for excretion of epinephrine, norepinephrine, and cortisol standardized with creatinine. Concentrations of urinary epinephrine were higher during June and July compared to the rest of the year, whereas concentrations of urinary cortisol were higher during December and January compared to the rest of the year. Excretion of norepinephrine was lower during working hours and higher during hours off work for June and July compared to the rest of the year. There was a high within- and between-subject variation, which could not be explained by menstrual cycle, behavioral, emotional, or cognitive stress reactions. CONCLUSIONS Despite high biological variation a reasonably low sample size, e.g. 10-50 individuals, is adequate for practical applicability, i.e. studying differences above 150%. The present study recommends to include the sampling time in the statistical evaluation of data and to be aware of the changes in diurnal variations over seasons. When single measurements are to be evaluated, reference intervals are recommended.

Cancer incidence among Danish workers exposed to trichloroethylene.

Human evidence regarding the carcinogenicity of the animal carcinogen trichloroethylene (TCE) is limited. We evaluated cancer occurrence among 803 Danish workers exposed to TCE, using historical files of individual air and urinary measurements of TCE-exposure. The standardized incidence ratio (SIR) for cancer overall was close to unity for both men and women who were exposed to TCE. Men had significantly elevated SIRs for non–Hodgkin’s lymphoma (SIR = 3.5;n = 8) and cancer of the esophagus (SIR = 4.2;n = 6). Among women, the SIR for cervical cancer was significantly increased (SIR = 3.8;n = 4). No clear dose-response relationship appeared for any of these cancers. We found no increased risk for kidney cancer. In summary, we found no overall increase in cancer risk among TCE-exposed workers in Denmark. For those cancer sites where excesses were noted, the small numbers of observed cases and the lack of dose-related effects hinder etiological conclusions.

Correlation between work process-related exposure to polycyclic aromatic hydrocarbons and urinary levels of α-naphthol, β-naphthylamine and 1-hydroxypyrene in iron foundry workers

SummaryIn two Danish iron foundries the concentration of polycyclic aromatic hydrocarbons (PAH) in 24 personal air samples of workers employed in selected processes, i.e. melters, melted iron transporters, casters, machine molders, hand molders, shake-out workers and finishing workers, were measured and correlated to levels of 1-hydroxypyrene, α-naphthol and β-naphthylamine in the urine of exposed workers. The highest total airborne PAH concentrations (sum of 15 selected PAH compounds: 9.6–11.2 μg/m3) were associated with casting, machine molding, and shake-out. The highest concentrations of the sum of six selected airborne carcinogenic PAH compounds were found for melting, casting and machine and hand molding. As seen in other working environments involving low-level PAH exposure, the content of naphthalene was high, in general exceeding 85% of the total content of PAH compounds. The present study demonstrates that 1-hydroxypyrene is a useful and direct biomarker of low-dose occupational exposure to PAH compounds. Molding and casting had the highest pyrene levels in iron foundries. Furthermore, the data shows that levels of β-naphthylamine in urine are significantly elevated in iron foundry workers. Hand molders, finishing workers and truck drivers tended to have the highest levels. Concerning α-naphthol the highest concentrations were measured in urine from casters and shake-out workers. With regard to epidemiologic studies demonstrating that molders and casters have a higher risk of lung cancer, the present study suggests that the elevated risk may be due to exposure to carcinogenic PAH compounds in iron foundries, particularly in some high-risk work processes, e.g. casting and molding. In addition, the present study suggests that biological monitoring of 1-hydroxypyrene and β-naphthylamine may be used to estimate the individual exposure, which seems to be correlated with exposure during individual work processes.

Effect of seafood consumption on the urinary level of total hydride-generating arsenic compounds. Instability of arsenobetaine and arsenocholine

Arsenobetaine and arsenocholine are considered to be non-toxic and are present as a relatively large proportion of total arsenic in seafoods, and they do not respond to hydride generation. The present study describes the effect of seafood consumption on the urinary concentration of hydride-generating arsenic compounds measured by a newly developed flow injection atomic absorption spectrometric (FI-AAS) method. Consumption of plaice, pighvar and tunny resulted in a 2-fold increase, and consumption of mussels produced a 6-fold increase in the urinary level of hydride-generating arsenic compounds. Hence, a person who has consumed mussels may be suspected of being occupationally or environmentally exposed, if the level of consumption of this seafood is unknown. As the FI-AAS method cannot be used to detect arsenobetaine and arsenocholine, the observed increase in urinary concentration of hydride-generating arsenic compounds after consumption of seafood must originate either from hydride-generating arsenic compounds in the seafood or from degraded arsenobetaine or arsenocholine. The present study has demonstrated that both arsenobetaine and arsenocholine are unstable when incubated in daylight in the presence of hydrogen peroxide, i.e., an oxidizing environment. Hence, it is tempting to speculate that arsenobetaine could be converted into hydride-generating arsenic compounds during storage or cooking of seafood. The feasibility of speciation methods based on high-performance liquid chromatographic (HPLC) separation and on-line analysis by inductively coupled plasma atomic emission spectrometry (ICP-AES) and FI-AAS was also investigated. The FI-AAS system is approximately 35 times more sensitive to the hydride-generating arsenic species than the ICP-AES system.(ABSTRACT TRUNCATED AT 250 WORDS)

Nickel-induced cytokine production from mononuclear cells in nickel-sensitive individuals and controls. Cytokine profiles in nickel-sensitive individuals with nickel allergy-related hand eczema before and after nickel challenge.

Abstract Exposure to nickel is a major cause of allergic contact dermatitis which is considered to be an inflammatory response induced by antigen-specific T cells. Here we describe the in vitro analysis of the nickel-specific T-cell-derived cytokine response of peripheral blood mononuclear cells from 35 nickel-allergic and 30 non-nickel-allergic individuals. Peripheral blood mononuclear cells were stimulated with 10 –4 and 10 –5 mol/l NiSO 4 for 6 days and then additionally with ionomycin and phorbol myristate acetate for 24 h. Culture supernatants were analysed for interleukin-4 (IL-4), IL-5, interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) by quantitative ELISA. The analysis showed that the synthesis of IL-4 and IL-5 but not of IFN-γ or TNF-α was significantly higher in the nickel-allergic individuals. The finding of preferential synthesis of Th2 cytokines was somewhat of a surprise, since previous studies have suggested a Th1 response in nickel-mediated allergic contact dermatitis. Subsequently, the nickel-allergic individuals were randomized to experimental exposure to nickel or vehicle in a double-blind design. A daily 10-min exposure of one finger to 10 ppm nickel solution for 1 week followed by 100 ppm for an additional week evoked a clinical response of hand eczema in the nickel-exposed group. Blood samples were drawn on days 7 and 14 after the start of this exposure to occupationally relevant concentrations of nickel. No statistically significant differences were observed in the nickel-induced in vitro cytokine response during the exposure period. Our results indicate the possibility that IL-4 and IL-5 are involved in the pathogenesis of nickel-mediated contact dermatitis.

Biomonitoring of genotoxic exposure among stainless steel welders

A biosurvey in the Danish metal industry measured the genotoxic exposure from stainless steel welding. The study comprised measurements of chromosomal aberrations (CA), sister-chromatid exchanges (SCE), unscheduled DNA synthesis (UDS) in peripheral lymphocytes and serum immunoglobulin G. Environmental monitoring of welding fumes and selected metal oxides, biomonitoring of chromium and nickel in serum and urine and mutagenic activity in urine, and evaluation of semen quality were also done. Manual metal arc (MMA) welding and tungsten inert gas (TIG) welding were the dominant welding processes. A higher frequency of chromosomal aberrations, classified as translocations, double minutes, exchanges and rings, was observed in stainless steel welders than in non-welders. SCE was lower in welders working with both MMA and TIG welding than in reference persons. N-Acetoxy-N-acetylaminofluorene (NA-AAF)-induced UDS was lower in 23 never-smoking welders than in 19 unexposed never-smokers. Smoking was a confounding factor resulting in significantly higher CA, SCE, NA-AAF binding to DNA and mutagenic activity in urine. Age was also a confounder: CA, SCE, NA-AAF binding to DNA and UDS increased significantly with age. No significant correlation between SCE and CA or between CA and UDS was found. UDS decreased significantly with increasing lymphocyte count and a higher lymphocyte count was seen in MMA welders than in reference persons and in smokers than in non-smokers. Differences in the composition among lymphocytes in exposed persons compared with non-exposed are suggested. MMA welding gave the highest exposure to chromium, an increased number of chromosomal aberrations and a decrease in SCE when compared with TIG welding. Consequently improvements in the occupational practice of stainless steel welding with MMA is recommended.

Validation of a high performance liquid chromatography analysis for the determination of noradrenaline and adrenaline in human urine with an on-line sample purification

A high performance liquid chromatography (HPLC) method with fluorescence detection including an on-line purification was established for determination of catecholamines in human urine. The method was evaluated using samples of pooled urine spiked with catecholamines and validated for measurements of catecholamines in urine of healthy individuals in a field study. The laboratory method evaluation study showed that the recovery of the method was 0.82 (confidence interval (CI): 0.79-0.86) and 0.92 (CI: 0.89-0.95) for noradrenaline and adrenaline, respectively. Thus, correction factors of 0.82(-1) and 0.92(-1) were applied to correct the measurement results for this systematic effect. Furthermore, an uncertainty budget was generated for the analytical method using the BIPM-approach recommended by the International Organization for Standardization. The relative uncertainty of the method was estimated to be 10-12%, which was consistent with the observed relative variability found in the method evaluation. The method was evaluated in accordance with EURACHEM Guidance Document No 1 concerning accreditation for chemical laboratories with respect to accuracy and precision. The field study showed that the standard deviation of the method was sufficiently low to distinguish between persons working with two different processes of garbage collection, i.e. collection using four wheeled containers versus collection using bins.

A 1982–1992 surveillance programme on Danish pottery painters. Biological levels and health effects following exposure to soluble or insoluble cobalt compounds in cobalt blue dyes

This paper provides a short overview of cobalt-related diseases with particular reference to the potential carcinogenicity of cobalt compounds, and a review of a 10-year surveillance programme on plate painters exposed to cobalt in two Danish porcelain factories. Clinical experience and epidemiological studies have demonstrated that cobalt exposure may lead to severely impaired lung function, i.e. hard metal lung disease and occupational cobalt-related asthma, contact dermatitis and cardiovascular effects. However, the evidence for the carcinogenicity of cobalt and cobalt compounds is considered inadequate (IARC, 1991). Most frequently, exposure to cobalt occurs simultaneously with exposure to other elements known to pose a health risk, (e.g. nickel, arsenic, chromium, tungsten). The importance of cobalt as sole causal agent in hard metal lung diseases, cardiomyopathy and cancer are still a matter of controversy. In the two Danish porcelain factories, cobalt blue underglaze dyes have been used since 1888. In contrast to the exposure experience of hard metal factories, the exposure of plate painters occurs with only low trace levels of other potentially harmful compounds such as the carcinogenic metals nickel, arsenic and chromium. Consequently, the nearly-pure cobalt exposure makes the plate painters an attractive group for studies on the health effects of cobalt. During the period 1982-1992 the surveillance programme showed a profound reduction in the urine level of cobalt (Co-U) from 100-fold to 10-fold above the median level of the unexposed control subjects. In the same period, the airborne cobalt exposure declined from 1356 nmol/m3 to 454 nmol/m3, the Danish occupational exposure limit being 845 nmol/m3. In 1982, when the cobalt exposure was above the occupational exposure limit, the plate painters showed a chronic impaired lung function. The obstructive effects may be similar to some of the effects observed in hard metal workers. In 1988, a study on the effect of cobalt exposure at low levels revealed no inhibitory effects on thyroid function, but the ratio between T4 and T3 increased, indicating that low cobalt exposure may have an impact on the metabolism of thyroid hormones. Parallel studies were conducted on the metabolism and excretion of cobalt. The gastrointestinal uptake of soluble CoCl was considerably higher than the uptake of insoluble cobalt(II) oxide. In addition, it was demonstrated that ingestion of controlled amounts of the soluble cobalt compound resulted in significantly higher concentrations of cobalt in urine and blood (Co-B) from females compared with males (P < 0.01). Future studies will involve epidemiology and genotoxicity to evaluate the previous and present cancer risk, and detailed process-related exposure assessment studies to select the methods most reliable for surveillance of low-dose cobalt exposure.