Reply to: A commentary on “Hair analysis for biomonitoring of environmental and occupational exposure to organic pollutants.”

In his comment on the review by Appenzeller and Tsatsakis, Doréa highlights the use of hair analysis for the biomonitoring of human exposure to mercury. Indeed, the relevance of this matrix for this specific purpose has been demonstrated in numerous works. We acknowledge that the review was voluntarily limited to “purely organic” pollutants, which excluded organic forms of metals such as methylmercury and ethylmercury. We also remind some important aspects related to the use of hair as a biomarker of exposure to mercury.     

Hair analysis for biomonitoring of environmental and occupational exposure to organic pollutants: state of the art, critical review and future needs

This paper presents the current state of the art in human hair analysis for the detection of organic pollutants associated with environmental and occupational exposure. The different chemical classes are reviewed with a special focus set on compounds that were only recently investigated. The importance of methods sensitivity and particularly the influence of this parameter on the results presented in previous publications is highlighted. This report also investigates the relevance of hair analysis as an indicator of subjects' level of exposure and underlines limitations that are still associated with this matrix. This study also presents a critical assessment of some specific aspects presented in the literature as well as future needs to strengthen the position of hair as a relevant biomarker of exposure to be used in epidemiological studies.     

Impact of a risk-benefit advisory on fish consumption and dietary exposure to methylmercury in France

We designed the CORAI (COnsumer Risk Advisory Inquiry) study to observe consumer reactions' after an advisory revealing risk of methylmercury contamination together with benefits of Long-Chain Poly Unsaturated Fatty Acids of the n-3 variety (LC n-3 PUFA). The message was very close to the ones commonly delivered by national food agencies and included recommendations for women of childbearing age and children below 15 years old. Two groups of subjects including consumers at risk were selected. Participants recorded the frequency of their fish consumption detailed by species for them and their family over a one-month period one month before, a month immediately after and 3 month after the advisory. Results were compared between consumers receiving the advisory and controls. Results show that the message revelation led to a significant decrease in total fish consumption which is greater for children below 6 years old than for the children between 6 and 15 years old and women. The consumption of the most contaminated fish quoted in the advisory, rarely consumed and poorly known by French consumers did not decrease in any group despite the advice to avoid their consumption. The consumption of other fish products quoted in the advisory but frequently consumed and better known, as canned tuna, did decrease and was a major contributor to the overall reduction of exposure for the advised group. Before the information, about 3% of women of childbearing age are exceeding the PTWI for MeHg and both the average and the high percentiles of the exposure to MeHg are decreasing significantly in the advised group. Regarding the number of subjects of the advised group exceeding the PTWI, they were 6, 3 and 2, respectively, in May, June and September. Accompanying questionnaires show that consumers imperfectly memorize most of the fish species quoted in the recommendation. This paper concludes that consumer advisory, which is a major tool for risk management, has a minimal effect under our experimental conditions to reduce the exposure of groups at risk. Messages to be carried to consumers should be carefully tested for long term memorization in order to become more effective.     

A longitudinal analysis of prenatal exposure to methylmercury and fatty acids in the Seychelles

Background

Maternal fish consumption during pregnancy exposes the fetus simultaneously to methylmercury (MeHg) and long chain polyunsaturated fatty acids (LCPUFA). Data from the Seychelles Child Development Nutrition Study (SCDNS) showed a negative association of MeHg with child development when children were 30 months of age, only when controlling for LCPUFA. Concomitantly, n − 3 LCPUFA were found to have a significant positive association only at 9 months. These findings suggest that the effects of MeHg and LCPUFA may vary with age over the first few years of life. We address this by including outcomes at two ages and adjusting for the child's age at testing.

Methods

A longitudinal analysis utilizing linear mixed models was performed to assess the associations of maternal hair total mercury (THg, a biomarker for MeHg) and maternal LCPUFA with children's Bayley Scales of Infant Development Psychomotor Developmental Index (BSID-II PDI) at 9 and 30 months of age, and to determine whether these associations change over time. Data from 228 children were included.

Results

Maternal hair MeHg had a negative effect on BSID PDI, while maternal n − 3 LCPUFA had a positive effect. These effects did not change significantly from 9 to 30 months in this analysis.

Conclusions

The longitudinal analysis provides increased power for estimating the relationships of prenatal MeHg and LCPUFA exposures during child development. Significant associations of these exposures in opposite directions confirm the importance of LCPUFA in development and the need to adjust for maternal nutrition when studying prenatal MeHg exposure.     

Human exposure to bisphenol A by biomonitoring: methods, results and assessment of environmental exposures

Human exposure to bisphenol A is controversially discussed. This review critically assesses methods for biomonitoring of bisphenol A exposures and reported concentrations of bisphenol A in blood and urine of non-occupationally ("environmentally") exposed humans. From the many methods published to assess bisphenol A concentrations in biological media, mass spectrometry-based methods are considered most appropriate due to high sensitivity, selectivity and precision. In human blood, based on the known toxicokinetics of bisphenol A in humans, the expected very low concentrations of bisphenol A due to rapid biotransformation and the very rapid excretion result in severe limitations in the use of reported blood levels of bisphenol A for exposure assessment. Due to the rapid and complete excretion of orally administered bisphenol A, urine samples are considered as the appropriate body fluid for bisphenol A exposure assessment. In urine samples from several cohorts, bisphenol A (as glucuronide) was present in average concentrations in the range of 1-3 microg/L suggesting that daily human exposure to bisphenol A is below 6 microg per person (<0.1 microg/kg bw/day) for the majority of the population.     

Urine and serum biomonitoring of exposure to environmental estrogens I: Bisphenol A in pregnant women

Despite its very low oral bioavailability and rapid elimination, multiple reports of unexpectedly high bisphenol A (BPA) concentrations in the serum of pregnant mothers or cord blood have raised questions about BPA exposures during pregnancy. Thirty healthy pregnant women recruited to the study were evaluated for total BPA exposure over a 30-h period comprising one-half day in the field and one day in a clinical setting. BPA and its metabolites were measured in serum and total BPA was measured in matching urine samples. The mean total exposure was similar to the 50^th percentile of exposure for U.S. women and pregnant women in a large North American cohort. Twenty volunteers had total daily exposures equal to or exceeding the U.S. mean, and six volunteers had exposures exceeding the 75th percentile. Women working as cashiers did not have higher total BPA exposure. BPA was detected in some serum samples (0.25–0.51 ng/ml), but showed no relationship to total BPA in corresponding urine samples, no relationship to total BPA exposure, and had unconjugated BPA fractions of 60–80%, consistent with established criteria for sample contamination. We conclude that typical exposures of North American pregnant women produce internal exposures to BPA in the picomolar range.     

Urine and serum biomonitoring of exposure to environmental estrogens II: Soy isoflavones and zearalenone in pregnant women

Urine and serum biomonitoring was used to measure internal exposure to selected dietary estrogens in a cohort of 30 pregnant women. Exposure was measured over a period comprising one-half day in the field (6 h) and one day in a clinic (24 h). Biomonitoring of the dietary phytoestrogens genistein (GEN), daidzein (DDZ) and equol (EQ), as well as the mycoestrogen, zearalenone (ZEN) and its congeners, was conducted using UPLC-MS/MS. Biomonitoring revealed evidence of internal exposure to naturally occurring dietary estrogens during pregnancy. Urinary concentrations of total GEN, DDZ and EQ were similar to levels reported for general adult U.S. population. Measurable concentrations of total (parent and metabolites) GEN, DDZ and EQ were present in 240, 207 and 2 of 270 serum samples, respectively. Six out of 30 subjects had measurable concentrations of unconjugated GEN and/or DDZ in serum between 0.6 and 7.1 nM. Urine to serum total isoflavone ratios for GEN, DDZ and EQ were 13, 47, and 180, respectively. ZEN and its reductive metabolite, α-zearalenol (α-ZEL), were present in pregnant women (11 out of 30 subjects) as conjugates at levels near the limit of quantification. The average total urinary concentration was 0.10 μg/L for ZEN and 0.11 μg/L for α-ZEL.     

Effects of postnatal exposure to methylmercury on spatial learning and memory and brain NMDA receptor mRNA expression in rats

The extreme vulnerability of developing nervous system to methylmercury (MeHg) is well documented. Still unclear is the consequence of different postnatal period exposure to MeHg. We investigated the critical postnatal phase when MeHg induced neurotoxicity in rats and the underlying mechanism. Rats were given 5 mg/(kg day) methylmercury chloride (MMC) orally on postnatal day (PND) 7, PND14, PND28, and PND60 for consecutive 7 days. A control group was treated with 0.9% sodium chloride solution 5 ml/(kg day) instead. On PND69, spatial learning and memory was evaluated by Morris water maze test. Behavior deficits were found in MMC-treated rats of PND7 and PND14 groups (p < 0.01). N-methyl-d-aspartate (NMDA) receptor 2 subunits mRNA expressions were evaluated 3 days after the last administration. In hippocampus, the mRNA expression of NR2A and NR2B decreased, but the NR2C expression increased in PND14 group following MMC-treatment (p < 0.01). In cerebral cortex, mRNA expression of NR2A decreased, with NR2C expression elevating in PND14 group following MMC-treatment (p < 0.05). These observations suggest that the postnatal exposure to MeHg during PND7–20 could cause neurobehavioral deficits which extend to adulthood. Furthermore, the abnormal expression of NMDAR 2 subunits might associate with the impairment.     

Effects of early life exposure to methylmercury in Daphnia pulex on standard and reduced food ration

As a well-known eco-toxicological model organism, Daphnia pulex may also offer advantages in human health research for assessing long-term effects of early life exposures to coupled stressors. Here, we examine consequences of early life exposure to methylmercury (MeHg) under standard and reduced food ration. We exposed Daphnia for 24 h in early life to varying concentrations of methylmercury(II) chloride (0, 200, 400, 800 and 1600 ng/L) and thereafter kept Daphnia on either a standard or a reduced food ration. The data suggests an additive effect of MeHg concentration and food ration on decreasing lifespan, although MeHg concentration does not affect survival linearly. Food ration and MeHg concentration were predictive of reduced reproduction, and there is some evidence of an interaction (p = 0.048). Multi-stressor work in alternative model systems may be useful for prioritizing research, taking into account potential antagonistic, additive or synergistic effects that nutritional status may have on chemical toxicity.     

A dynamic system for single and repeated exposure of airway epithelial cells to gaseous pollutants

In vitro models are promising approaches to investigate the adverse effects and the mode of action of air pollutants on the respiratory tract. We designed a dynamic system that allows the single or repeated exposure of cultured cells to two major indoor air gaseous pollutants, formaldehyde (HCHO) and nitrogen dioxide (NO₂), alone or as a mixture. In this system, the Calu-3 human bronchial epithelial cell line was exposed at the air–liquid interface (ALI) or submerged by culture medium to synthetic air or to target concentrations of HCHO and/or NO₂ once or on 4 consecutive days before assessment of cell viability and necrosis, IL-6 and IL-8 release and trans-epithelial electrical resistance. Our data showed that whereas the ALI method can be used for single short-term exposures only, the submerged method provides the possibility to expose Calu-3 cells in a repeated manner. As well, we found that repeated exposures of the cells to HCHO and NO₂ at concentrations that can be found indoors triggered a significant decrease in cell metabolism and an increase in IL-8 release that were not evoked by a single exposure. Thus, our work highlights the fact that the development of systems and methods that allow repeated exposures of cultured cells to gaseous compounds in mixtures is of major interest to evaluate the impact of air pollution on the respiratory tract.     

The role of interleukin-6 in pulmonary inflammation and injury induced by exposure to environmental air pollutants.

This study was designed to examine the role of the cytokine interleukin-6 (IL-6) in environmental air pollutant-induced pulmonary inflammation, injury, and repair. IL-6 knockout (KO) mice and wild-type (WT) mice were exposed to filtered air; aged and diluted cigarette smoke (ADSS), a surrogate for environmental tobacco smoke; ozone; or ADSS followed by ozone (ADSS/ozone). The proportion of monocytes and neutrophils recovered by bronchoalveolar lavage (BAL) as well as the level of total protein in BAL fluid were significantly increased in both IL-6 KO and WT mice following exposure to ozone or to ADSS/ozone. However, bromodeoxyuridine (BrdU) labeling within terminal bronchiolar epithelium and proximal alveolar regions in IL-6 KO mice exposed to ozone or to ADSS/ozone was significantly reduced compared with IL-6 sufficient mice (WT). WT mice treated with IL-6 antibodies also demonstrated a reduction in BrdU cell labeling similar to that observed in IL-6 KO mice following exposure to ozone or ADSS/ozone. Clara cell secretory protein (CCSP) abundance, a marker of Clara cell maturation and function, was markedly reduced in the terminal bronchiolar epithelium of WT mice following exposure to ADSS and/or ozone, whereas CCSP abundance was unchanged in IL-6 KO mice. We conclude that endogenous IL-6 in mice plays a critical role in the progress of lung inflammation/injury, but CCSP may also play a role to protect the lungs of mice exposed to toxic air pollutants. Data from this study further suggest that IL-6 antibody treatment modalities may be a means to attenuate pulmonary inflammation and injury.     

A model of human nasal epithelial cells adapted for direct and repeated exposure to airborne pollutants

Airway epithelium lining the nasal cavity plays a pivotal role in respiratory tract defense and protection mechanisms. Air pollution induces alterations linked to airway diseases such as asthma. Only very few in vitro studies to date have succeeded in reproducing physiological conditions relevant to cellular type and chronic atmospheric pollution exposure. We therefore, set up an in vitro model of human Airway Epithelial Cells of Nasal origin (hAECN) close to real human cell functionality, specifically adapted to study the biological effects of exposure to indoor gaseous pollution at the environmental level. hAECN were exposed under air–liquid interface, one, two, or three-times at 24 h intervals for 1 h, to air or formaldehyde (200 μg/m³), an indoor air gaseous pollutant. All experiments were ended at day 4, when both cellular viability and cytokine production were assessed. Optimal adherence and confluence of cells were obtained 96 h after cell seeding onto collagen IV-precoated insert. Direct and repeated exposure to formaldehyde did not produce any cellular damage or IL-6 production change, although weak lower IL-8 production was observed only after the third exposure. Our model is significantly better than previous ones due to cell type and the repeated exposure protocol.     

Hair analysis for drugs of abuse XIII. Effect of structural factors on incorporation of drugs into hair: the incorporation rates of amphetamine analogs

 In order to clarify the incorporation mech-anism of drugs from blood into hair, seven effects of structural factors on the incorporation rate (ICR) were studied using 32 amphetamine analogs: (1) effect of a straight chained N-alkyl group; (2) effect of benzene and furan ring at N-position; (3) effect of aliphatic and aromatic hydroxy groups; (4) effect of triple bond group at N-position; (5) effect of N-acyl group and ketone group; (6) effect of methylenedioxy and methoxy groups on benzene ring; and (7) comparison between phenyltertiarybutylamines and phenylisopropylamines. After shaving the back hair and i.p. administration of drugs to Dark-Agouti rats (5 mg/kg, 10 days, n=3), the areas under the concentration versus time curve (AUCs) of drugs in the plasma and the concentrations in hair newly grown for 4 weeks were determined by gas chromatography-mass spectrometry. The ICRs represented by the ratios of hair concentrations to AUCs were compared with those of amphetamine (AP) and methamphetamine (MA). The ICRs of N-alkyl AP increased depending on the length of carbon branches from proton to propyl (C₃>C₂>C₁>H) at N-position. The compounds containing a benzene or furan ring at the N-position (benzphetamine, clobenzorex, norbenzphetamine, prenylamine, furfenorex, and norfurfenorex) had much higher ICRs than those of AP or MA, suggesting that a benzene or furan ring increases their ICRs. The ICRs of deprenyl, nordeprenyl, and fenproporex were significantly low, implying that triple bonds such as of a propargyl or cyano group serve as a negative factor for the ICRs. An ephedrine group (ephedrine, methylephedrine, phenylpropanolamine) showed slightly lower ICRs than the corresponding amphetamine group. However, a hydroxy group on benzene ring apparently decreased the ICRs. Methoxy and methylenedioxy groups on benzene ring distinctly increased their ICRs. The lack of basicity such as N-formyl MA, N-acetyl AP, and N-acetyl MA dramatically lowered their ICRs to zero or nearly zero. Moreover, the compounds containing a ketone group adjacent to benzene ring, e.g., cathinone and amfepramone, showed very low ICRs. Chlorine on propyl group (mefenorex) or benzene ring (clobenzorex) seemed to act positively for the ICR. These results showed that structural factors related to the lipophilicity and basicity clearly affect the drug incorporation into hair from blood. Received: 4 January 1996/Accepted: 12 March 1996     

A systematic review of occupational exposure to synthetic vitreous fibers and mesothelioma

Objective We investigated whether available epidemiological and toxicological data suggest an increased risk of mesothelioma among workers exposed to synthetic vitreous fibers (SVF). Methods We conducted a systematic review of epidemiological studies on the risk of mesothelioma among workers exposed to SVF, and toxicological studies on SVF and mesothelioma. Results Seven cohort studies were conducted among workers employed in production of rock/slag wool, glass wool, or continuous glass filament in the United States, Canada, and Europe. Of the six deaths from mesothelioma identified in these studies, three had exposure to asbestos. A review of death certificates in a study of rock wool production workers identified one additional probable death. A formal comparison with expected deaths is not feasible. Four community-based case–control studies were identified, of which three reported an increased risk among SVF-exposed workers. The number of cases not exposed to asbestos was less, and residual confounding from asbestos exposure misclassification may explain the association in these studies. The toxicology review of SVF suggested that they present a low hazard mostly due to their low biopersistence, typically with a half-life in rat studies of tens of days compared to amphibole asbestos which has a half-life of 400–500 days. Conclusions The combined evidence from epidemiology and toxicology provide little evidence that exposure to SVF increases the risk of mesothelioma.     

A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance.     

Evaluation and identification of priority air pollutants for environmental management on the basis of risk analysis in Russia

Since 1997, more than 30 health-risk analyses were conducted using Russian data sets. These studies demonstrated that air pollution is the most important environmental contributor toward morbidity and mortality risk in Russia, with 90% of the total human health risk coming from the criteria pollutants total suspended particulate (TSP), SO2, and NO(x). This article contributes to the ongoing discussion of the magnitude of this health issue in Russia by providing an estimate of both the mortality rate attributed to airborne pollutants and the associated economic damages. The 90% confidence interval of mortality is 46,000-132,000, and the associated economic damages are between 2.6 and 6.5% of gross domestic product (GDP). The largest source of uncertainty in mortality is the concentration-response parameter, accounting for 50-60% of the total variability in the estimate. The point estimate of 87,000 implies that mortality due to airborne pollutants is threefold higher than reported due to tuberculosis, twofold due to transportation accidents, and about the same as that from suicide and homicide combined. By 2002 there was enough evidence regarding potential health hazard and air pollution exposure in Russia to start environmental management reform. In 2004 Russia officially adopted guidelines for health risk analysis associated with air pollution. The next step is to use this health-risk assessment approach as a lead for sensible reforms of the emissions-permit system and environmental finance.     

Relationship between prenatal exposure to polychlorinated biphenyls and birth weight: A systematic analysis of published epidemiological studies through a standardization of biomonitoring data

Impact of prenatal PCB exposure on birth weight was investigated in various children cohorts and findings of published studies show inconsistencies. Because a direct comparison of results obtained from different studies remains difficult, the “biological concentration–birth weight” relationship is not clearly established. The objective of this research was to perform a systematic analysis of published epidemiological data to reassess relationship between prenatal PCB exposure and low birth weight, using toxicokinetic considerations and a novel standardization procedure of biological concentration data across studies. A systematic analysis of 20 epidemiological studies published up to 2011 on this topic was conducted. This was achieved through a standardization of reported exposure data in terms of total PCBs per kg of lipids in maternal plasma. Systematic analysis of the “standardized biological concentration–birth weight” relationship across studies was then conducted through the application of Hill criteria. Combining results of all 20 reviewed studies did not allow to establish an association between prenatal exposure to PCBs at the described levels and abnormal birth weight (<2500 g). Our approach provides a framework for the use of published data to establish “PCB biological concentration–response” relationships.     

Critical analysis of biomonitoring endpoints for measuring exposure to polymeric diphenyl-methane-4,4'-diisocyanate (MDI) in rats: a comparison of markers of exposure and markers of effect

The object of this study was to compare the relative sensitivity of markers of exposure and effects in the lung of rats exposed to polymeric diphenyl-methane-4,4'-diisocyanate (pMDI) aerosol. Rats were repeatedly exposed to 12.9 mg pMDI/m(3) (6 h/day, 5 days/week for 14 days; exposure was from days 0--17 followed by a post-exposure period to day 35). Markers of exposure were determined in bronchoalveolar lavage (BAL), blood (haemoglobin, plasma proteins), and urine on days 1, 4, 11, 18, 21, 28 and 35. Markers of effects were determined at the same time points and focused on changes in BAL constituents. In BAL, a maximum increase of total protein occurred following the first exposure and levelled off subsequently whilst BAL cell-related endpoints increased in a time-dependent manner. The kinetics of formation and elimination of adducts differed appreciably from one dosimeter to another. Whilst haemoglobin adducts were integrated by the cumulative exposures, the incremental yield of adduct formation appeared to be dependent on pulmonary as well as yet unknown erythrocyte-related factors. Plasma protein adducts attained a plateau after 1 week of exposure. MDI-related metabolite levels in urine did not show any time-dependent changes during the entire exposure period and declined rapidly during the post-exposure period. Thus, the kinetics of the fractional loading and clearance of pulmonary and extrapulmonary dosimeters did not parallel each other, nor was there a clear correlation with the markers of effects. In summary, it is concluded that biomonitoring is a powerful tool for the comparative dosimetry of well-defined exposure regimens. However, especially for irritant agents demonstrating portal-of-entry effects, markers related to 'total body burden' may not necessarily predict the absence or presence of local responses occurring within the target organ, namely the lung. In all compartments, dosimeters related to higher oligomers of MDI demonstrated low bioavailability, i.e. their recovery was appreciably lower than expected.