Immunological and cytotoxicological responses of the Asian clam,Corbicula fluminea (M.), experimentally exposed to cadmium

Abstract

Bivalve molluscs, as filter-feeding organisms, are known to accumulate metals that can produce deleterious effects on organisms. The phagocytic activity of haemocytes and lysosomal alterations in the digestive gland cells were measured in the freshwater Asian clam exposed to cadmium, in order to assess the possible use of immunocompetence and lysosomal responses as biomarkers of freshwater quality. Clams were exposed in the laboratory to nominal concentrations of 3, 10, 21.4, 46.5 and 100 µg l^?1 of cadmium and sampled after 7, 15 and 30 days of exposure. The results show a decrease of phagocytic activity after only 7 days of exposure to 10 µg l^?1 of cadmium. This response was also observed as the exposure time was increased. Lysosomes in the digestive cells increased in size and number after 7 days of exposure as cadmium concentration increased. After 30 days of exposure, a decrease in size and number indicated a change in the response to the metal from concentrations of 46.5 µg l^?1 of cadmium. A dose and time response both in phagocytic activity of haemocytes and lysosomal structure demonstrated a possible use of these biomarkers in freshwater biomonitoring.     

Estimation of urinary cotinine cut-off points distinguishing non-smokers, passive and active smokers

An objective assessment of exposure to tobacco smoke may be accomplished by means of examining particular biomarkers in body fluids. The most common biomarker of tobacco smoke exposure is urinary, or serum, cotinine. In order to distinguish non-smokers from passive smokers and passive smokers from active smokers, it is necessary to estimate cotinine cut-off points. The objective of this article was to apply statistical distribution of urinary cotinine concentration to estimate cut-off points distinguishing the three above-mentioned groups. The examined group consisted of 327 volunteers (187 women and 140 men) who were ethnically homogenous inhabitants of the same urban agglomeration (Sosnowiec, Poland). The values which enabled differentiation of the examined population into groups and subgroups were as follows: 50 µg l^-1 (differentiation of non-smokers from passive smokers), 170 µg l^-1 (to divide the group of passive smokers into two subgroups: minimally and highly exposed to environmental tobacco smoke), 550 µg l^-1 (differentiation of passive smokers from active smokers), and 2100 µg l^-1 (to divide group of active smokers into two subgroups: minimally and highly exposed to tobacco smoke). The results suggest that statistical distribution of urinary cotinine concentration is useful for estimating urinary cotinine cut-off points and for assessing the smoking status of persons exposed to tobacco smoke.     

Beta-naphthoflavone inhibits the induction of hepatic oestrogen-dependent proteins by 17alpha-ethynylestradiol in mosquitofish (Gambusia holbrooki)

The interactive effects of an aryl hydrocarbon receptor (AhR) agonist and of a xenoestrogen on biomarker responses were studied in the liver of male mosquitofish (Gambusia holbrooki). Hepatic 7-ethoxyresorufin O-deethylase (EROD) enzymatic activity was measured as a biomarker of exposure to the model AhR agonist beta-naphthoflavone (bNF). Hepatic proteins indicating the exposure of males to the synthetic oestrogen 17alpha-ethynylestradiol (EE2) were monitored by Western blot analysis using immunoserum prepared for this study. After a semi-static exposure only to waterborne EE2, Western blot analysis of liver homogenate revealed the induction of two protein bands (a double band at 205 kDa and a single band at 125 kDa). The interaction between bNF and EE2 was investigated by analysing, on the one hand, EROD activity and, on the other hand, immunoreactivity corresponding to the two oestrogen-dependent protein bands in the liver of fish exposed to different concentrations of bNF for 2 days, then to the same concentrations of bNF plus 0.1 µg l^-1 EE2 for 5 days. EE2 changed neither the basal activity of EROD nor its rate of induction with 1.0 and 4.0 µg l^-1 bNF. On the other hand, the induction of oestrogen-dependent proteins with 0.1 µg l^-1 EE2 was inhibited by exposure to 4.0 µg l^-1 bNF. These results together with literature data suggest that field monitoring of xenoestrogen contamination through the analysis of oestrogen-dependent protein in male fish as a biomarker should take into account the possible negative interference of AhR agonists.     

Urinary homovanillic acid and serum prolactin levels in children with low environmental exposure to lead

Current evidence suggests that the neurotoxic effects of lead may partially be mediated through interference with the dopaminergic system. The aim of this study was to assess the levels of two peripheral dopaminergic markers- serum prolactin (Pro-S) and urinary homovanillic acid (HVA-U) - in children living around two lead smelters, who are presumed to be exposed to high environmental lead pollution (n = 200), and compare their results with 200 age- and sex-matched controls living in an area unpolluted by heavy metals, giving a total of 400 children (200 boys and 200 girls). The influence of lead exposure on HVA-U and Pro-S was assessed by stepwise multiple regression, testing lead concentrations in blood (Pb-B), age, sex and area of residence as predictors. Though lead levels were significantly higher in boys and in the lead-polluted environment, mean Pb-B values were relatively low, indicating a low uptake of lead in the contaminated environment (39.5 µg l^-1, range 4.6-165µgl^-1, n = 200), and no significant correlation could be found with either Pro-S or HVA-U. However, when the subgroup of 121 children with Pb-B levels above 50 µg l^-1 were considered, a weak positive correlation was found between Pb-B and HVA-U (r² = 0.04, p = 0.03), whilst in the even smaller subgroup of 15 children with Pb-B levels above 100 µg l^-1, Pro-S appeared to be positively correlated with Pb-B, though the numbers of children were too small for the correlation to reach statistical significance (p = 0.095). These weak associations, probably not important in biological terms, indicate that Pro-S and HVA-U are not useful biomarkers at present exposure levels to lead in the environment. Nevertheless, the finding of subtle biochemical alterations in the dopaminergic system at Pb-B levels of around 100µgl^-1 supports the recommended setting of the action level at this value.     

Biomarker responses in juvenile rainbow trout (Oncorhynchus mykiss) after single and combined exposure to low doses of cadmium, zinc, PCB77 and 17β-oestradiol

The objective of this study was to examine (i) the biochemical responses of rainbow trout exposed to sublethal water concentrations of the metals cadmium (Cd) (1.5 μg l^-1) and zinc (Zn) (150 μg l^-1); and (ii) the potential combined effects when applied in mixture (Cd/Zn) with and without co-exposure to model organic chemicals 3,3',4,4'-tetrachlorobiphenyl (PCB77) (1 mg kg^-1) and 17β-oestradiol (E2) (0.5 mg kg^-1). After 21 days of exposure, several biomarkers were assessed in the liver (enzymatic and non-enzymatic antioxidants, heat shock proteins [HSP70 and HSP60], ethoxyresorufin-O-deethylase [EROD]) and in the plasma (vitellogenin [Vtg], aminotransferases). Plasma aminotransferases were not affected, whereas the other biomarkers showed different patterns of response depending on the treatment. For example, Cd, and Zn to a lesser extent, induced an adaptive response in the liver shown by an increase in antioxidant defences (total glutathione [GSH], superoxide dismutase, Trolox equivalent antioxidant capacity [TEAC]), without any impairment of GSH redox status or induction of heat shock proteins. Antagonistic effects were observed in GSH-related biomarkers after Cd/Zn exposure. PCB77 strongly induced EROD activity, HSP70 and TEAC. Co-exposure with metals did not modulate significantly the effects of PCB77. E2 induced Vtg and inhibited liver antioxidants and basal EROD activity. These inhibitory effects were suppressed in fishes exposed to E2 + Cd/Zn, suggesting additive effects of E2 and metals. In addition, E2-induced Vtg was not altered by metals. Multivariate analyses confirmed some correlation between the biomarkers. The use of complementary biomarkers is necessary to discriminate different treatments and to highlight interactive effects.     

The potential role of microzooplankton in a northwestern Australian pelagic food web

The role of microzooplankton in waters adjacent to Australia's North West Cape (21°49'S 114°14'E) was studied during the austral summers 1997/1998 and 1998/1999. We estimated microzooplankton abundance and biomass at a shallow (∼20 m) shelf station and at a shelf break station (∼80 m). Microzooplankton were placed into six categories: four ciliate groups (strombidiids, strobilidiids, tintinnids, “other ciliates”), dinoflagellates, and sarcodines. Total microzooplankton abundances ranged between 0.14×10³ l^-1 and 3.4×10³ l^-1. The most abundant groups were the dinoflagellates (mean 459±73 standard error l^-1) and strombidiids (mean 334±42 standard error l^-1). Total microzooplankton biomass ranged between 0.03 and 1.70 µg C l^-1 (mean 0.33±0.05 standard error l^-1). Redundancy analysis indicated differences in microzooplankton community composition between stations and sampling years but no differences with sampling depth. The microzooplankton community showed considerable variability between adjacent sampling dates, reinforcing the conclusion of earlier studies that this area is a dynamic environment. Ciliate production on the shelf was estimated to be 1.05 µg C l^-1day^-1 (∼20 mg C m^-2day^-1) and 0.79 µg C l^-1day^-1(∼70 mg C m^-2day^-1) at the shelf break. Ciliate production near North West Cape was two- to six-fold higher than the rate of secondary production by juvenile copepods. Despite this, ciliate grazing appears to account for only ∼5% of primary production and ciliates do not appear to be a major conduit between primary producers and higher trophic levels in these waters.     

Urinary hydroxylated metabolites of polycyclic aromatic hydrocarbons as biomarkers of exposure in asphalt workers

Background. Fumes and vapours released during laying of hot asphalt mix have been recognised as a major source of exposure for asphalt workers. Objectives. We investigated the relationships between inhalation exposure to asphalt emissions and urinary biomarkers of polycyclic aromatic hydrocarbons (PAHs) in asphalt workers (AW, n=75) and in ground construction workers (CW, n=37). Methods. Total polyaromatic compounds (PAC) and 15 priority PAHs in inhaled air were measured by personal sampling. Hydroxylated PAH metabolites (OH-PAHs) (2-naphthol, 2-hydroxyfluorene, 3-hydroxyphenanthrene, 1-hydroxypyrene, 6-hydroxychrysene and 3-hydroxybenzo[a]pyrene) were determined in urine spot samples collected in three different times during the work week. Results. Median vapour-phase PAC (5.5 µg m^-3), PAHs (≤50 ng m^-3) and OH-PAHs (0.08-1.11 µg l^-1) were significantly higher in AW than in CW, except in the cases of air naphthalene and 2-naphthol. Airborne levels of particle-phase contaminants were similar in the two groups and much lower than vapour-phase levels; metabolites of particulate PAHs were never found in quantifiable amounts. An appreciable increase in OH-PAH levels during the work day and work week was found in AW; median levels for 2-hydroxyfluorene, 3-hydroxyphenanthrene and 1-hydroxypyrene were, respectively, 0.29, 0.08 and 0.18 at baseline; 0.50, 0.18 and 0.29, pre-shift; 1.11, 0.44 and 0.44 µg l^-1, post-shift. Each OH-PAH exhibited a characteristic profile of increase, reflecting differences in half-lives of the parent compounds. In non-smoking subjects, positive correlations were found between vapour-phase PAC or PAHs and OH-PAHs both in pre- and post-shift samples (0.34 ≤ r≤69). Smokers exhibited 2-5-fold higher OH-PAHs than non-smokers, at any time and at both workplaces. Conclusions. Our results suggest that OH-PAHs are useful biomarkers for monitoring exposure to asphalt emissions. The work-related exposure to PAC and PAHs was low in all AW, but urinary metabolites reflected exposure satisfactorily.     

Effects of water hardness and alkalinity on the toxicity of uranium to a tropical freshwater hydra (Hydra viridissima)

In tropical Australian freshwaters, uranium (U) is of potential ecotoxicological concern, largely as a consequence of mining activities. Although the toxicity of uranium to Australian freshwater biota is comprehensive, by world standards, few data are available on the effects of physicochemical variables, such as hardness, alkalinity, pH and organic matter, on uranium speciation and bioavailability. This study determined the individual effects of water hardness (6.6, 165 and 330 mg l^-1 as CaCO₃) and alkalinity (4.0 and 102 mg l^-1 as CaCO₃), at a constant pH (6.0), on the toxicity (96 h population growth) of uranium to Hydra viridissima (green hydra). A 50-fold increase in hardness (Ca and Mg concentration) resulted in a 92% (two-fold) decrease in the toxicity of uranium to H. viridissima [i.e . an increase in the EC₅₀ value and 95% confidence interval from 114 (107-121) to 219 (192-246) µg l^-1]. Conversely, at a constant hardness (165 mg l-1 as CaCO₃), the toxicity of uranium to H. viridissima was not significantly (P > 0.05) affected by a 25-fold increase in alkalinity (carbonate concentration) [i.e. EC₅₀ values of 177 (166-188) and 171 (150-192) µg l^-1 at 4.0 and 102 mg l^-1 as CaCO₃, respectively]. A knowledge of the relationship between water chemistry variables, including hardness and alkalinity, and uranium toxicity is useful for predicting the potential ecological detriment in aquatic systems, and can be used to relax national water quality guidelines on a site-specific basis.     

An epidemiological study of reproductive function biomarkers in male welders

In a cross-sectional study, the serum concentrations of inhibin B and prolactin of 96 male current welders were compared with the concentrations measured in 96 age-matched referents. Also, 23 patients who were all former welders diagnosed as having welding-related manganism were studied. The current welders' geometric mean (GM) airborne exposure to manganese (Mn) was 121 µg m^-3 (range 7-2320). The serum concentrations of prolactin adjusted for age and smoking habits (GM 193 mIU l^-1 vs. 166 mIU l^-1; p=0.047) and inhibin B adjusted for alcohol consumption (arithmetic mean (AM) 151 ng l^-1 vs. 123 ng l^-1; p=0.001) were higher in the welders compared with the referents. The whole blood Mn concentration was associated with the serum prolactin concentrations. Tobacco smoking resulted in lower serum prolactin concentrations. The GM serum prolactin concentrations of the patients did not significantly differ from that of the referents, but their AM serum inhibin B concentration was statistically significantly lower. The results may suggest an effect of Mn on the pituitary that is reversible upon cessation of exposure. Lower inhibin B concentrations in the patients could point to a functional impairment of the testicular Sertoli cells, that may be caused by a welding fume component or other factors in their work environment.     

Clara cell protein (CC16) in serum and bronchoalveolar lavage fluid of subjects exposed to asbestos

The Clara cell protein (CC16) is a small and readily diffusible protein of 16kDa secreted by bronchiolar Clara cells in the distal airspaces. These epithelial cells are altered in several pulmonary pathological processes induced by various lung toxicants. In the search for a new biomarker of asbestos-induced lung impairment, we used a sensitive immunoassay to determine the levels of CC16 in bronchoalveolar fluid (BALF) and serum of subjects exposed to asbestos compared with a group of healthy controls. In the BALF of asbestos-exposed subjects there was an insignificant trend towards CC16 elevation compared with controls, with a (mean ±SD of 0.81 ±0.65mg l^-1 for asbestos-exposed subjects (n = 23) versus 0.39 ±0.19mg l^-1 for controls (n = 11) (p = 0.09). In serum, CC16 concentration was significantly increased among asbestos-exposed subjects, with values of 27.2 ±24.0 µg l^-1 for asbestos-exposed subjects (n = 34) versus 16.1 ±7.6 µg l^-1 for controls (n = 34) (p = 0.01). Regarding the effects of smoking, there were significant differences between generally lower CC16 levels in serum and BALF (p = 0.05 and 0.001, respectively) of smokers compared with the higher levels in non-smokers. Serum CC16 levels positively correlated with those in BALF, which is consistent with a diffusional transfer of CC16 from the bronchoalveolar space into the serum. No association, however, emerged between the levels of CC16 in serum or BALF and either the duration of asbestos exposure or the severity of the lung impairment as assessed by chest X-ray. These findings suggest that exposure to asbestos elicits early changes in the local and, importantly, also the systemic levels of CC16. This pneumoprotein therefore appears as a promising non-invasive biomarker of asbestos-induced lung injury and occupational disease in both smoking and non-smoking exposed subjects.     

Hepatic CYP1A induction in rainbow trout (Oncorhynchus mykiss) after exposure to benzo[a]pyrene in water

Juvenile rainbow trout were exposed to unlabelled benzo[a]pyrene BaP and ³H benzo a pyrene (³H BaP), in a static exposure system for 2 days. The initial concentration was 30 μg l^-1 and 0.625 μCi l^-1, corresponding to 6 mg kg^-1 body weight and 125 μCi kg^-1 body weight. Hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity was measured during the exposure and depuration periods, elucidating the time course pattern of CYP1A induction. Maximum induction (11-fold) of EROD activity was observed on day 2 after addition of BaP to the water. Tissue distribution of ³H-BaP was studied by liquid scintillation counting and whole body autoradiography. The concentration of ³H-BaP-derived radioactivity was highest in the bile at all sampling times. High levels of radiolabelled compound were also present in the gills, liver and the olfactory organ. There was an overall decrease in all tissues during the depuration period. The elimination of ³H-BaP-derived radioactivity from the gills, however, was slow compared with liver and blood (6.2 days vs 2.7 and 2.9 days, respectively).     

Activities of glutamate dehydrogenase and aspartate and alanine aminotransferases in freshwater snails Helisoma duryi and Lymnaea natalensis exposed to copper

In this paper we investigate the potential of glutamate dehydrogenase (GDH) and aspartate and alanine aminotransferases (AST and ALT) as biomarkers of water pollution due to copper in the freshwater snails Helisoma duryi and Lymnaea natalensis. Snails were dosed with copper(II) ion concentrations of 0.01, 0.1 and 1 mg kg^-1 breeding water for a period of 96 h, after which those surviving were shelled. The copper content in the breeding water, in whole snail tissue and in the snail shells was determined at the end of the period of exposure. For enzyme determinations, whole snail tissue was first homogenized and fractionated by centrifugation at 500 g to remove the nuclei. The resulting supernatant was then centrifuged at 10 000 g to give a pellet fraction representing the mitochondrial fraction and a supernatant representing the cytosolic fraction. Copper was very toxic to both snail species at concentrations above 0.2 mg l^-1, with only 3% of the Helisoma and 12% of the Lymnaea surviving at concentrations of approximately 1 mg l^-1. The copper content in the shells and tissues of snails rose with increasing copper concentration in the breeding water, and was 2.1- to 4.9-fold in snails exposed to copper ion at a dose of 1 mg kg^-1 water compared with undosed snails. Similarly, the activities of GDH and AST rose by up to 4.7-fold in the homogenate and the mitochondrial and cytosolic fractions with increasing concentrations of copper. These activities, however, fell at copper concentrations of approximately 1 mg l^-1, which coincided with massive death of snails. Mitochondrial ALT disappeared at copper ion concentrations of approximately 0.2 mg l^-1 for Lymnaea and 1 mg l^-1 for Helisoma, possibly indicating mitochondrial degeneration. These results show that GDH, AST and ALT have the potential to be biomarkers of suplethal copper pollution in these two snail species, since their activities were significantly altered by low copper concentrations.     

Ascorbate and glutathione contents in duckweed, Lemna minor, as biomarkers of the stress generated by copper, folpet and diuron

Glutathione and ascorbate are essential components of the general antioxidative strategy to overcome oxidative stress due to environmental constraints such as pollution. The variation of glutathione and ascorbate contents in duckweed (Lemna minor) was investigated after a 48 h exposure to copper, diuron and folpet under laboratory conditions in order to determine whether changes in their level could serve as suitable and early biomarkers of pollution. One could observe that diuron and folpet caused the glutathione level to increase, its redox status remaining unchanged, while copper led to a depletion of this antioxidant and to an increase in its oxidation rate. When duckweed was contaminated by folpet and the metal, an increase of the ascorbate pool size occurred from concentrations as low as 1 mg l^-1 and 50 μg l^-1 respectively. While the ascorbate pool became more oxidized because of exposure to copper concentrations ≤ 200 μg l^-1, folpet caused an increase in its reduction rate. Diuron was responsible for depletion of ascorbate, the redox status of which remained unchanged. Because it is an adaptation to stress and a defence process, the increase in the antioxidant pool size was proposed as a biomarker of exposure to an unsafe environment. Since depletion of antioxidant and an increase in its oxidation rate weakened cellular defences and indicated a precarious state, they could constitute early indicators of toxicity. So they were proposed as potential biomarkers of toxicity. It was concluded that the antioxidant content in duckweed might serve as a useful biomarker for monitoring water quality.     

Chronotoxicity of a Copper (II) Complex with a Linear Tetradentate Ligand in Mice

In vitro copper (II) complex presents antimitotic effects. In this work, we have studied the in vivo seasonal toxic effects of copper (II), ligand (H₂L) and the complex [Cu(H₂L)(H₂O)₂]Cl₂·4H₂O in male Swiss mice. During spring, an i.p. injection of CuCl₂ in aqueous NaCl (9 g·l^-1) up to 0.05 µmol·kg^-1 b.w. (body weight) killed 60% of the rodents after 6 days. LD100 was up to 0.3 µmol·kg^-1; H₂L was well tolerated, while the complex was 30% lethal with 50 µmol·kg^-1. In autumn, mice were less sensitive to CuCl₂, and both ligand and complex were equally tolerated and this leads to the conclusion that, in vivo, chronotoxicities of copper (II) and complex in NaCl aqueous solutions are quite different in spring and autumn seasons.     

Simultaneous determination of urinary 1- and 2-naphthols, 3- and 9-phenanthrols, and 1-pyrenol in coke oven workers

A method was developed for simultaneous quantification of urinary 1- and 2-naphthols, 3- and 9-phenanthrols and 1-pyrenol using gas chromatography with mass spectrometry (GC-MS). This method was applied to urine samples from coke oven workers (n =28) and controls (n =22) from Northern China. Geometric mean levels of urinary 1-naphthol (58.8 μg l^-1), 2-naphthol (34.1 μg l^-1), 3-phenanthrol (7.35 μg l^-1), 9-phenanthrol (1.28 μg l^-1) and 1-pyrenol (25.4 μg l^-1) were significantly higher among coke oven workers than controls. All the substances tested were highest among top-of-oven workers, who had 15-fold higher 1-naphthol, eight-fold higher 2-naphthol and 20-fold higher 1-pyrenol levels compared with controls. Using multiple linear regression models, 72.5% of the variation in 1- and 2-naphthol and 82.8% of the variation in 1-pyrenol were explained by the concentration of naphthalene or pyrene in the urine, the work category and the smoking intensity. Cigarette consumption significantly contributed to levels of urinary 1-pyrenol and naphthols, particularly 2-naphthol. A negative relationship between work category and the ratio of naphthols/1-pyrenol was observed among smokers. Our results suggest that urinary naphthols and phenanthrols reflect polycyclic aromatic hydrocarbon (PAH) exposure as well as the widely used 1-pyrenol, and that interactions between cigarette smoking and PAH exposure result in different patterns of metabolism for individual PAHs.     

Exhaled breath condensate as matrix for toluene detection: A preliminary study

The study was designed to investigate whether exhaled breath condensate, obtained by cooling exhaled air in spontaneous breathing, could be a suitable matrix for toluene quantitative analyses. Nine healthy subjects were exposed for a short period (20 min) to a known concentration of toluene. Exhaled breath condensate samples were collected before and at the end of the exposure, while the environmental concentration of toluene was continuously monitored. Toluene was analysed by head-space gas-chromatography mass spectrometry, and assay repeatability was also estimated in vitro. Baseline and post-exposure measurement of hippuric acid, the urinary toluene metabolite, was performed to assess current toluene exposure. Before the exposure toluene concentrations in the exhaled breath condensate were lower than the detectable limit in all subjects, while after the exposure toluene was detectable with a median value 0.35 µg l^-1 (range 0.15-0.55 µg l^-1) in all the exhaled breath condensate samples. As compared with the standard calibration in distilled water, the curves obtained by exhaled breath condensate were linear and comparable with the range examined in vivo for toluene. A significant correlation was found between the environmental toluene levels and toluene in the exhaled breath condensate at the end of exposure. Furthermore, a significant relationship between increased exhaled breath condensate toluene levels and urinary hippuric acid after the exposure was found. In conclusion, exhaled breath condensate is a promising matrix for toluene assessment, although its application in humans requires further investigations.     

Determination of free and total S-phenylmercapturic acid by HPLC/MS/MS in the biological monitoring of benzene exposure

Urinary S-phenylmercapturic acid (SPMA) is a biomarker suggested by the American Conference of Governmental Industrial Hygienists (ACGIH) for assessing occupational exposure to benzene. A possible cause of the miscorrelation between environmental monitoring and biological monitoring for benzene exposure, which many authors complain about, is the existence of a urinary metabolite that turns into SPMA by acid hydrolysis. Forty urine samples were tested to determine which concentration value would correspond to the ACGIH Biological Exposure Index (BEI) of 25 µg g^-1 creatinine if exposure assessment was based on the determination of SPMA after quantitative hydrolysis of its precursor. An aliquot of each sample was hydrolysed with 9 M H₂SO₄, a second one was brought to pH 2 and a third one was used as it was (free SPMA). SPMA was determined by high-performance liquid chromatography/tandem mass spectrometric technique (HPLC/MS/MS) using an internal standard. The analytical method was validated in the range 0.5-50 µg l^-1. The average SPMA in pH 2 samples is 45-60% of the total, while free SPMA varies from 1% to 66%. The hydrolysis of pre-SPMA reduces the likelihood of variability in the results by reducing pH differences in urine samples and increasing the amount of measured SPMA. The BEI limit value would be about 50 µg g^-1 creatinine.     

Cadmium exposure of rainbow trout, Salmo gairdneri Richardson: effects on immune functions

Differential leucocyte counts, phagocytosis, humoral antibody response and the in vitro blasto-genetic response to mitogens (lipopolysaccharide and Concanavalin A) and to an antigen ( Vibrio anguillarum ) were studied in rainbow trout exposed to 0,0.7 or 3.6 μg Cd 1⁻¹ for 12 weeks.
Although the fish did not exhibit any clinical or histological changes, cadmium exposure was found to affect two of the immune parameters measured. The cellular response of fish immunized with V. anguillarum to the homologous antigen was significantly lower for splenocytes obtained from fish exposed to cadmium for 9 weeks (3.6 μg Cd 1⁻¹ group) than for splenocytes obtained from non-exposed fish. Conversely, the humoral antibody response to V. anguillarum O-antigen was higher in the 3.6 μg Cd 1⁻¹ group than in the non-exposed group. Protective immunity of fish vaccinated against V. anguillarum was equally as good in the cadmium-exposed group as in the non-exposed group. No cadmium-induced changes in differential leucocyte counts or in the proportions of phagocytic cells were observed.     

Validation of urinary thiocyanate as a biomarker of tobacco smoking

Thiocyanate ion (SCN) is the major detoxication product of cyanide, which is converted to SCN by a thiosulphate sulphurtransferase, mainly in hepatic mitochondria. Low-level cyanide exposure for man is caused by factors such as dietary intake of cyanogenic glucosides, tobacco smoking, drug administration and occupational exposure to organic nitriles. Urinary SCN concentration was determined through a commercial kit for the analysis of cyanide in water. Spot urine samples were collected at 7:30 h and 12:30 h, from 99 healthy male white-collar office workers (non-smokers n=72, smokers n=27). Comparison of SCN excretion values did not show any difference between the morning and midday samples. The SCN median value of non-smokers was 24 μmol l^-1 (range 9-24 μmol l^-1) and was statistically different from that of smokers (SCN = 92 μmol l^-1, range 33-275 μmol l^-1) (