Human Whole Blood Cultures Research Articles

Porins of Pseudomonas aeruginosa induce release of tumor necrosis factor alpha and interleukin-6 by human leukocytes.

The aim of this study was to examine the ability of Pseudomonas aeruginosa components to induce release of cytokines from human leukocytes. Human whole-blood cultures were incubated with several concentrations of purified P. aeruginosa products, including porins, exomucopolysaccharide, lipopolysaccharide, and toxin A. Supernatants were assayed for tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) activities. All of the P. aeruginosa components except toxin A were able to stimulate the release of both cytokines. On a weight basis, porins were as effective as lipopolysaccharide and significantly more effective than exomucopolysaccharide in inducing IL-6 release (P < 0.05). Moreover, porins were more potent than either exomucopolysaccharide or lipopolysaccharide in inducing TNF-alpha release (P < 0.05). Further experiments using isolated leukocytes suggested that monocytes were the cell population predominantly responsible for the production of both cytokines. These data indicate that P. aeruginosa porins are able to induce significant cytokine production. These components may be responsible for the chronically overactive inflammatory response associated with persistent lung infection in cystic fibrosis patients.

Effects of indomethacin and arachidonic acid on sister chromatid exchange induction by styrene and styrene-7,8-oxide

Styrene is converted into styrene-7,8-oxide in human lymphocyte cultures, in a reaction probably mediated by oxyhemoglobin. As a consequence, styrene induces sister-chromatid exchanges (SCEs) in whole-blood lymphocyte cultures without exogenous metabolic activation systems. Another metabolic pathway that could be involved in the metabolism of styrene is cooxidation by prostaglandin-endoperoxide synthase (PES). To study the role of PES in the metabolism of styrene, human whole-blood lymphocyte cultures were treated for the entire culture time of 72 h with styrene (0.5 and 1 mM) or styrene-7,8-oxide (50 and 100 μM), in the presence and absence of 75 or 150 μM indomethacin (an inhibitor of PES) and arachidonic acid (substrate of PES). Indomethacin potentiated SCE induction by both styrene and styrene-7,8-oxide; a slight but statistically significant enhancement (16–32%; p < 0.05−0 p < 0.001) was observed in all treatments with styrene and at 150 μM indomethacin in the case of styrene-7,8-oxide. At 150 μM, arachidonic acid induced a 15–20% suppression ( p < 0.01) in SCE induction by both styrene (1 mM only) and styrene-7,8-oxide (100 μM only). Indomethacin or arachidonic acid did not alone influence the frequency of SCEs. The results suggest that PES acts as an inactivation route for styrene and styrene-7,8-oxide in human whole-blood lymphocyte cultures, possibly through PES-mediated binding to glutathione.

Effects of indomethaci and arachidonic acid on sister chromatid exchange induction by styrene and styrene-7,8-oxide

Styrene is converted into styrene-7,8-oxide in human lymphocyte cultures, in a reaction probably mediated by oxyhemoglobin. As a consequence, styrene induces sister-chromatid exchanges (SCEs) in whole-blood lymphocyte cultures without exogenous metabolic activation systems. Another metabolic pathway that could be involved in the metabolism of styrene is cooxidation by prostaglandin-endoperoxide synthase (PES). To study the role of PES in the metabolism of styrene, human whole-blood lymphocyte cultures were treated for the entire culture time of 72-h with styrene (0.5 and 1 mM) or styrene-7,8-oxide (50 and 10 μM), in the presence and absence of 75 or 150 μM indomethacin (an inhibitor of PES) and arachidonic acid (substrate of PES). Indomethacin potentiated SCE induction by both styrene and styrene-7,8-oxide; a slight but statistically significant enhancement (16–32%; p < 0.05 – p < 0.001) was observed in all treatments with styrene and at 150 μM indomethacin in the case of styrene-7,8-oxide. At 150 μM, arachidonic acid induced a 15–20% suppression ( p < 0.01) in SCE induction by both styrene (1 mM only) and styrene-7,8-oxide (100 μM only). Indomethacin or arachidonic acid did not alone influence the frequency of SCEs. The results suggest that PES acts as an inactivation rout for styrene and styrene-7,8-oxide in human whole-blood lymphocyte cultures, possibly through PES-mediated binding to glutathione.

Cytokine-inducing glycolipids in the lipoteichoic acid fraction from Enterococcus hirae ATCC 9790

Five high molecular weight glycolipids capable of stimulating human peripheral whole-blood cell cultures to cause interleukin 6 (IL-6) and tumor necrosis factor (TNF)-α induction were isolated from one of the lipoteichoic acid fractions (LTA-2) extracted from Enterococcus hirae ATCC 9790 (Tsutsui et al., (1991) FEMS Microbiol. Immunol. 76, 211–218) by a combination of hydrophobic interaction and anion-exchange chromatographies. This purification procedure resulted in a remarkable increase in the cytokine-inducing activities on the weight basis of isolated glycolipids (a maximum of 36- and 17-fold increases of IL-6 and TNF-α induction, respectively). The total yield of these bioactive glycolipids amounted to 6 wt% of the parent LTA-2 fraction, while the recovery rate in terms of the cytokine-inducing activities was estimated to be sufficient. The chemical composition and the profile, using SDS-PAGE, revealed that all of the isolated bioactive components were high molecular weight glycolipids, which were distinct from each other and from the parent LTA-2 fraction. These findings suggest that the IL-6 and TNF-α-inducing activities previously noted in the parent LTA-2 fraction are not attributable to a chemical entity, the structure of which had been proposed elsewhere (Fischer, W. (1990) in Glycolipids, Phosphoglycolipids and Sulfoglycolipids (Kates, M. ed.) pp. 123–234, Plenum Press, New York), but to the other high molecular weight glycolipids described here.

Cytokine-inducing glycolipids in the lipoteichoic acid fraction fromEnterococcus hiraeATCC 9790

Five high molecular weight glycolipids capable of stimulating human peripheral whole-blood cell cultures to cause interleukin 6 (IL-6) and tumor necrosis factor (TNF)-alpha induction were isolated from one of the lipoteichoic acid fractions (LTA-2) extracted from Enterococcus hirae ATCC 9790 (Tsutsui et al., (1991) FEMS Microbiol. Immunol. 76, 211-218) by a combination of hydrophobic interaction and anion-exchange chromatographies. This purification procedure resulted in a remarkable increase in the cytokine-inducing activities on the weight basis of isolated glycolipids (a maximum of 36- and 17-fold increases of IL-6 and TNF-alpha induction, respectively). The total yield of these bioactive glycolipids amounted to 6 wt% of the parent LTA-2 fraction, while the recovery rate in terms of the cytokine-inducing activities was estimated to be sufficient. The chemical composition and the profile, using SDS-PAGE, revealed that all of the isolated bioactive components were high molecular weight glycolipids, which were distinct from each other and from the parent LTA-2 fraction. These findings suggest that the IL-6 and TNF-alpha-inducing activities previously noted in the parent LTA-2 fraction are not attributable to a chemical entity, the structure of which had been proposed elsewhere (Fischer, W. (1990) in Glycolipids, Phosphoglycolipids and Sulfoglycolipids (Kates, M. ed.) pp. 123-234, Plenum Press, New York), but to the other high molecular weight glycolipids described here.

Effects of highly toxic organochlorine compounds retained in human body on induction of sister chromatid exchanges in cultured human lymphocytes

In this study, we investigated the effect of mixture of the organochlorine compounds, which very resembled their contamination of healthy Japanese people in its composition, on the induction of sister chromatid exchanges (SCEs) in human whole-blood cultures in order to clarify their genotoxicity as a whole. The following results were obtained. Regardless of the presence or absence of 7,8-benzoflavone (ANF) in the blood culture system, we could observe a fairly good dose-response relationship between the concentration of the mixture of organochlorine compounds and the induction of SCEs/cell. In particular, we found that 50% effective concentration of the mixture of the organochlorine compounds was considered to be only about 3 times greater level over the average concentration in the healthy people, namely 70ppt as 2,3,7,8-TCDD, in the absence of ANF and about 8 times more than that in the presence of ANF.

Cytokine induction by extracellular products of oral viridans group streptococci.

During an etiological study of Kawasaki disease (mucocutaneous lymph node syndrome [MCLS]), we found that dominant viridans streptococcal strains on tooth surfaces and in the throat of both MCLS patients and non-MCLS control children formed erythrogenic and biologically active, extracellular products. In this study, we demonstrated that erythrogenic culture supernatant concentrates of representative strains (two Streptococcus mitis and two Streptococcus oralis), when injected intravenously, induced serum tumor necrosis factor alpha, interleukin-6 (IL-6), and gamma interferon in muramyldipeptide- or Propionibacterium acnes-primed C3H/HeN mice. The concentrates also induced tumor necrosis factor alpha, IL-6, and thymocyte-activating factor (essentially IL-1) in murine peritoneal macrophage, human monocyte, and human whole-blood cultures. An erythrogenic, heat-labile extracellular protein fraction (F-1) that was concentrated from the culture supernatants of a representative S. mitis strain exhibited the above-mentioned cytokine-inducing activity. This partially purified F-1 fraction also induced thymocyte-activating factor and IL-6 in human umbilical vascular endothelial cell and gingival fibroblast cultures.

Mechanisms of the formation of micronuclei in human lymphocyte cultures exposed to mitomycin C

The micronucleus (MN) test is one of the cytogenetic indicators reflecting DNA damage. Experiments have been carried out using human whole-blood cultures to determine the effects of sampling times and to study the fate of micronuclei in the MN frequencies following exposure to mitomycin C (MMC). Cells were pulse-treated for 1h with MMC at G0, and then sampled at 6h in intervals up to 96h after the stimulation of cultures with phytohemagglutinin (PHA). Results showed that MMC-treatment induced an approximate 6h proliferation delay per cell cycle, and that the frequencies of MMC-induced MN increased at 60h and then stayed almost constant after that time. These data indicate that the formation of MN appeared after the time when the cell-cycle had doubled. In subsequent experiments, after incubation periods of 72h and 96h, lymphocyte preparations were prepared using 2 different methods, the conventional method and the cytokinetics-block (CB) micronucleus method. Results showed that the frequencies of MN did not change between 72h and 96h using either method. These results suggest that some MN remain in the daughter cells. The CB method was developed to overcome the kinetic problems inheritant in the use of human lymphocytes by Fenech and Morley. Comparison of the results obtained with the conventional method and the CB method found that the conventional method underestimates this effect.

Chromosome aberrations and sister-chromatid exchanges induced by technical grade toluene diisocyanate and methylenediphenyl diisocyanate in cultured human lymphocytes

Technical-grade toluene diisocyanate (TDI; 80% 2,4-isomer and 20% 2,6-isomer) and 4,4'-methylenediphenyl diisocyanate (MDI; about 45% MD1, 25% 4,4 ′-methylenediphenyl triisocyanate and 30% unspecified compounds of higher molecular weight), used as hardeners in the production of Polyurethane, induced chromosome aberrations after a 24 h treatment in the absence of metabolic activation in human whole-blood lymphocyte cultures, MDI at all doses tested (0.54–4.30 μl/ml) and TDI (0.019–0.150 μl/ml) at the highest two doses (0.075 and 0.150 μl/ml). In the presence of rat liver S9 mix (1.5 h treatment), both mixtures significantly increased aberrations at only one of the doses used, MDI at the highest dose (4.30 μl/ml) and TDI at the second highest dose (0.038 μl/ml). MDI also marginally increased sister-chromatid exchanges at the highest dose available (2.17 μl/ml) with and without (48 h treatment) S9 mix. On addition to culture medium, both TDI and MDI formed polymers, which were seen as small particles on the microscopic slides. At high doses the presence of these polymers made metaphase analysis impossible.

Induction of micronuclei by vinyl acetate in mouse bone marrow cells and cultured human lymphocytes

A dose-dependent increase in micronucleated polychromatic erythrocytes was observed in the bone marrow of male C57B1/6 mice 30 h after a single intraperitoneal injection of vinyl acetate (250, 500, 1000 or 2000 mg/kg b.wt.; (9–14 animals per group). The effect was statistically significant at 1000 mg/kg (1.33±0.29 vs. 0.6±0.0% in olive oil-treated controls) and at 2000 mg/kg (1.57±0.19%) of vinyl acetate. These doses were fatal to 6 (100 mg/kg) and 8 (2000 mg/kg) out of 14 animals in both groups. The ratio of polychromatic to normochromatic cells decreased as a function of vinyl acetate dose. Cyclophosphamide (20 mg/kg), used as a positive control chemical, induced a clear increase in micronucleated polychromatic erythrocytes (2.07±0.20%). None of the treatments affected the number of micronuclei in normochromatic erythrocytes. In human whole-blood lymphocyte cultures, micronucleus induction by a 48-h treatment with vinyl acetate (0.125, 0.25, 0.5, 1 and mM; 24 h after culture initiation) was studied in lymphocytes with preserved cytoplasm from smear prepared by a method involving the removal of erythrocytes at harvest by sodium cyanide treatment to improve preparation quality. The frequency of micronucleated lymptocytes reached a peak at 0.5 mM (3.2±1.0% vs. 0.9±0.1% in control cultures) and 1 mM (3.1±0.7%), with a decline at 2 mM probably because of a toxic effect resulting in mitotic inhibition.

Variation in sister chromatid exchange frequencies between human and pig whole blood, plasma leukocyte, and mononuclear leukocyte cultures.

Sister chromatid exchange (SCE) induction by ultraviolet (UV) light was studied in both human and pig whole blood cultures (WBC) and plasma leukocyte cultures (PLC). No variation in SCE frequency was observed between pig WBC and PLC in control as well as in treated cells. Conversely, SCE frequencies of human PLC were consistently higher than those of WBC in control and UV-exposed cells. Thus, red blood cells (RBCs) do not influence the sensitivity of lymphocytes to UV light exposure, and there must be some different culture condition(s) in the induction of SCEs between human WBC and PLC but not in swine lymphocyte cultures. Since the BrdUrd/lymphocyte ratio of WBC was halved in PLC, the effect of BrdUrd concentration in inducing the SCE baseline frequency of PLC may be ruled out. Also, Ficoll-Hypaque-separated human mononuclear leukocytes in culture (MLC), but not pig MLC, showed a two-fold increase in SCE frequency over WBC values. Thus, neither the cell separation technique nor polymorphonuclear leukocytes had a significant role in the elevated SCE frequency of human PLC or MLC. Experiments where human RBCs were titrated into human PLC showed that the induction of an elevated SCE frequency of PLC was suppressed in a dose-dependent manner by the presence of RBCs in the culture medium. Since the incorporation of pig or human RBCs into human PLC as well as into MLC reduced the SCE frequency to that of WBC, a common component and/or function existing in these cells is suggested. Analysis of different RBC components showed that RBCs, specifically RBC ghosts, release a diffusible but not dialyzable "corrective" factor into culture medium that is able to reduce the SCE frequencies of PLC. Antioxidant enzymes such as catalase and horseradish peroxidase were unable to reduce the SCE frequency of human PLC to WBC values.

Sister-chromatid exchanges and cell-cycle kinetics in human lymphocyte cultures exposed to alkylating mutagens: apparent deformity in dose-response relationships

Experiments have been carried out using human whole-blood cultures to determine the effects of sampling times and of the duration of 5-bromodeoxyuridine (BrdUrd) treatment before fixation on sister-chromatid exchanged (SCE) frequencies following exposure to mitomycin C (MMC). Cells were pulse treated for 1 h with 3 × 10 −6 M MMC at G 1, and then sampled at 4-h intervals up to 88 h after stimulation of cultures with phytohemagglutinin (PHA). Results showed that this MMC treatment induced a 5–6-h proliferation delay per cell cycle, and that SCE frequencies first increased with time of fixation, peaking at 68 h, and then decreased. When cells were similarly treated with MMC, but subsequently exposed to BrdUrd for various times before fixation of cultures at 72 h, the SCE frequencies markedly increased with increasing durations of BrdUrd incubation times. These data indicate that, in mutagen-treated cultures, lymphocytes having relatively longer cell-cyle times show a higher mean frequency of SCEs. In a subsequent experiment, cells were treated for 1 h with increasing doses of MMC or 4-nitroquinoline 1-oxide (4NQO) at 0, 24, or 48 h, and then fixed at 72 h after PHA stimulation. Results showed that the optimal treatment times at which the agents could most efficiently produce SCEs were different for MMC and 4NQO, and that the dose-response curves tended to ‘bend down’ at very high doses; that is, treatments with very high doses induced smaller than expected numbers of SCEs. However, cells similarly treated with very high doses showed a higher, expected frequency of SCEs when sampled at 84 h, but again had a lower than expected SCE frequency when fixed at 96 h. The results indicate that there is an optimal time for sampling at which one can observe the maximum increase in SCE frequencies following mutagen exposure, and strongly that the higher the dose, the later the optimal sampling time. Because of the apparent deformity of dose-repsonse curves obtained after various treatments and sampling times, it seems necessary that extra fixation-time points be included in test protocols soa as to avoid negatives or confirm possible positives.

Genetic toxicity of styrene and some of its derivatives.

Styrene, an important plastic monomer, is mutagenic after metabolic activation in several test systems. Probably because of an unfavorable activation : inactivation ratio, some mutagenicity assays have not, however, found styrene mutagenic. Styrene is converted by microsomal monooxygenases in vivo to styrene-7,8-oxide, which is a well-known mutagen. Arene oxides have also been proposed as the reactive metabolites of styrene, but the significance of these compounds is not yet fully understood. Only few derivatives of styrene have been tested for mutagenicity. The results are characterized by difficulties in metabolic activation. Many styrene-7,8-oxide analogues substituted at the phenyl ring are electrophilic reactants and mutagenic in vitro. Human whole-blood lymphocyte cultures have a peculiar feature, ie, styrene and many of its analogues substituted at the ring or vinyl chain induce sister chromatid exchanges in the cultured cells without exogenous metabolizing systems. This activation is brought about by erythrocytes present in the cultures and probably results from the conversion of styrenes to styrene-7,8-oxides.

Induction of sister-chromatid exchanges by styrene analogues in cultured human lymphocytes

Styrene and 11 styrene analogues were tested for their ability to induce sister-chromatid exchanges (SCEs) by a 48-h treatment in human whole-blood lymphocyte cultures (72 h). Styrene, its methyl-substituted derivatives (substituted at 2-, 3-, 4-, 3,5- or β-position) and two styrene oxides (3,5-dimethylstyrene-7,8-oxide and 4-nitrostyrene-7,8-oxide) induced a distinct dose-dependent increase in SCEs. Also, 4-methylstyrene-7,8-oxide and α-methylstyrene showed a positive effect, but were not able to double the mean number of SCEs/cell at the concentration ranges available. Ethylbenzene had a marginal effect on SCEs at the highest dose tested. 2-Phenylethanol did not increase SCEs. The results indicate that styrene and methylstyrenes are converted into reactive metabolites in the whole-blood lymphocyte cultures. The negative or weak effects of styrene analogues without a double bond in the side-chain (ethylbenzene and 2-phenylethanol) suggest that the reactive metabolites are derived from the conversion of the vinyl group and are styrene-7,8-oxides.

Sister-chromatid exchange induction by sodium selenite: Plasma protein-bound selenium is not the active SCE-inducing metabolite of Na 2SeO 3

Sodium selenite (Na 2SeO 3) is an anticarcinogenic/antimutagenic/anticlastogenic agent that under certain incubation conditions induces sister-chromatid exchanges (SCEs), unscheduled DNA synthesis, and chromosome aberrations. Previous work has shown that SCE induction by Na 2SeO 3 depends on the presence of red blood cells (RBCs). Therefore, Na 2SeO 3 uptake and release by RBCs was studied in the present report as was the effect of plasma protein-bound selenium, the end product of Na 2SeO 3 metabolism by RBCs, on the SCE frequency of purified lymphocyte cultures. Na 2SeO 3 uptake by human whole blood, RBCs in plasma, RBCs in fetal calf serum, and RBCs in Hanks' balanced salt solution occurs rapidly, reaching a maximum after 2m in (37°C). Release of Na 2SeO 3 from RBCs depends on the presence of plasma proteins to which the metabolized selenium becomes bound. In spite of the fact that plasma protein-bound selenium is the major product of Na 2SeO 3 metabolism by RBCs, the SCE frequency of purified lymphocyte cultures was unchanged when plasma protein-bound selenium (7.90 × 10 −6 and 1.19 × 10 −5 M) was added to the medium for the final 19 h of incubation. Further study showed that Na 2SeO 3 could induce SCEs in blood cultures that had been washed to remove plasma proteins and incubated in medium 199, a maintenance medium lacking fetal calf serum. These findings indicate that a Na 2SeO 3 metabolite other than plasma protein-bound selenium is responsible for Na 2SeO 3's SCE-inducing ability in human whole-blood cultures.

Selenite prevents the induction of sister-chromatid exchanges by methyl mercury and mercuric chloride in human whole-blood cultures

The protective effect of sodium selenite (Na 2SeO 3) against the cytogenetic toxicity of methyl mercury (CH 3HgCl) and mercuric chloride (HgCl 2) were investigated on human whole-blood cultures in relation to induction of sister-chromatid exchange (SCE). Both mercurials caused a dose-dependent increase in SCEs, methyl mercury being about 5 times more potent than mercuric chloride. Sodium selenite also induced SCEs. However, the simultaneous addition of selenite (1 × 10 −7−3 × 10 −5 M) to cell cultures containing either methyl mercury (3 × 10 −6 M) or mercuric chloride (1 × 10 −5 M) prevented the induction of SCEs by the mercurial in a clear dose-related manner. When selenite and mercurial were simultaneously added at a molar ratio of 1:2 Na 2SeO 3:CH 3HgCl, or 1:1 Na 2SeO 3:HgCl 2, cells from treated cultures showed no increase in the SCE frequency. These results indicate that selenite and mercury mutually antagonize their ability to cause DNA damage leading to the formation of SCEs. The formation of bis(methylmercuric)selenide, (CH 3Hg) 2Se, from Na 2SeO 3 and CH 3HgCl, or a high molecular complex consisting of glutathione-Se-Hg from Na 2SeO 3 and HgCl 2 involving the participation of glutathione in RBCs might play a key role in this antagonism between mercury and selenium.

Characterization of Feline Whole-blood Cultures and Determination of the Frequency of Radiation-induced Dicentrics in Human and Feline Lymphocytes

The Ham's F-10 and PHA culture system was applied to whole feline blood and cell-cycle characteristics such as DNA synthesis and mitotic indices were studied. The results are comparable to those obtained from human whole-blood cultures. The yields of dicentrics were also determined in lymphocytes from X-irradiated human and feline blood. The ratio between the experimental yields of dicentrics in human as compared to feline lymphocytes was 1:0.27.