Alfred Bernard

Cloning and characterization of AOEB166, a novel mammalian antioxidant enzyme of the peroxiredoxin family.

Using two-dimensional electrophoresis, we have recently identified in human bronchoalveolar lavage fluid a novel protein, termed B166, with a molecular mass of 17 kDa. Here, we report the cloning of human and rat cDNAs encoding B166, which has been renamed AOEB166 for antioxidantenzyme B166. Indeed, the deduced amino acid sequence reveals that AOEB166 represents a new mammalian subfamily of AhpC/TSA peroxiredoxin antioxidant enzymes. Human AOEB166 shares 63% similarity with Escherichia coli AhpC22 alkyl hydroperoxide reductase and 66% similarity with a recently identifiedSaccharomyces cerevisiae alkyl hydroperoxide reductase/thioredoxin peroxidase. Moreover, recombinant AOEB166 expressed in E. coli exhibits a peroxidase activity, and an antioxidant activity comparable with that of catalase was demonstrated with the glutamine synthetase protection assay against dithiothreitol/Fe3+/O2 oxidation. The analysis of AOEB166 mRNA distribution in 30 different human tissues and in 10 cell lines shows that the gene is widely expressed in the body. Of interest, the analysis of N- and C-terminal domains of both human and rat AOEB166 reveals amino acid sequences presenting features of mitochondrial and peroxisomal targeting sequences. Furthermore, human AOEB166 expressed as a fusion protein with GFP in HepG2 cell line is sorted to these organelles. Finally, acute inflammation induced in rat lung by lipopolysaccharide is associated with an increase of AOEB166 mRNA levels in lung, suggesting a protective role for AOEB166 in oxidative and inflammatory processes.

Lung hyperpermeability and asthma prevalence in schoolchildren: Unexpected associations with the attendance at indoor chlorinated swimming pools

Aims: To study whether exposure to nitrogen trichloride in indoor chlorinated pools may affect the respiratory epithelium of children and increase the risk of some lung diseases such as asthma. Methods: In 226 healthy children, serum surfactant associated proteins A and B (SP-A and SP-B), 16 kDa Clara cell protein (CC16), and IgE were measured. Lung specific proteins were measured in the serum of 16 children and 13 adults before and after exposure to NCl3 in an indoor chlorinated pool. Relations between pool attendance and asthma prevalence were studied in 1881 children. Asthma was screened with the exercise induced bronchoconstriction test (EIB). Results: Pool attendance was the most consistent predictor of lung epithelium permeability. A positive dose-effect relation was found with cumulated pool attendance and serum SP-A and SP-B. Serum IgE was unrelated to pool attendance, but correlated positively with lung hyperpermeability as assessed by serum SP-B. Changes in serum levels of lung proteins were reproduced in children and adults attending an indoor pool. Serum SP-A and SP-B were already significantly increased after one hour on the pool side without swimming. Positive EIB and total asthma prevalence were significantly correlated with cumulated pool attendance indices. Conclusions: Regular attendance at chlorinated pools by young children is associated with an exposure dependent increase in lung epithelium permeability and increase in the risk of developing asthma, especially in association with other risk factors. We therefore postulate that the increasing exposure of children to chlorination products in indoor pools might be an important cause of the rising incidence of childhood asthma and allergic diseases in industrialised countries. Further epidemiological studies should be undertaken to test this hypothesis.

Renal and Neurologic Effects of Cadmium, Lead, Mercury, and Arsenic in Children: Evidence of Early Effects and Multiple Interactions at Environmental Exposure Levels

Lead, cadmium, mercury, and arsenic are common environmental pollutants in industrialized countries, but their combined impact on children’s health is little known. We studied their effects on two main targets, the renal and dopaminergic systems, in > 800 children during a cross-sectional European survey. Control and exposed children were recruited from those living around historical nonferrous smelters in France, the Czech Republic, and Poland. Children provided blood and urine samples for the determination of the metals and sensitive renal or neurologic biomarkers. Serum concentrations of creatinine, cystatin C, and β2-microglobulin were negatively correlated with blood lead levels (PbB), suggesting an early renal hyperfiltration that averaged 7% in the upper quartile of PbB levels (> 55 μg/L; mean, 78.4 μg/L). The urinary excretion of retinol-binding protein, Clara cell protein, and N-acetyl-β-d-glucosaminidase was associated mainly with cadmium levels in blood or urine and with urinary mercury. All four metals influenced the dopaminergic markers serum prolactin and urinary homovanillic acid, with complex interactions brought to light. Heavy metals polluting the environment can cause subtle effects on children’s renal and dopaminergic systems without clear evidence of a threshold, which reinforces the need to control and regulate potential sources of contamination by heavy metals.

Markers of early renal changes induced by industrial pollutants. II. Application to workers exposed to lead.

The present study has been carried out in the framework of a collaborative research project on the development of new markers of nephrotoxicity. A battery of more than 20 potential indicators of renal changes has been applied to 50 workers exposed to lead (Pb) and 50 control subjects. After application of selection criteria 41 exposed and 41 control workers were eventually retained for the final statistical analysis. The average blood Pb concentration of exposed workers was 480 micrograms/l and their mean duration of exposure was 14 years. The battery of tests included parameters capable of detecting functional deficits (for example, urinary proteins of low or high molecular weight), biochemical alterations (for example, urinary eicosanoids, glycosaminoglycans, sialic acid) or cell damage (for example, urinary tubular antigens or enzymes) at different sites of the nephron or the kidney. The most outstanding effect found in workers exposed to Pb was an interference with the renal synthesis of eicosanoids, resulting in lower urinary excretion of 6-keto-PGF1 alpha and an enhanced excretion of thromboxane (TXB2). The health significance of these biochemical alterations, detectable at low exposure to Pb is unknown. As they were not associated with any sign of renal dysfunction, they may represent reversible biochemical effects or only contribute to the degradation of the renal function from the onset of clinical Pb nephropathy. The urinary excretion of some tubular antigens was also positively associated with duration of exposure to Pb. Another effect of Pb that might deserve further study is a significant increase in urinary sialic acid concentration.

Renal excretion of proteins and enzymes in workers exposed to cadmium

Abstract. The proteinuria rate and the relative clearances of β2‐microglobulin, orosomucoid, albumin, transferrin and IgG were measured in forty‐two workers exposed to cadmium and in seventy‐seven control workers. A tubular type proteinuria with an increased excretion of β2‐microglobulin and often also a glomerular type proteinuria with an increased excretion of orosomucoid, albumin, transferrin and IgG were observed mainly in workers exposed to cadmium for more than 25 years and whose cadmium concentration in blood exceeded 1 μg Cd/100 ml and that in urine 10 μg Cd/g creatinine. The glomerular dysfunction was also suggested by an increased plasma level of β2‐microglobulin and creatinine. Both tubular and glomerular impairments occurred with the same prevalence and were not necessarily associated. The increased release of β‐galactosidase by the kidney suggested that cadmium can damage some epithelial cells.

In vivo measurement of liver and kidney cadmium in workers exposed to this metal: Its significance with respect to cadmium in blood and urine

Abstract The cadmium concentration in liver (CdL) and in kidney is measured in vivo by neutron capture γ-ray analysis in 309 male workers occupied in two Belgian zinc-cadmium plants. At the same time, blood cadmium (CdB) and the urinary excretion of β 2 -microglobulin, albumin, total protein, calcium, and cadmium (CdU) is also determined. Among the 264 subjects retained for the statistical analysis 236 are still active at the plants (group A) while 28 others are retired Cd workers or workers removed from Cd exposure (group R). Group A comprises 149 subjects with normal renal function who are engaged in jobs not directly related to Cd production (subgroup Ala) and 87 Cd workers daily occupied with Cd production of whom 72 subjects (subgroup Alb) have not and 15 (subgroup A2) have sign(s) of renal dysfunction. Group R is subdivided into subgroups R1 ( n = 10) and R2 ( n = 18), subjects without and with renal dysfunction, respectively. Examination of the cumulative frequency distributions and the correlations between the various biological parameters in the different subgroups leads to the following conclusions: (a) calciuria is not much different among the subgroups, (b) CdB mainly reflects recent exposure to cadmium in the absence of Cd-induced renal damage, (c) CdU follows the body burden of cadmium but increases proportionately much more in workers with renal dysfunction particularly when signs of tubular dysfunction are present, (d) CdL is proportional to duration and intensity of Cd exposure in workers without as well as with renal dysfunction, (e) renal cortical cadmium (CdKc) is higher in subgroup Alb and Rl than in subgroup Ala but does not differ between Cd workers without (subgroup Alb) and with (subgroups A2 and R2) renal dysfunction. The latter finding can be explained by a progressive decrease of CdKc after the onset of the renal damage. This hypothesis is supported by the observations that in Cd workers from subgroup R2. CdKc negatively correlates with years of past exposure to Cd ( r = −0.59), that Cd workers with renal dysfunction (subgroups A2 and R2) excrete significantly more cadmium in urine, and that they show a much lower slope of CdKc versus CdL than those with normal renal function (subgroup Alb). The results of this investigation suggest that there exists a range of critical CdKc levels , i.e., approximately from 160 to 285 ppm. Beyond a CdKc of 285 ppm the probability is very high that all persons will show sign(s) of renal dysfunction. It has been found that kidney dysfunction is likely to develop in workers with CdL between 30 and 60 ppm and that almost all the Cd workers with CdL above 60 ppm evidence renal dysfunction. This study also demonstrates that in the absence of kidney dysfunction. CdU is correlated with the body burden of cadmium ( r = 0.59), but that CdB is not. On the basis of the interrelationships among CdL, CdKc, CdU, and the indicators of renal function, it can be concluded that the probability of developing Cd-induced renal dysfunction in male Cd workers appears to be very low when the critical CdU level of 10 μg/g creatinine is not regularly exceeded. This CdU level corresponds to an average cadmium body burden of 160–170 mg.

Food contamination by PCBs and dioxins

In February 1999, a poisoning episode broke out in several poultry farms in Belgium. The Belgian authorities took immediate safeguards to protect public health and implemented a large-scale food-monitoring programme. Here we analyse the scale of the contamination and assess the likelihood of its adversely affecting the health of the general population.

Chlorinated pool attendance, atopy, and the risk of asthma during childhood.

The pool chlorine hypothesis postulates that the rise in childhood asthma in the developed world could result at least partly from the increasing exposure of children to toxic gases and aerosols contaminating the air of indoor chlorinated pools. To further assess this hypothesis, we explored the relationships between childhood asthma, atopy, and cumulated pool attendance (CPA). We studied 341 schoolchildren 10–13 years of age who attended at a variable rate the same public pool in Brussels (trichloramine in air, 0.3–0.5 mg/m3). Examination of the children included a questionnaire, an exercise-induced bronchoconstriction (EIB) test, and the measurement of exhaled nitric oxide (eNO) and total and aeroallergen-specific serum IgE. CPA by children (range, 0–1,818 hr) emerged among the most consistent predictors of asthma (doctor diagnosed or screened with the EIB test) and of elevated eNO, ranking immediately after atopy and family history of asthma or hay fever. Although the risk of elevated eNO increased with CPA [odds ratio (OR) = 1.30; 95% confidence interval (CI), 1.10–1.43] independently of total or specific serum IgE, the probability of developing asthma increased with CPA only in children with serum IgE > 100 kIU/L (OR for each 100-hr increase in CPA = 1.79; 95% CI, 1.07–2.72). All these effects were dose related and most strongly linked to pool attendance before 6–7 years of age. Use of indoor chlorinated pools especially by young children interacts with atopic status to promote the development of childhood asthma. These findings further support the hypothesis implicating pool chlorine in the rise of childhood asthma in industrialized countries.

Dioxin/polychlorinated biphenyl body burden, diabetes and endometriosis: findings in a population-based study in Belgium.

Dioxins and polychlorinated biphenyls (PCBs) are persistent organic pollutants widely distributed in the food chain, which is the main source of human exposure. Their effects on human health at background exposure levels are still poorly understood. Recent epidemiological evidence suggests a possible association between these pollutants and diabetes. We report here the results of a population-based study in Belgium on 257 (142 women and 115 men) environmentally exposed subjects, including 10 cases of endometriosis and nine cases of diabetes. Seventeen 2,3,7,8-polychlorinated dibenzodioxins/dibenzofurans (PCDD/Fs or dioxins), four coplanar PCBs (International Union of Pure and Applied Chemistry [IUPAC] nos 77, 81, 126 and 169) and 12 PCB markers (IUPAC nos 3, 8, 28, 52, 101, 118, 138, 153, 180, 194, 206 and 209) were quantified in serum fat from fasting blood samples in order to estimate the body burden of these pollutants. Whilst no difference was found between women with endometriosis and their controls, diabetic patients had significantly increased serum levels of dioxins, coplanar PCBs and the 12 PCB markers. After adjustment for age and other covariates, serum total toxic equivalent activity (sum of PCDD/Fs and coplanar PCBs) and 12 PCB marker concentrations in diabetics were 62% (p=0.0005) and 39% (p=0.0067) higher, respectively, than in controls. The risk of diabetes was significantly increased in subjects in the top decile for adjusted concentrations of dioxins (odds ratio 5.1, 95% confidence interval [CI] 1.18–21.7), coplanar PCBs (odds ratio 13.3, 95% CI 3.31–53.2) or 12 PCB markers (odds ratio 7.6, 95% CI 1.58–36.3). These findings warrant further studies to assess the significance of the associations between diabetes and environmental exposure to polychlorinated pollutants.

Clara cell secretory protein (CC16): characteristics and perspectives as lung peripheral biomarker

Clara cell protein (CC16) is a 15.8‐kDa homodimeric protein secreted in large amounts in airways by the non‐ciliated bronchiolar Clara cells. This protein increasingly appears to protect the respiratory tract against oxidative stress and inflammation. In vitro, CC16 has been shown to modulate the production and/or the activity of various mediators of the inflammatory response including PLA2, interferon‐γ and tumour necrosis factor‐α. CC16 has also been found to inhibit fibroblast migration or to bind various endogenous or exogenous substances such as polychlorobiphenyls (PCBs). This protective role is confirmed by studies on transgenic mice, showing that CC16 deficiency is associated with an increased susceptibility of the lung to viral infections and oxidative stress. In humans, a polymorphism of the CC16 gene, localized to a region linked to airway diseases, has recently been discovered in association with an increased risk of developing childhood asthma. Finally, CC16 also presents a major interest as a peripheral marker for assessing the integrity of the lung epithelium. The determination of CC16 in serum is a new non‐invasive test to detect Clara cell damage or an increased epithelial permeability in various acute and chronic lung disorders.