In vitro assessment of the toxicity of metal compounds

A review of in vitro mutagenesis assessment of metal compounds in mammalian and nonmammalian test systems has been compiled. Prokaryotic assays are ineffective or inconsistent in their detection of most metals as mutagens, with the notable exception of hexavalent chromium. Mammalian assay systems appear to be similarly inappropriate for the screening of metal compounds based upon the limited number of studies that have employed those compounds having known carcinogenic activity. Although of limited value as screening tests for the detection of potentially carcinogenic metal compounds, the well-characterized in vitro mutagenesis systems may prove to be of significant value as a means to elucidate mechanisms of metal genotoxicity.     

High affinity specific antiestrogen binding sites are concentrated in rough microsomal membranes of rat liver

Saturation and competitive binding analyses demonstrated the presence of a high affinity (K_D = 0.92 nM), specific antiestrogen binding site (AEBS) in rat liver microsomes and at least 75% of total liver AEBS was recovered in this fraction. When microsomes were further separated into smooth and rough fractions, AEBS was concentrated in the latter. Subsequent dissociation of ribosomes from the rough membranes revealed that AEBS was associated with the membrane and not the ribosomal fraction. Antiestrogen binding activity could not be extracted from membranes with 1 M KCl or 0.5 M acetic acid but could be solubilized with sodium cholate. These data indicate that AEBS is an integral membrane component of the rough microsomal fraction of rat liver.     

Mechanisms of peroxidase oxidation of o-dianisidine, 3,3′,5,5′-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate

Peroxidase oxidation of o-dianisidine, 3,3′,5,5′-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate (SDS), an anionic surfactant, was spectrophotometrically studied. It was found that 0.1–100 mM SDS concentrations stabilize intermediates formed in the peroxidase oxidation of these substrates. The cause of the stabilization is an electrostatic interaction between positively charged intermediates and negatively charged surfactant.     

Monoclonal antibodies against beta-galactoside-binding lectin of chick embryo recognizes conformational change of the antigen

Monoclonal antibodies against chick embryonic beta-galactoside-binding lectin were obtained. One of the monoclonal antibodies was ineffective in Western blotting and seemed to be unable to bind the SDS-denatured lectin. When the native lectin was dotted on a nitrocellulose filter and subjected to denaturation by treatment with SDS, urea or heat, binding of this antibody no longer occurred, though other monoclonal antibodies bound normally. This antibody seems to have been raised against an epitope which is destroyed upon denaturation.     

Synthesis by Enzymatic Catalysis VI—An Investigation of Chemical Modification of HRP by Fourier Transform Infra-Red Vibrational Spectroscopy

Horseradish peroxidase was chemically conjugated on its carbohydrate moieties with short aliphatic chains (C₈ and C₁₆). An analytical method using FT.IR spectroscopy was developed to analyze this alteration in enzyme structure. This method is non-destructive, and can be applied directly to samples of the reaction mixture. More general applications of this technique are described and discussed.     

Antagonistic and synergistic interactions between aluminum and manganese on growth of Vigna unguiculata at low ionic strength

Concentrations of soluble aluminum (Al) and manganese (Mn) frequently reach phytotoxic levels in acid soils. While dose response relationships for these metals are well documented, the effects of combined exposure have received less attention. We have examined the effect of combinations of Al and Mn on growth and metal accumulation in Vigna unguiculata (L.) Walp. grown in solution culture under conditions of low ionic strength (conductivities typically < 100 µS cm⁻¹). The nature of interaction between these metals varied with the specific physiological response, the part of the plant investigated, and the relative amount of stress imposed. Analysis of growth data provided evidence for amelioration of metal toxicity (antagonistic effects), although this effect was dose dependent. Analysis of metal content data provided evidence for antagonistic and synergistic (exacerbation of toxicity) effects, again depending on dose. Analysis of foliar symptoms also provided evidence for antagonisms and synergisms, with the nature of the response dependent on the specific physiological response and specific plant part investigated. In contrast with previous reports, evidence for antagonistic, synergistic, and multiplicative effects on growth, metal uptake, and expression of foliar symptoms have been obtained under physiologically and environmentally relevant conditions. These results suggest a more detailed analysis of the potential for interactions between metals in the environment is required.     

Horseradish peroxidase: a reliable or a misleading tool for the investigations on the origin of the proteins of the aqueous humor?

The concept of the blood-aqueous barrier is largely based on the use of horseradish peroxidase (HRP). The present investigation was designed to check its reliability as a macromolecular tracer, especially with regard to the transport of plasma proteins. Rabbits were killed 5 min to 24 h after being intravenously injected with HRP. The tracer diffused rapidly, reaching the aqueous humor of the eye in 3 min or less and was detected at high concentration in the narrow space between the outer epithelial layer of the ciliary epithelium and the wall of the pervious capillaries in the stroma of the processes. HRP appeared to migrate from the blood to the posterior chamber, permeating the tight junctions, viz., the anatomical basis of the blood-aqueous barrier. It was detected at higher concentration at the anterior surface of the iris, at short time intervals; this was interpreted as penetration of the tracer from the aqueous humor of the anterior chamber. The choroid was also labeled in continuation with the reaction in the stroma of the pars plana of the ciliary body which, in turn, sometimes reached the iris root. Therefore, the pervious blood vessels of the choroid could be a source of macromolecules for the iris root. HRP also induced the formation of lysosomes in the ciliary epithelium. This can hardly be accepted as the way in which plasma proteins are physiologically transported to the aqueous humor. However, the pathway of HRP migration over short time intervals seems to be in agreement with previous research indicating that the entrance of serum albumin into the posterior chamber is the first step of its incorporation into the aqueous humor. Received: 7 June 1996 / Accepted: 15 January 1997     

Photo-oxidative damage in the ripening tomato fruit: Protective role of superoxide dismutase

Factors relating to photo-oxidative damage in tomatoes were investigated during maturation of the fruit and upon induction of sunscald. Superoxide dismutase (SOD) activity passed through a minimum at the mature-green and breaker stages of ripening and availability of zinc and copper did not appear to be a limiting factor in the synthesis of the enzyme. Iron levels were maximal and total carotenoid concentrations were lowest during the same mature-green and breaker stages of maturation, while chlorophyll was starting to decrease but was still present in large amounts. Peroxidase activity decreased steadily during ripening. Artificial induction of tolerance to photodynamic damage by controlled heat treatment was accompanied by an increase in SOD activity, while carotenoid levels and peroxidase activity did not change. These findings support the thesis that the previously reported susceptibility of tomatoes to photodynamic damage, i.e. sunscald, during the mature-green and breaker stages of maturation is related to enhanced formation of superoxide ions, at a time when chloroplast structure begins to break down. SOD, by scavenging the superoxide, appears to supplement the protective action of carotenoids against photo-oxidative injury.     

Location of crosslinks in chemically stabilized horseradish peroxidase: implications for design of crosslinks.

The bifunctional compound, ethylene-glycol bis(N-hydroxysuccinimidylsuccinate) (EGNHS), stabilizes horseradish peroxidase C (HRP) by reaction with the enzyme's lysine residues. In this study we compare native and modified HRP by proteolytic fragmentation, peptide sequencing, and mass spectroscopy, and identify the sites of modification. Most significantly, EGNHS is shown to form a crosslink between Lys232 and Lys241 of HRP and modifies Lys174 without formation of a crosslink. These findings are in agreement with the lysine side-chain reactivities predicted from the surface accessibility of the amino groups, and the maximal span of 16 A of the EGNHS crosslinker.     

Zinc,copper, selenium,and glutathione peroxidase in blood of 11-yr-old dunedin,New Zealand children

Blood was obtained from 564 11-yr-old children who had participated since birth in a multidisciplinary health and development study. Serum zinc concentration did not differ between the boys and the girls (mean±SD: 91=17 μg/100 mL,n=453). Five-6% of serum zinc values were low; although there was a weak correlation with height, none of the boys with low values were below the 10th percentile for height for this group. Serum copper concentration (112±24 μg/100 mL,n=454) was unrelated to sex, height, weight, body mass index, socioeconomic status (SES), or iron status. Blood selenium concentration (49±10 ng/mL,n=564) was lower than previously reported for Dunedin children; it was higher in children in the lower SES categories. The data represent normal values for healthy, 11-yr-old NZ children.