Suspension Assay Research Articles

Staphylococcus aureus phagocytosis : A new cytofluorometric method using FITC and paraformaldehyde

A new method for measuring the uptake of Staphylococcus aureus by human polymorphonuclear neutrophils (PMN) using flow cytometry (FCM) is described. Bacteria were labeled with fluorescein isothiocyanate (FITC) and incubated with PMN in a suspension assay. At the end of the assay, phagocytosis was arrested by the addition of cold paraformaldehyde. Thereafter, phagocytosis was quantitated by FCM, using crystal violet to distinguish adherent versus ingested bacteria. The method was validated in multiple samples by reference to our microscopic technique; correlations of phagocytic indices were in good agreement. The FCM method was also found to be reproducible and provided a mean to detect low phagocyte values. Another feature of the approach is that FCM readings can be delayed without any appreciable alterations in the results.

An SV40 mutant oncoprotein has a nuclear location

T147 is an SV40 mutant that makes a normal small t antigen and a large T antigen that is only 147 amino acids long. We have introduced a second mutation into the genome of T147 which eliminates its ability to encode small t antigen. We show that this double mutant is able to transform C3H10T1 /2 mouse cells in a focus assay and F111 rat cells in an agar suspension assay, demonstrating that the transforming domain of T antigen is located within its amino-terminal 147 amino acids. We also show that the T147 mutant T antigen, like wild-type T antigen, has a nuclear location. However, in contrast to wild-type T antigen, which is also found in the plasma membranes of wild-type transformed cells, we fail to detect any mutant T antigen associated with the plasma membranes of T147 transformants.

Metabolism of mutagenic polycyclic aromatic hydrocarbons by photosynthetic algal species

Polycyclic aromatic hydrocarbons (PAH) known to produce carcinogenic and mutagenic effects have been shown to contaminate waters, sediments and soils. While it is accepted that metabolites of these compounds are responsible for most of their biological effects in mammals, their metabolism, and to a large extent their bioactivity, in aquatic plants have not been explored. Cultures of photosynthetic algal species were assayed for their ability to metabolize benzo[ a]pyrene (BaP), a carcinogenic PAH under conditions which either permitted (white light) or disallowed (gold light) photooxidation of the compound. Growth of Selenastrum capricornutum, a fresh-water green alga, was completely inhibited when incubated in white light with 160 μg BaP/1 medium. By contrast concentration at the upper limit of BaP solubility in aqueous medium had not effect on algal growth when gold light was used. BaP quinones and phenol derivatives were found to inhibit growth of Selenastrum under white light incubation. BaP phototoxicity and metabolism were observed to be species-specific. All 3 tested species of the order Chlorococcales were growth-inhibited by BaP in white light whereas neither the green alga Chlamydomonas reinhardtii nor a blue-green, a yellow-green or an euglenoid alga responded in this fashion. Assays of radiolabeled BaP metabolism in Selenastrum showed that the majority of radioactivity associated with BaP was found in media as opposed to algal cell pellets, that the extent of metabolism was BaP concentration dependent, and that the proportion of various metabolites detected was a function of the light source. After gold light incubation, BaP diols predominated while after white light treatment at equal BaP concentrations, the 3,6-quinone was found in the highest concentration. Extracted material from algal cell pellets and from media was tested for mutagenicity in a forward mutation suspension assay in Salmonella typhimurium using resistance to 8-azaguanine for selection. Direct-acting mutagens were detected in extracted media from incubation of Selenastrum with 400 μg BaP/1 for 1 day in gold light. Extracts of media from algae incubated in gold light from 1 to 4 days with 1200 μg BaP/1 were found to have direct-acting mutagens as well as those requiring further metabolism.

Inhibitory effect of phenolic compounds on aflatoxin B 1 metabolism and induced mutagenesis

The interaction between phenolic compounds and the food-borne carcinogenic mycotoxin, aflatoxin B 1 (AFB 1), was examined. 6 phenolic compounds (gallic acid, chlorogenic acid, caffeic acid, dopamine, p-hydroxybenzoic acid and salicyclic acid) inhibited AFB 1-induced mutagenesis in Salmonella typhimurium strain TA98 in a suspension assay in the presence of rat-liver microsomes (S9). The inhibitory effect was observed when the phenolic compound and the mutagen (AFB 1 plus S9) were administered concurrently, but not when exposure to the mutagen was followed by the phenolic compound. The concentrations of the phenolic compounds used were not mutagenic to S. typhimurium strain TA98 and had no effect on the survival of the bacteria. The inhibition of AFB 1 metabolism was studied using high-pressure liquid chromatography. Increasing the concentration of all 6 phenolic compounds resulted in a dose-dependent reduction of both major AFB 1 metabolite peaks. The results are consistent with the hypothesis that (1) the phenolic compounds do not react covalently with AFB 1, and (2) the inhibitory effect of phenolic compounds on AFB 1-induced mutagenesis may be due to the inhibition of the activation enzymes.

The Escherichia coli K-12 SOS chromotest agar spot test for simple, rapid detection of genotoxic agents

The Escherichia coli K-12 SOS chromotest is a colorimetric (β-galactosidase induction) system for detecting genotoxic chemicals as agents which induce filamentation in response to DNA damage. The chromotest was modified from a liquid suspension assay to a simple, convenient agar spot test, which was performed in the manner of a related colorimetric prophage induction assay (BIA). Chromotest agar dishes yielded optimal results after 16–18 h incubation, presumably because of the agar growth characteristics of tester strain PQ37. Of 44 tested chemicals, nitro aromatics, cytotoxic/antitumor agents, polycyclic hydrocarbons and aflatoxins showed good activity. Alkylating agents such as MNNG and MMS were active only at high concentrations. Compounds active in both the chromotest and BIA were active at 10–100-fold lower concentrations in the chromotest. The chromotest appeared to be less effective than the Salmonella Ames mutagenicity test in the detection of diverse classes of chemical carcinogens. The chromotest may be a useful alternative to the BIA in the study of particular classes of genotoxic compounds.

Activation of mutagens by hepatocytes and liver 9000 × g supernatant from human origin in the Salmonella typhimurium mutagenicity assay : Comparison with rat liver preparations

The mutagenicity of 10 known genotoxic compounds, of several chemical classes, was measured in Salmonella typhimurium mutagenicity assays comprising isolated human hepatocytes or human liver 9000 × g supernatant (S9)from 4 different individuals, as activating system. The mutagenic activity of several compounds as determined with the Salmonella/hepatocyte suspension assay showed obvious differences when compared with the values obtained in the Salmonella/S9 plate assay. For instance, the mutagenic activity of BZ, DMN and DEN appeared to be much higher in the hepatocyte assay than in the S9 assay. However, 2-AF and 2-AAF were activated more effectively into mutagens in the S9 assay than in the hepatocyte assay. 2-AF was slightly more mutagenic than 2-AAF in the hepatocyte assay, whereas it was far more mutagenic than 2-AAF in the S9 assay. DMN was found more mutagenic than DEN in the hepatocyte assay, whereas in the S9 assay DEN appeared to be slightly more mutagenic. Furthermore, great interindividual differences in the metabolic activation of certain compounds, e.g. BZ and DMN, were observed in the hepatocyte suspension assay, whereas these variations were less evident in the S9 plate assay. Comparison of the mutagenicity data obtained with the human liver preparations, with those obtained with rat liver preparations, showed great interspecies differences in the capacity to activate certain chemicals into mutagens. The use of human liver preparations, in particular isolated human hepatocytes, may be of great value in studies on inter- and intraspecies variations in metabolic activation of genotoxic agents.

The mutagenicity of sodium bisulfite on base‐substitution strains of Salmonella typhimurium

The mutagenic effects of sodium bisulfite on Salmonella typhimurium LT2 strains carrying the hisG46 allele were investigated. Treating the cells with 1 M sodium bisulfite in 0.2 M acetate buffer (pH 5.2) for various time intervals at 37 degrees C resulted in an increase in the yield of mutants above the spontaneous level. The fold enhancement of the mutant yield and the fold enhancement of the mutant frequency were greatest for the parent strain hisG46 with wild type DNA repair; the derived strains with reduced repair capacity responded to a lesser degree. While sodium bisulfite was mutagenic when tested in liquid suspension assays, strain hisG46 did not respond when the standard Ames plate incorporation assay was used. The greater mutagenic response in repair-proficient strains suggests that normal repair capacities may favor the mutagenic activity of sodium bisulfite. Transduction studies demonstrated that mutations of his+ were closely linked to the hisD gene, and probably were in the structural hisG gene itself.

Heparan sulfate proteoglycans from mouse mammary epithelial cells. Basal extracellular proteoglycan binds specifically to native type I collagen fibrils.

Mouse mammary epithelial cells (NMuMG cells) deposit at their basal surfaces an extracellular heparan sulfate-rich proteoglycan that binds to type I collagen. The binding of the purified proteoglycan to collagen was studied by (i) a solid phase assay, (ii) a suspension assay using preformed collagen fibrils, and (iii) a collagen fibril affinity column. The binding interaction occurs at physiological pH and ionic strength and can be inhibited only by salt concentrations that greatly exceed those found physiologically. Binding requires the intact proteoglycan since the protein-free glycosaminoglycan chains will not bind under the conditions of these assays. However, binding is mediated through the heparan sulfate chains as it can be inhibited by block-sulfated polysaccharides, including heparin. Binding requires native collagen structure which may be optimal when the collagen is in a fibrillar configuration. Binding sites on collagen fibrils are saturable, high affinity (Kd approximately 10(-10) M), and selective for heparin-like glycosaminoglycans. Because a culture substratum of type I collagen fibrils causes NMuMG cells to accumulate heparan sulfate proteoglycan into a basal lamina-like layer, binding of heparan sulfate proteoglycans to type I collagen may lead to the formation of a basal lamina and may link the basal lamina to the connective tissue matrix, an association found in basement membranes.

The effect of the environment on the immunofluorescence staining of bacteria

Quantitative direct immunofluorescence (IF) assays of Bacillus anthracis, B. cereus and B. subtilis spores, and of Escherichia coli and Serratia Marcescens were performed with a microfluorometer to measure the fluorescence of individual stained bacteria. Bacteria were either stained after fixation on multispot slides (the slide assay) or they were stained in suspension and subsequently immobilised on slides (the suspension-slide method) or on membrane filters (the suspension-filter method). A similar degree of variation of bacterial fluorescence was encountered in the slide and suspension-slide methods, suggesting that the fluorescence variation in the slide method was not the result of variable interference by the glass support. Results in the suspension-filter assays revealed additional variation due to the membrane filter itself. The IF staining of Bacillus spores was strongly influenced by the concentration of normal serum buffer in the reaction mixture. It is tentatively concluded that the specific binding of antibody was altered. Mean bacterial fluorescence levels in the Bacillus slide assays were usually markedly lower than in suspension assays performed under the same buffer conditions. The implications of this are discussed.

Metabolic activation and bacterial mutagenicity of aflatoxin B 1 in two different test systems

The metabolism of aflatoxin B 1 (AFB 1 by liver post-mitochondrial supernatant fraction from phenobarbitone treated male albino rats in liquid suspension and soft-agar plate incorporation bacterial mutation assay systems has been studied. The AFB 1 concentration used was in the range of those added to bacterial mutagenicity tests with this mycotoxin. Three oxidative metabolites of AFB 1 viz the Tris derivative of AFB 1 8,9-diol (derived from the 8,9-epoxide), aflatoxin Q 1 (AFQ 1) and M 1 (AFM 1) were observed. The metabolite profile and time course of formation were qualitatively similar in both assay systems. The rate and overall formation of metabolites in the soft-agar system was approximately one half that in the liquid suspension system which was reflected in decreased AFB 1 induced bacterial mutation in the former system. The AFB 1 metabolite profile in these in vitro systems did not mirror completely the reported in vivo profile seen in the rat, as aflatoxin P 1 (AFP 1) and a glutathione conjugate were not detected. No evidence was found for a prolongation of metabolism in the soft-agar system.

Phagocytic killing of Neisseria gonorrhoeae by human monocytes.

The ability of human monocytes to phagocytize and kill nonpiliated opaque (T3) and transparent (T4) gonococci was investigated in a tumbling tube suspension assay. A serum-sensitive strain, F62, and a serum-resistant strain, FA19, were studied. CFU remaining after incubation with monocytes were used to assess the extent of killing. The data show that 50% of T3 and T4 gonococci of both strains were killed by monocytes over a 2-h period. Serum was necessary for the killing of transparent gonococci of both strains as well as for FA19 T3. Concentrations of serum ranging from 0.5 to 10% were equally effective, and heat-labile components were required. Killing of F62 T3, however, occurred in the absence of serum. An increased ratio of bacteria to monocytes decreased the rate of killing. A 30-min preopsonization of gonococci in 10% serum resulted in an enhanced rate of killing. Monocytes were able to kill plate-grown, but not log-phase, organisms. Disruption of the monocytes by sonication to release internalized bacteria did not increase the number of viable organisms. The addition of 10 micrograms of cytochalasin B per ml completely inhibited the reduction in colony numbers over time. These data indicate that freshly isolated human monocytes are capable of phagocytizing and killing nonpiliated gonococci.

A differential DNA-repair test using mixtures of E. coli K12 strains in liquid suspension and animal-mediated assays

The feasibility of performing tests for repairable DNA damage in animal assay procedures was investigated by using repair-proficient and repair-deficient derivatives of E. coli K12 strain 343/113, including mutations in the uvrB, recA, polA and dam genes. To avoid variations in the relative recovery of viable cells from different samples, the strains were further marked with auxotrophic growth requirements, so that mixtures could be treated and the survival of each strain determined individually on media containing the corresponding growth factors. Spot tests were performed with the various strains to re-assess the necessity of using a combination of repair deficiencies, when genotoxic agents of differing mode of action are to be detected. Liquid suspension tests on mixtures of the different strains, furthermore, confirmed that the survival of the individual strains can be determined separately on selective media after treatment with methyl methanesulfonate (MMS) and methyl nitrosourea (MNU). These tests were also used to demonstrate that dimethyl nitrosamine (DMNA) is activated by Aroclor-1254-induced rat-liver S9 fractions to genotoxic products, as measured by the low survival of a recA derivative compared with the repair-proficient wild-type strain. Intrasanguineous host-mediated assays using the present derivatives of E. coli K12/343/113 showed that the various strains, injected simultaneously into mice, could be recovered in amounts sufficient for the individual determination of the relative survival in liver, spleen, lungs, kidneys, pancreas and the blood stream of the host animals. Using a mixture of the repair-proficient parent and the recA derivative inoculated into mice that were subsequently treated with MMS, NMU or DMNA, we found that these chemicals induce a larger decrease in survival in the recA strain as compared with the wild-type in cells recovered from the liver and the spleen. The order of genotoxic potency so determined was DMNA > MNU > MMS; this is similar to the ranking of the carcinogenicity of these compounds in rodents and probably also reflects the various degrees of DNA alkylation in cells of the livers of the treated animals. The general usefulness of the host-mediated differential DNA repair assay for detecting genotoxic factors in various organs of animals remains to be assessed by using chemical mutagens of different modes of action.

Mutagenic Activity of Phthalate Esters in Bacterial Liquid Suspension Assays

The mutagenic activities of several phthalate esters have been evaluated in an 8-azaguanine resistance assay in Salmonella typhimurium. Three phthalate esters were found to be mutagenic: dimethyl phthalate, diethyl phthalate and di-n-butyl phthalate. A number of other phthalate esters were not found to be mutagenic, including di(2-ethylhexyl) phthalate, di-n-octyl phthalate, diallyl phthalate, diisobutyl phthalate and diisodecyl phthalate. A metabolite of di(2-ethylhexyl) phthalate, 2-ethylhexanol, was also noted to be mutagenic. The mutagenic activity of this agent and others in this series was dose dependent but weak. No dose-response curve exceeded more than 3.5 times background at maximally testable concentrations. A liquid suspension histidine reversion assay of dimethyl phthalate showed levels of mutagenic activity similar to that observed in the azaguanine resistance assay. The data suggest a need for further investigation of the mutagenic potential of these agents in other assay systems.

Mutagenic activity of phthalate esters in bacterial liquid suspension assays.

The mutagenic activities of several phthalate esters have been evaluated in an 8-azaguanine resistance assay in Salmonella typhimurium. Three phthalate esters were found to be mutagenic: dimethyl phthalate, diethyl phthalate and di-n-butyl phthalate. A number of other phthalate esters were not found to be mutagenic, including di(2-ethylhexyl) phthalate, di-n-octyl phthalate, diallyl phthalate, diisobutyl phthalate and diisodecyl phthalate. A metabolite of di(2-ethylhexyl) phthalate, 2-ethylhexanol, was also noted to be mutagenic. The mutagenic activity of this agent and others in this series was dose dependent but weak. No dose-response curve exceeded more than 3.5 times background at maximally testable concentrations. A liquid suspension histidine reversion assay of dimethyl phthalate showed levels of mutagenic activity similar to that observed in the azaguanine resistance assay. The data suggest a need for further investigation of the mutagenic potential of these agents in other assay systems.

The specific binding of androgens and the subsequent distribution of androgen-receptor complexes within MCF-7 human breast cancer cells

Using a whole cell suspension assay technique, we have examined both the specific binding of testosterone (T) and dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) (DHT) and the subsequent distribution of androgen binding activity within MCF-7 human breast cancer cells. We have observed that both androgens are bound with high affinity to the same single class of receptor which is present at a concentration of approximately 8,000 sites per cell. The incubation of cells with either T or DHT was followed by the nuclear accumulation of specifically bound ligand which increased to maximal values within 1 h and then decreased thereafter. However, only about 30% of the total cellular specific binding activity observed with either androgen was localized within the nuclear compartment at any time during a 4 h incubation. Further examination of the extranuclear binding component suggested that a substantial portion of this activity was localized to the particulate fraction of the cytoplasm. The results of these studies suggest that both T and DHT are capable of exerting biological activity within MCF-7 cells, and raise the interesting possibility that androgen-receptor complexes may participate in the direct regulation of mitochondrial and/or microsormal function in this system.

Beta-Adrenergic stimulation of the release of ACTH- and LPH-related peptides from the pars intermedia of the rat pituitary gland.

The intermediate lobe of the rat pituitary gland produces a series of peptides related to ACTH and LPH. The spontaneous and isoproterenol-stimulated release of such peptides was studied during in vitro superfusion of rat neurointermediate lobes with Krebs-Ringer medium. Products released into the superfusion medium were quantified by direct measurement or after chromatography on Sephadex G-50. ACTH bioactivity was determined by use of adrenal cortical cell suspension assay. In addition, NH2-terminal ACTH, CO2H-terminal ACTH, alpha-MSH and beta-endorphin radioimmunoassays were used. The results show that 1. neurointermediate lobes of rats secret spontaneously various ACTH- and LPH-related peptides in amounts proportional to the amounts in which these peptides are found in extracts of the neurointermediate lobe; 2. the beta-adrenergic agonist, isoproterenol, stimulated the spontaneous release of various peptides, including alpha-MSH, ACTH, CLIP, glycosylated CLIP, and beta-endorphin-like peptides; 3. isoproterenol induced a dose-dependent (10(-9)-10(-7) M), parallel increase in the release of alpha-MSH and ACTH following similar time courses and showing identical EC50 values (about 10(-8)M). Although the spontaneous release of alpha-MSH and ACTH from rat neurointermediate lobes is not strictly coupled under the conditions used in this study, isoproterenol seems to affect the spontaneous release of these peptides to the same relative extent.

Mutagenicities of styrene oxide derivatives on bacterial test systems: Relationship between mutagenic potencies and chemical reactivity

The reaction products of deoxyguanosine with 3,4-dimethyl-, p-methyl-, m-methyl-, m-methoxy-, p-bromo-, m-chloro- and unsubstituted styrene oxide were isolated and characterized. The reaction followed second-order kinetics. The reactive site was the N-7 site of guanine. The normal isomer (attack at CH 2 of styrene oxides) and the abnormal isomer (attack at CH) were obtained. The Hammett reaction constant (ϱ) for the normal reaction was +0.42 and that for the abnormal reaction was −1.1. The difference of acid dissociation constant, rate of imidazole ring opening and rate of glycosidic bond cleavage was not found between the normal adduct and the abnormal adduct. Styrene oxides have been tested for toxicity and mutation in a liquid suspension assay using Escherichia coli WP2 and some of its repair deficient derivatives. Salmonella typhimurium TA100 was also employed. The mutation frequency of 3,4-dimethylstyrene oxide was higher in CM571 than in WP2. The greatest mutability was found with the WP2uvrA strain. 3,4-Dimethylstyrene oxide also acted as an inducer of E. coli K-12 (λ). The mutagenicity of 3,4-dimethylstyrene oxide may depend on a combination of the recA-dependent and recA-independent action mechanisms. The mutagenicities of these compounds in WP2 uvrA and TA100 increased in the order: m-chlorostyrene oxide = p-bromostyrene oxide < m-methoxystyrene oxide < styrene oxide < m-methylstyrene oxide < p-methylstyrene oxide < 3,4-dimethylstyrene oxide. There was no correlation between the mutagenicities and the total reaction rates. On the other hand, the reaction rates of these compounds for the abnormal reaction correlated with their mutagenicities. The results indicate that the mutagenic effect of a chemical is not dependent simply on the quantity of the adducts, but may vary with the structure of its adducts.

An examination of the quantitative suspension assay for mutagenesis with strains of Salmonella typhimurium.

The effects of cell toxicity on the mutagenic response obtained in a quantitative suspension assay using the strains of Salmonella typhimurium developed by Ames et al [Mutat Res 31:347--364, 1975] were examined. Cell toxicity was shown to produce false-positive results when the cells were plated immediately with trace amounts of histidine in the overlay agar. Replacement of the overlay agar with one lacking histidine produced false-negative results when the cells were plated immediately after exposure to a frame-shift-type mutagen. The inclusion of an overnight growth period in brain-heart infusion broth before plating the cells on minimal medium permitted incorporation of mutations into the genome and also yielded constant titers. The addition of the regrowth period is shown to be necessary for obtaining valid results in a suspension assay.

Induction of mitotic recombination by certain hair-dye chemicals in Saccharomyces cerevisiae

A number of procedures were used to test for the potential of 5 hair-dye chemicals, 4-nitro- o-phenylenediamine, 2-nitro- p-phenylenediamine, m-phenylenediamine, 2,4-diaminoanisole sulfate and 2,5-diaminoanisole sulfate, to induce genetic damage in yeast strains D3 and D4 of Saccharomyces cerevisiae. Various plate-test procedures, short-term suspension assays in phosphate buffer and suspension assays with liver enzyme activation all proved to be ineffective for demonstrating genetic effects of these chemicals. Only suspension assays in which the yeast cells were treated with the test chemical under growing conditions for up to 72 h were effective in demonstrating the genetic activity of 4-nitro- o-phenylenediamine and 2,4-diaminoanisole sulfate. The implications of these results for testing of mutagens in yeast systems are discussed along with other supportive evidence from the literature.

Influence of subcellular fractions of mammalian testes on the mutagenic activity of nitrofurans toward Escherichia coli; presence of a co-mutagen-like factor.

The activation of nitrofurans to mutagenic intermediates by testicular tissue was investigated. AF-2 and nitrofurazone were tested in a microsomal suspension assay with strain E. coli K-12 343/113 as indicator and subcellular fractions from rabbit testes. Different mutation patterns were observed in the presence or absence of testicular homogenate, indicating the presence of different mutagenic intermediates. The frequency of arg+ reversion increased proportionally to the homogenate concentration suggesting that the nitrofurans were activated by testicular components to intermediates that induced base-pair substitutions. Other experiments showed that a component of low molecular weight, present in the soluble fraction of homogenates of testes from rabbits, rats and monkeys, was responsible for the increased mutation frequency. It is concluded that this "co-mutagen-like" factor either alters the metabolism of nitrofurans in E. coli and/or promotes the formation of base-pair substitution-type mutations. This direct interaction between a nonenzymic component of mammalian testes and the mutation induction/expression process in E. coli suggests the role of co-mutagen-like factors in the sensitivity of testes to nitrofurans.