Binding of beryllium to nuclear acidic proteins.

In vitro beryllium (Be) binding to rat liver nuclei has been reassessed (KAss = 2.0 X 10(6) M: n = 17 nmol Be/mg protein). Be also binds to rat liver nucleoli (KAss approx. 4 X 10(6) M: n = 10 nmol Be/mg protein). Examination of rat liver chromatin fractionated on a hydroxyapatite column shows that Be does not bind to histone or to the non-histone protein eluted by 0.05 M sodium phosphate. Be is strongly bound to the non-histone proteins eluted by 0.2 M sodium phosphate (KAss = 1.1 X 10(6) M: n = 55 nmol Be/mg protein) and also to the same extent to the fraction containing DNA which is subsequently eluted from the column. Evidence is provided that the latter binding is not due to DNA. The fractions containing the Be-binding proteins also contain the proteins which are phosphorylated to the greater extent.     

Immunogenetic factors in beryllium sensitization and chronic beryllium disease

Exposure to beryllium in the workplace can cause beryllium sensitization and chronic beryllium disease. Sensitization to beryllium can be detected in the laboratory using the beryllium lymphocyte proliferation test. It was shown that anti-HLA antibodies could block the beryllium-specific response in the beryllium lymphocyte proliferation test, thereby implicating HLA genes in chronic beryllium disease. A supratypic genetic marker, HLA-DPB1*E69, was found to be strongly associated with immunologic sensitization to beryllium and chronic beryllium disease in beryllium workers. However, there are 40 HLA-DPB1 gene variants that have E69 but that also have other DNA sequence variations. The purpose of the study was to evaluate the evidence for potential differential susceptibility that may be associated with the physical characteristics of HLA protein molecules for which different HLA-DPB1*E69 variants code; that is, do some HLA-DPB1*E69 variants convey higher risk of beryllium sensitization and chronic beryllium disease than others. To do this, two approaches were pursued: first, detailed analysis of the findings from the published literature was performed, and second, computational chemistry was used to seek clues concerning the physical properties of the HLA protein molecules for which these alleles code. Among the 40 HLA-DPB1 gene variants that code for E69, molecular epidemiological studies have suggested a risk hierarchy, where some variants appear to convey low to moderate risk of chronic beryllium disease (e.g., HLA-DPB1*0201, approximately 3-fold increased risk), some convey an intermediate risk (e.g., HLA-DPB1*1901, approximately 5-fold) and others convey high risk (e.g., HLA-DPB1*1701, >10-fold). Molecular modeling has been used to further investigate a potential mechanistic basis for these observations. We found a strong correlation between the hierarchical order of risk of chronic beryllium disease associated with specific alleles and the predicted surface electrostatic potential and charge of the corresponding isotypes. Therefore, when alleles were grouped by the relative negative charge on the molecules for which they code, the data suggest that those alleles associated with the most negatively charged proteins carry the greatest risk of beryllium sensitization and disease.     

Ferritin. Binding of beryllium and other divalent metal ions

Rat liver homogenates in 0.1 M Tris, pH 7.5, were heated to 80 degrees C, cooled immediately, and centrifuged at 24,000 X g, and 7Be2+ was added to the supernatant. Twenty-five per cent of the radioactivity was bound to a single protein. It was purified to homogeneity and identified to be ferritin as judged by different criteria. These were sucrose density gradient centrifugation, electrophoresis in polyacrylamide gel of the native or sodium dodecyl sulfate-treated protein, reactivity to antibodies, isoelectric focusing, and total amino acid composition. Comparative study of the ability of ferritin or apoferritin to bind Cd2+, Zn2+, Cu2+, and Be2+ was conducted by using a gel equilibrium technique, Centifree micropartition technique, and microcentrifuge desalting technique. Ferritin could be saturated with Cd2+ or Zn2+ or Cu2+ but not with Be2+ even after 800 g atoms of Be2+ were bound. None of the bound Be2+ was dialyzable at 4 degrees C in 0.05 Tris acetate buffer, pH 8.5, but at pH 6.5 over 80% of the bound metal ion was dialyzed after 72 h. By contrast, apoferritin bound similar amounts of all four metal ions, some of which were dialyzable. By spectrophotometric titrations at pH 6.5 of Be2+ with sulfosalicylic acid (SSA), BeKDSSA was calculated to be 5.0 X 10(-6) M and by competition of sulfosalicyclic acid and ferritin for Be2+ the BeKDferritin was calculated to be 6.8 X 10(-6) M.     

Influence of MHC class II in susceptibility to beryllium sensitization and chronic beryllium disease

A glutamic acid at residue 69(Glu(69)) in the HLA-DPB1 gene (Glu(69)) is associated with chronic beryllium disease (CBD) and possibly beryllium sensitization (BeS). This study tested the hypothesis that MHC class II polymorphisms are important in susceptibility to BeS and CBD and that the Glu(69) variant is related to markers of disease severity. Genomic DNA was obtained from BeS (n = 50), CBD (n = 104), and beryllium-exposed nondiseased (Be-nondiseased) (n = 125) subjects. HLA-DPB1, -DRB1, and -DQB1 genotypes were determined by (sequence-specific primers) PCR. Disease severity was assessed by pulmonary function and exercise testing. A higher frequency of the DPB1 Glu(69) gene was found in CBD and BeS compared with the Be-nondiseased subjects, with odds ratios of 10.1 for CBD vs Be-nondiseased and 9.5 for BeS vs Be-nondiseased. The majority of BeS and CBD subjects displayed non-0201 Glu(69) alleles. Glu(69) homozygosity was higher in the CBD subjects, while BeS subjects were intermediate and Be-nondiseased lowest. DRB1*01 and DQB1*05 phenotypes were reduced in CBD vs Be-nondiseased subjects, while DRB1*13 and DQB1*06 were associated with CBD in the absence of Glu(69). Markers of disease severity, including a lower forced vital capacity, diffusion capacity for carbon monoxide, PaO(2) at rest, maximum workload on exercise testing, and a higher arterial-alveolar gradient at rest, were associated with Glu(69) homozygosity. We conclude that DPB1 Glu69 is a marker of sensitization and not specific for disease. Glu(69) homozygosity acts as a functional marker associated with markers of CBD severity.     

Genotoxicity of beryllium, gallium and antimony in short-term assays.

The genotoxicity of beryllium, gallium and antimony compounds was studied with the rec, Salmonella mutagenicity and SCE assays. In the rec assay, all the salts of the metals, BeCl2, Be(NO3)2, GaCl3, Ga(NO3)3, SbCl3, SbCl5, and an oxide, Sb2O3, had DNA-damaging activity. None of the compounds was mutagenic to Salmonella. In the SCE assays using V79 cells, 2 antimony(III) compounds, SbCl3 and Sb2O3, and 2 beryllium compounds, BeCl2 and Be(NO3)2, induced SCEs significantly. Sb2O3, slightly soluble in water, was positive in both the rec assay and the SCE assay at very low doses.     

Effects produced on isolated rat heart mitochondria by beryllium sulfate.

Beryllium sulfate increases the rate of oxygen uptake by isolated rat heart mitochondria. This effect is proportional to the Be²⁺ concentration. Beryllium binds to a limited number of sites on the isolated mitochondria and stimulates the mitochondrial ATPase. These effects, which appear to be related to an uncoupling of respiration, cause swelling of the mitochondria.     

The inhibition of phosphoglucomutase by beryllium

1. The inhibition of phosphoglucomutase by beryllium has been examined. 2. Inhibition by beryllium does not occur unless a complex-forming agent such as cysteine or imidazole is present. It is therefore similar to activation by magnesium. 3. The inhibition is progressive and the rate follows first-order kinetics, which may be defined by a bimolecular rate constant. 4. In the presence of magnesium the rate of inhibition is less. By using a fixed time for inhibition competition between beryllium and magnesium may be demonstrated. After inhibition has taken place, the addition of magnesium does not reverse it.     

The inhibition of enzymes by beryllium

1. The action of beryllium on the following enzymes has been examined: alkaline phosphatase (Escherichia coli and kidney), acid phosphatase, phosphoprotein phosphatase, apyrase (potato), adenosine triphosphatase (liver nuclei, liver mitochondria, brain microsomes), glucose 6-phosphatase, polysaccharide phosphorylases a and b, phosphoglucomutase, hexokinase, phosphoglyceromutase, ribonuclease, A-esterase (rabbit serum), cholinesterase (horse serum), chymotrypsin. Alkaline phosphatase and phosphoglucomutase are inhibited by 1mum-beryllium sulphate whereas the other enzymes are largely unaffected by 1mm-beryllium sulphate. 2. Possible mechanisms for the inhibition of phosphoglucomutase and alkaline phosphatase are discussed.     

HLA-DP allele-specific T cell responses to beryllium account for DP-associated susceptibility to chronic beryllium disease

Occupational exposure to small molecules, such as metals, is frequently associated with hypersensitivity reactions. Chronic beryllium (Be) disease (CBD) is a multisystem granulomatous disease that primarily affects the lung, and occurs in approximately 3% of individuals exposed to this element. Immunogenetic studies have demonstrated a strong association between CBD and possession of alleles of HLA-DP containing glutamic acid (Glu) at position 69 in the HLA-DP beta-chain. T cell clones were raised from three patients with CBD in whom exposure occurred 10 and 30 years previously. Of 25 Be-specific clones that were obtained, all were restricted by HLA-DP alleles with Glu at DP beta69. Furthermore, the proliferative responses of the clones were absolutely dependent upon DP beta Glu(69) in that a single amino acid substitution at this position abolished the response. As befits a disease whose pathogenesis involves a delayed type hypersensitivity response, the large majority of Be-specific clones secreted IFN-gamma (Th1) and little or no IL-4 (Th2) cytokines. This study provides insights into the molecular basis of DP2-associated susceptibility to CBD.     

Interaction of morphocycline with beryllium ions]

Behaviour of morphocycline (H5R) and its complex with beryllium ions in acqueous solutions was studied fluorimetrically. The ionization constants of H5R were estimated at pH 1.5-15 according to the data of fluorimetric determination with respect to OH-group: pK1 6.40, pK2 8.25, pK3 10.65, pK4 11.30. Two constants characterizing the deprotonization process with respect to the carbonylic group and nitrogen were also estimated: pK01--1.0 (greater than C = 0) and pK02 4.75 (--N=). Formation of an intensively fluorescing compound less than [Be3(OH)3(H2O2)5]2HR greater than 2+ was observed at pH 6.0-7.0. The cause of such intensive fluorescence was deformation of ion [Be3(OH)3(H2O)6]3+ because of its exclusion from the coordinating sphere of one molecule of water during the complex formation and decreasing of level H comes from II as compared to the morphocycline level II comes from n. A procedure for detecting morphocycline in the blood of humans and animals was developed.