Hemolysis is associated with altered heparan sulfate of the endothelial glycocalyx and with local complement activation in thrombotic microangiopathies.

The complement system plays a key role in the pathophysiology of kidney thrombotic microangiopathies (TMA), as illustrated by atypical hemolytic uremic syndrome. But complement abnormalities are not the only drivers of TMA lesions. Among other potential pathophysiological actors, we hypothesized that alteration of heparan sulfate (HS) in the endothelial glycocalyx could be important. To evaluate this, we analyzed clinical and histological features of kidney biopsies from a monocentric, retrospective cohort of 72 patients with TMA, particularly for HS integrity and markers of local complement activation. The role of heme (a major product of hemolysis) as an HS-degrading agent in vitro, and the impact of altering endothelial cell (ECs) HS on their ability to locally activate complement were studied. Compared with a positive control, glomerular HS staining was lower in 57 (79%) patients with TMA, moderately reduced in 20 (28%), and strongly reduced in 37 (51%) of these 57 cases. Strongly reduced HS density was significantly associated with both hemolysis at the time of biopsy and local complement activation (C3 and/or C5b-9 deposits). Using primary endothelial cells (HUVECs, Glomerular ECs), we observed decreased HS expression after short-term exposure to heme, and that artificial HS degradation by exposure to heparinase was associated with local complement activation. Further, prolonged exposure to heme modulated expression of several key genes of glycocalyx metabolism involved in coagulation regulation (C5-EPI, HS6ST1, HS3ST1). Thus, our study highlights the impact of hemolysis on the integrity of endothelial HS, both in patients and in endothelial cell models. Hence, acute alteration of HS may be a mechanism of heme-induced complement activation.

Current sources of lead exposure and their relative contributions to the blood lead levels in the general adult population of Northern France: The IMEPOGE Study, 2008-2010.

There is justification for limiting lead (Pb) exposure as much as possible, given its impact on health at low concentrations. Consequently, the aim of this study was to measure blood lead levels (BLL) and examine exposure factors related to BLL variations in the general adult population of northern France, a current and past industrial area. Two thousand inhabitants of northern France, aged between 20 and 59 years, were recruited using the quota method with caution. Blood lead levels were quantified by inductively coupled plasma-mass spectroscopy (ICP-MS), and variation factors were studied separately in men and women using multivariate stepwise linear and logistic regression models. The geometric mean of the BLL was 18.8 µg/L (95% confidence interval [CI]: 18.3-19.3). Occupational factors affected BLL only in men and represented 14% of total explained variance of BLL. External occupational factors significantly increasing mean levels of BLL were tobacco, consumption of some beverages (wine, coffee, tea, and/or tap water), raw vegetables, housing characteristics (built prior to 1948, Pb piping in the home) and do-it-yourself or leisure activities (paint stripping or rifle shooting). Consumption habits accounted together for 25% and 18% of the total explained variance, respectively, in men and women. Industrial environment did not significantly contribute to BLL variations. Blood lead levels observed in the general population of this industrial part of France did not appear to be excessively elevated compared to values found internationally. Nonetheless, these BLL remain a public health issue in regard to nonthreshold toxicity attributed to Pb.

Sniff nasal inspiratory pressure in the longitudinal assessment of young Duchenne muscular dystrophy children.

Traditional measures of respiratory function in children with Duchenne muscular dystrophy (DMD) are based on maximal inspiratory pressure (PImax) and vital capacity (VC). Sniff nasal inspiratory pressure (SNIP) measurements are easily performed by young children with neuromuscular disorders. The clinical value of SNIP in the longitudinal assessment of respiratory weakness remains to be assessed. The objective of the present study was to assess longitudinally the changes in SNIP, PImax and VC with age in DMD children. We hypothesised that their longitudinal assessment would show an earlier decline in SNIP than VC. A 3-year, prospective follow-up, at 6-month intervals of, 33 steroid-naïve, 5-20-year-old DMD patients was analysed using a linear mixed model. SNIP measurements were reliable (within-session coefficient of variation 8%). SNIP and VC increased until 10.5 and 12.5 years of age, respectively, and declined thereafter, while PImax did not change with age. SNIP was an earlier marker of decline in respiratory muscle strength (at 10.5 years) than VC (at 12.5 years) in young DMD patients. SNIP longitudinal assessment is useful in the detection of inspiratory strength decline in young DMD patients when VC values remain within normal values and as an outcome measure in clinical trials for emerging therapeutics in young DMD patients from the age of 5 years.

Stability of trace element concentrations in frozen urine - Effect on different elements of more than 10 years at - 80 °C.

BACKGROUND: Analysis of urinary trace elements is widely used in Human Biology, especially in occupational and environmental biomonitoring. Collections of urine samples are of great interest for those studying trace elements but many of them are actually unused, in part perhaps because of a lack of knowledge about the stability of trace element concentrations under such storage conditions. The aim of this study was to evaluate the impact of a long-term frozen storage on the measurement of the urinary concentration of 10 trace elements. METHOD: Forty-eight urinary samples were re-analysed by inductively coupled plasma mass spectrometry (ICP-MS) for the quantification of As, Cd, Co, Cr, Mn, Ni, Pb, Sb, Tl, and Zn, after 11-13 years of frozen storage at - 80 °C. RESULTS: A slight water loss likely occurred, even at - 80 °C, but seems to be compensated by using creatinine-adjusted concentrations. Concentrations of As, Cd, Pb, Tl, Zn, and possibly Mn, did not suffer from major modification during storage, while the plastic container likely contaminated samples with Sb. The technological evolution over 13 years may have affected some results, especially those with lower concentrations, and must be taken into account when comparing data over time. CONCLUSION: This study provides some promising preliminary data on the stability of trace element concentrations during long-term frozen storage, and some evidence that urine samples in existing biobanks remain a valuable resource, even if they were collected many years ago.

The receptor for advanced glycation end products is a sensor for cell-free heme.

Heme's interaction with Toll-like receptor 4 (TLR4) does not fully explain the proinflammatory properties of this hemoglobin-derived molecule during intravascular hemolysis. The receptor for advanced glycation end products (RAGE) shares many features with TLR4 such as common ligands and proinflammatory, prothrombotic, and pro-oxidative signaling pathways, prompting us to study its involvement as a heme sensor. Stable RAGE-heme complexes with micromolar affinity were detected as heme-mediated RAGE oligomerization. The heme-binding site was located in the V domain of RAGE. This interaction was Fe3+ -dependent and competitive with carboxymethyllysine, another RAGE ligand. We confirmed a strong basal gene expression of RAGE in mouse lungs. After intraperitoneal heme injection, pulmonary TNF-α, IL1ß, and tissue factor gene expression levels increased in WT mice but were significantly lower in their RAGE-/- littermates. This may be related to the lower activation of ERK1/2 and Akt observed in the lungs of heme-treated, RAGE-/- mice. Overall, heme binds to RAGE with micromolar affinity and could promote proinflammatory and prothrombotic signaling in vivo, suggesting that this interaction could be implicated in heme-overload conditions.

Development of a nanoparticulate formulation of diminazene to treat African trypanosomiasis.

There is a real need to develop new therapeutic strategies for African trypanosomiasis infections. In our study, we developed a new drug delivery system of diminazene (DMZ), a trypanocidal drug registered for veterinary use. This drug candidate presents a limited efficacy, a poor affinity for brain tissue and instability. The development of colloidal formulations based on a porous cationic nanoparticle with an oily core ((70)DGNP(+)), has potentially two advantages: stabilization of the drug and potential targeting of the parasite. We analyzed two processes of drug loading: in process (DMZ was added during the preparation of (70)DGNP(+) at 80 °C) and post-loading (DMZ was mixed with a (70)DGNP(+) solution at room temperature). Poor stability of the drug was observed using the in process technique. When using the post-loading technique over 80% drug entrapment efficiency was obtained at a ratio of DMZ:phospholipids (wt:wt) < 5%. Moreover, DMZ loaded into (70)DGNP(+) was found to be protected against oxidation and was stable for at least six months at 4 °C. Finally, in vitro tests on T.b. brucei showed an increased efficacy of DMZ loaded in (70)DGNP(+).

Integrated analysis of halogenated organic pollutants in sub-millilitre volumes of venous and umbilical cord blood sera.

A rapid, robust and economical method for the analysis of persistent halogenated organic compounds in small volumes of human serum and umbilical cord blood is described. The pollutants studied cover a broad range of molecules of contemporary epidemiological and legislative concern, including polychlorobiphenyls (PCBs), polychlorobenzenes (CBs), hexachlorocyclohexanes (HCHs), DDTs, polychlorostyrenes (PCSs) and polybromodiphenyl ethers (PBDEs). Extraction and clean-up with n-hexane and concentrated sulphuric acid was followed with analysis by gas chromatography coupled to electron capture (GC-ECD) and GC coupled to negative ion chemical ionisation mass spectrometry (GC-NICI-MS). The advantages of this method rest in the broad range of analytes and its simplicity and robustness, while the use of concentrated sulphuric acid extraction/clean-up destroys viruses that may be present in the samples. Small volumes of reference serum between 50 and 1000 microL were extracted and the limits of detection/quantification and repeatability were determined. Recoveries of spiked compounds for the extraction of small volumes (> or = 300 microL) of the spiked reference serum were between 90% and 120%. The coefficients of variation of repeatability ranged from 0.1-14%, depending on the compound. Samples of 4-year-old serum and umbilical cord blood (n = 73 and 40, respectively) from a population inhabiting a village near a chloro-alkali plant were screened for the above-mentioned halogenated pollutants using this method and the results are briefly described.

Knockout of receptor for advanced glycation end-products attenuates age-related renal lesions.

Pro-aging effects of endogenous advanced glycation end-products (AGEs) have been reported, and there is increasing interest in the pro-inflammatory and -fibrotic effects of their binding to RAGE (the main AGE receptor). The role of dietary AGEs in aging remains ill-defined, but the predominantly renal accumulation of dietary carboxymethyllysine (CML) suggests the kidneys may be particularly affected. We studied the impact of RAGE invalidation and a CML-enriched diet on renal aging. Two-month-old male, wild-type (WT) and RAGE-/- C57Bl/6 mice were fed a control or a CML-enriched diet (200 µg CML/gfood ) for 18 months. Compared to controls, we observed higher CML levels in the kidneys of both CML WT and CML RAGE-/- mice, with a predominantly tubular localization. The CML-rich diet had no significant impact on the studied renal parameters, whereby only a trend to worsening glomerular sclerosis was detected. Irrespective of diet, RAGE-/- mice were significantly protected against nephrosclerosis lesions (hyalinosis, tubular atrophy, fibrosis and glomerular sclerosis) and renal senile apolipoprotein A-II (ApoA-II) amyloidosis (p < 0.001). A positive linear correlation between sclerosis score and ApoA-II amyloidosis score (r = 0.92) was observed. Compared with old WT mice, old RAGE-/- mice exhibited lower expression of inflammation markers and activation of AKT, and greater expression of Sod2 and SIRT1. Overall, nephrosclerosis lesions and senile amyloidosis were significantly reduced in RAGE-/- mice, indicating a protective effect of RAGE deletion with respect to renal aging. This could be due to reduced inflammation and oxidative stress in RAGE-/- mice, suggesting RAGE is an important receptor in so-called inflamm-aging.

Blood and urinary levels of metals and metalloids in the general adult population of Northern France: The IMEPOGE study, 2008-2010.

BACKGROUND: The assessment of human chemical risks related to occupational or environmental exposure to pollutants requires the use of both accurate exposure indicators and reference values. The objective of this study was to evaluate the blood and urinary levels of various metals and metalloids in a sample of adults aged 20-59 years of the general population of Northern France, a formerly heavily industrialised area that retains some industrial activity. METHODS: A cross-sectional study was conducted between 2008 and 2010, enrolling 2000 residents of Northern France. The quota method was used to guarantee the representativeness of the participants on a sex, age, social category and smoking status basis, according to the census done by the French National Institute of Statistics and Economic Studies. The levels of 14 metals: aluminium (Al), antimony (Sb), total arsenic (As), beryllium (Be), cadmium (Cd), cobalt (Co), chromium (Cr), mercury (Hg), manganese (Mn), nickel (Ni), lead (Pb), thallium (Tl), vanadium (V) and zinc (Zn) were quantified by ICP-MS in urine and blood samples. RESULTS: A total of 982 men and 1018 women participated, allowing the analysis of 1992 blood and 1910 urine samples. Some metal(loid)s were detected in over 99% of the blood (Cd, Co, Mn, Ni, Pb) and urine (As, Co, Pb, Zn) samples and the remaining metals in 84-99% of the samples, with the exception of blood V (19%), blood Be (57%) and urine Be (58%). Mean blood levels of Pb and Zn were significantly higher in men, and Mn, Co and Cr in women. In urine, mean Pb, Tl and Sb concentrations were significantly higher in men, and Al and Co in women. Current smokers had significantly higher mean levels of blood Cd and Pb and lower blood Co, Mn and Hg. In urine (adjusted on urinary creatinine), the smokers had higher mean levels of Cd, Pb, V and Zn and lower mean levels of As, Co, and Hg. Overall, the mean urinary levels of most metal(loid)s found in the general population of Northern France were higher than those found in the French national survey for the same period except for urinary V. Mean blood lead level was markedly less than that of the French national population. CONCLUSION: This first biomonitoring survey of a large number of metal(loid)s in the general population of Northern France provides useful information on exposure levels to toxic elements and highlights the specificity of the regional environment. These data could be used, in complement to the national human biomonitoring reference values, for the interpretation of biomonitoring results.

Beryllium in exhaled breath condensate as a biomarker of occupational exposure in a primary aluminum production plant.

OBJECTIVE: Low beryllium exposure can induce pulmonary granulomatosis, so called berylliosis. For occupational health monitoring, it is more relevant to assess the internal dose of Be received by the lungs than urinary or atmospheric Be. Exhaled breath condensate (EBC) is a matrix collected non-invasively that derives from the airway lining fluid. EBC beryllium (Be) levels were evaluated as a marker of occupational exposure in a primary aluminum production plant. METHODS: We collected urine and EBC from controls and workers recently exposed to beryllium in the pot room and the anode repair sectors, and calculated a cumulative beryllium exposure index (CBEI) summing the number of years of employment in each task and multiplying by the estimated average beryllium exposure for the task. Concentrations of beryllium and aluminum were measured in EBC (Be-EBC and Al-EBC) and in urine (Be-U and Al-U) by ICP-MS. RESULTS AND CONCLUSION: We have shown that it was possible to measure Be and Al in workers' EBC. Compared with controls and after adjustment for smoking status, levels of Be-EBC and Al-EBC were higher in pot room workers and exposed subjects, respectively. Due to its relationship with CBEI, but not with Be-U, it appears that Be-EBC could be a promising marker of occupational exposure and provide additional toxicokinetic information in occupational health studies.

Evolution of availability of curcumin inside poly-lactic-co-glycolic acid nanoparticles: impact on antioxidant and antinitrosant properties.

PURPOSE: Curcumin exhibits antioxidant properties potentially beneficial for human health; however, its use in clinical applications is limited by its poor solubility and relative instability. Nanoparticles exhibit interesting features for the efficient distribution and delivery of curcumin into cells, and could also increase curcumin stability in biological systems. There is a paucity of information regarding the evolution of the antioxidant properties of nanoparticle-encapsulated curcumin. METHOD: We described a simple method of curcumin encapsulation in poly-lactic-co-glycolic acid (PLGA) nanoparticles without the use of detergent. We assessed, in epithelial cells and in an acellular model, the evolution of direct antioxidant and antinitrosant properties of free versus PLGA-encapsulated curcumin after storage under different conditions (light vs darkness, 4°C vs 25°C vs 37°C). RESULTS: In epithelial cells, endocytosis and efflux pump inhibitors showed that the increased antioxidant activity of PLGA-encapsulated curcumin relied on bypassing the efflux pump system. Acellular assays showed that the antioxidant effect of curcumin was greater when loaded in PLGA nanoparticles. Furthermore, we observed that light decreased, though heat restored, antioxidant activity of PLGA-encapsulated curcumin, probably by modulating the accessibility of curcumin to reactive oxygen species, an observation supported by results from quenching experiments. Moreover, we demonstrated a direct antinitrosant activity of curcumin, enhanced by PLGA encapsulation, which was increased by light exposure. CONCLUSION: These results suggest that the antioxidant and antinitrosant activities of encapsulated curcumin are light sensitive and that nanoparticle modifications over time and with temperature may facilitate curcumin contact with reactive oxygen species. These results highlight the importance of understanding effects of nanoparticle maturation on an encapsulated drug's activity.

Organochlorine exposure and colorectal cancer risk.

Organochlorine compounds have been linked to increased risk of several cancers. Despite reductions in their use and fugitive release, they remain one of the most important groups of persistent pollutants to which humans are exposed, primarily through dietary intake. We designed a case-control study to assess the risk of colorectal cancer with exposure to these chemicals, and their potential interactions with genetic alterations in the tumors. A subsample of cases (n = 132) and hospital controls (n = 76) was selected from a larger case-control study in Barcelona, Catalonia, Spain. We measured concentrations in serum of several organochlorines by gas chromatography. We assessed point mutations in K-ras and p53 genes in tissue samples by polymerase chain reaction/single-strand conformation polymorphism and assessed expression of p53 protein by immunohistochemical methods. An elevated risk of colorectal cancer was associated with higher serum concentrations of mono-ortho polychlorinated biphenyl (PCB) congeners 28 and 118. The odds ratio for these mono-ortho PCBs for middle and higher tertile were, respectively, 1.82 [95% confidence interval (CI), 0.90-3.70] and 2.94 (95% CI, 1.39-6.20). Alpha-hexachlorocyclohexane, hexachlorobenzene, and p,p'-DDE (4,4'-dichlorodiphenyltrichloroethene) showed nonsignificant increases in risk. Risk associated with mono-ortho PCBs was slightly higher for tumors with mutations in the p53 gene but was not modified by mutations in K-ras. Mono-ortho PCBs were further associated with transversion-type mutations in both genes. These results generate the hypothesis that exposure to mono-ortho PCBs contributes to human colorectal cancer development. The trend and magnitude of the association, as well as the observation of a molecular fingerprint in tumors, raise the possibility that this finding may be causal.

A solvent-free method for spiking terrestrial algae (Desmococcus spp.) with pyrene for use in bioassays.

Two methods for spiking terrestrial algae (Desmococcus spp., used as a foodstuff in bioassays with Collembola) with pyrene were tested; a "traditional" method that used a carrier solution of pyrene in acetone, and a solvent-free method developed using the principles of partition driven administration (PDA). The PDA method used a pre-spiked, highly-contaminated C18 disk as the source for pyrene, suspended and rotated in an aqueous algal suspension. The pyrene partitioned from the C18 disk into the aqueous phase, and a concentration of 18.4 +/- 0.7 microg pyrene g(-1)dwt. algae (mean +/- sd) was achieved after 18 h exposure by this method, with good reproducibility also observed for lower concentrations achieved over shorter exposure periods. The acetone-spiked algae lost much of its cell integrity and a significant amount of pigment, while cell integrity was maintained after 120 h spiking using the PDA method. Results from a short bioassay with Orchesella cincta (Collembola, Insecta) showed that animals fed acetone-spiked algae had slower growth rates and higher concentrations of pyrene metabolites than animals fed PDA-spiked algae. It is speculated that this was the result of the poor quality of the acetone-spiked food, and that the difference in food quality between treatments stimulated changes in body composition that may have affected the production of pyrene metabolites. The PDA spiking method is expected to be suitable for introducing a range of persistent organic pollutants into other types of sample matrix.

Pyrene metabolism in the springtail Orchesella cincta L. (Collembola, Entomobryidae).

A novel, solvent-free spiking method was used to contaminate foodstuff (Desmococcus spp. algae) with pyrene for bioassays with the springtail Orchesella cincta L. (Collembola, Entomobryidae). The primary metabolite 1-hydroxypyrene and two conjugates (pyrene-1-glucoside and one tentatively identified as pyrene-1-glucosidemalonate) were quantified in the insects by high-performance liquid chromatography (HPLC) with fluorescence detection. Individuals from an unpolluted site (SUS population) were capable of rapid metabolism of pyrene, reaching an apparent steady state after 24 h with a composite assimilation/transformation rate of approximately 25 pmol pyrene x g(-1) fresh weight x h(-1) at an exposure level of approximately 18 microg pyrene x g(-1) dry weight algae. A cadmium (Cd)-tolerant population (TOL), genetically distinct from SUS, was exposed in parallel and the populations' pyrene metabolism compared in order to gain insight into any potential cost of Cd-tolerance. The TOL animals assimilated and/or performed phase 1 transformation of pyrene approximately twice as fast as SUS (approximately 56 pmol x g(-1) x h(-1)), but there was no significant difference in the elimination rate constants between SUS and TOL animals. Attributing this difference to any specific cost of Cd-tolerance requires more work and may he coincidental to metal tolerance, but pyrene metabolism in these distinct genotypes, subjected to different selection pressures, was nevertheless significantly different.

Manganese in exhaled breath condensate: a new marker of exposure to welding fumes.

OBJECTIVE: To evaluate manganese in exhaled breath condensate (Mn-EBC) as an indicator of exposure to fumes from metal inert gas welding process. METHODS: We collected EBC and urine from 17 welders and 16 unexposed control subjects after 5 days exposure. Concentrations of manganese (Mn), nickel (Ni), iron (Fe) and chromium (Cr) were measured in EBC and urine samples and correlated with cumulative exposure indices for the working week (CIW) and for the total welding years (WY), based on duration of welding activity and atmospheric metal measurements. RESULTS: Concentrations of Mn and Ni in EBC were significantly higher among welders than controls whereas this difference was not significant for Mn in urine. Levels of Mn and Ni in EBC were not correlated with their respective levels in urine. The linear regressions found significant positive coefficients between Mn-EBC, Ni-EBC, Ni-U and Cr-U concentrations and the cumulative exposure indices. Taking into account tobacco use, statistical analysis showed the same trends except for the relationship between Mn-U and CIW. CONCLUSION: This pilot study showed that Mn-EBC, as well as Ni-EBC, can serve as reliable indices of occupational exposure to welding fumes and provide complimentary toxicokinetic information to that provided by urine analyses.

1-Hydroxypyrene and 3-hydroxybenzo[a]pyrene as biomarkers of exposure to PAH in various environmental exposure situations.

OBJECTIVE: To evaluate the utility of urinary 3-hydroxybenzo[a]pyrene (3-OH-B[a]P) as a biomarker of exposure to polycyclic aromatic hydrocarbons (PAHs) in various environmental exposure scenarios alongside the more usually studied 1-hydroxypyrene (1-OH-Pyr). METHODS: Two groups of 15 and 10 non smoking, healthy men and women, were exposed for approximately 6h to ambient air at two outdoor locations close to metallurgical industries, and at one indoor location in an urban setting. Atmospheric measurements of 16 "priority" PAHs were carried out during each exposure. Urinary 1-OH-Pyr and 3-OH-B[a]P were also analysed, samples being taken the morning before exposure, at the end of exposure, then 4 and 15 h after the end of exposure. Urinary 1-OH-Pyr and 3-OH-B[a]P were analysed using high performance liquid chromatograph with fluorescence detection. Limits of detection (LOD) were 0.092 nmol/L and 0.28 pmol/L for 1-OH-Pyr and 3-OH-B[a]P respectively. RESULTS: Average ambient air concentrations varied from 0.27 to 2.87 ng/m(3) for pyrene, 0.04 to 1.20 ng/m(3) for B[a]P, and from 70.0 to 910.6 ng/m(3) for the sum of the 16 PAH (SigmaPAHs). Concentrations of 1-OH-Pyr were not correlated with atmospheric concentrations of PAHs to which subjects were exposed, nor with the concentrations of 3-OH-B[a]P. Nearly 80% of measurements of 3-OH-B[a]P were lower than the LOD and no relationship between atmospheric concentrations and urinary metabolites was observable. However, the percentage of post-exposure values of 3-OH-B[a]P greater than the LOD increased significantly with the median of atmospheric concentrations of Pyr, B[a]P and SigmaPAH at the different sites (test of linear trend, p<0.02 in all cases). CONCLUSION: Although we used very sensitive techniques for the measurements of urinary metabolites, especially for 3-OH-B[a]P, neither 1-OH-Pyr nor 3-OH-B[a]P were an unequivocal biomarker of exposure to atmospheric PAHs in environmental exposure scenarios relevant to the general population. It would be interesting to investigate other urinary monohydroxy PAH metabolites in this context.

Urinary PAH metabolites as biomarkers of exposure in aquatic environments.

While immunoassays have been extensively applied to evaluate environmental contamination, to date they have rarely been used for the analysis of biological fluids outside of human medicine. These media are important because pollutants such as polycyclic aromatic hydrocarbons (PAHs) and their metabolites become concentrated in tissues, body fluids, and excreta, thereby offering a measure of exposure to biologically available contaminants. Such analyses also provide a nondestructive tool for monitoring exposure. Crabs (Carcinus maenas) were exposed to phenanthrene and pyrene (separately) at concentrations ranging from 0 to 200 microg L(-1). After 48 h, urine samples were taken and analyzed by immunoassay and UV-fluorescence spectrophotometry. Urinary levels (calibrated against hydroxylated metabolites) proved to be dose dependent for both compounds, and good agreementwas demonstrated between the immunoassay and the fluorescence techniques. The cross reactivity of the immunochemical technique (ELISA) for pyrene and hydroxy-metabolites was lower than for phenanthrene. HPLC analyses demonstrated that urine from the crabs exposed to pyrene contained mainly conjugate PAH metabolites whose concentrations (the sum of the four main pyrene metabolites/conjugates) showed very good agreement with the ELISA (r2 > 0.94) and fluorescence (r2 > 0.91) data. Environmental samples were also analyzed by ELISA and UV-fluorescence, and both techniques detected PAH (mainly petrogenic) contamination in the urine samples from a polluted harbor. These data demonstrate the potential of urine analyses by ELISA and UV-fluorescence to measure exposure of crabs to PAH.