Purified Lymphocyte Cultures Research Articles

Chromosomal response of human lymphocytes to streptozotocin

The induction of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) by the methylating agent streptozotocin (STZ) and the effect of this compound on mitotic index (MI) and cell cycle progression in human lymphocytes were investigated. Unstimulated (G 0) or cycling lymphocytes derived from whole blood or purified lymphocyte cultures were pulse-treated with increasing doses of STZ for 0.5–24 h. Induction of CAs by STZ was only observed in cycling lymphocytes derived from whole blood cultures (WBC) ( P<0.05). On the contrary, STZ produced a significant and dose–response increase in the yield of SCEs in unstimulated as well as cycling lymphocytes ( P<0.05). In addition, STZ induced a dose-dependent decrease in the MI but had a slight effect on cell cycle progression. These results suggest that SCEs are the most sensitive endpoint for evaluating the chromosomal effects of STZ on these cells.

Immunomodulatory effects of interferon beta-1a in multiple sclerosis

Several studies have established a role for interferon beta (IFNβ) as a treatment for relapsing–remitting multiple sclerosis (MS). IFNβ has been reported to decrease the relapse rate, relapse severity, progression of disability and development of new brain lesions. Its mechanisms of action, however, remain unclear. We hypothesize that immunomodulatory effects of IFNβ may underlie its clinical efficacy. We used intracellular cytokine flow cytometry to analyze the effects of IFNβ-1a on expression of the anti-inflammatory cytokine, IL-10, and its effects on major co-stimulatory molecules in MS patients. We found that peripheral blood mononuclear cells (PBMC) produced more IL-10 following in vitro or in vivo treatment with IFNβ-1a. The primary cellular sources of IL-10 were monocytes and CD4 + T lymphocytes. IL-10 production in response to IFNβ-1a was increased in unseparated PBMC compared to purified lymphocyte cultures, indicating that interaction between monocytes and lymphocytes may influence IL-10 production in response to IFNβ-1a. Using flow cytometry, we monitored the ex vivo expression of two major co-stimulatory pairs-B7/CD28 and CD40/CD40L-before and after intramuscular IFNβ-1a treatment of MS patients. IFNβ-1a lowered the expression of B7.1 on circulating B cells and increased B7.2 expression on monocytes. CD40 expression on B cells was down-regulated, but CD40 on monocytes was up-regulated by IFNβ-1a treatment. These data suggest that co-stimulatory molecules are modulated by IFNβ, providing a possible mechanism for its in vivo immune regulatory effects.

Influence of culture time on the frequency and contents of human lymphocyte micronuclei with and without cytochalasin B

The effects of culture time (52, 64 and 76 h) and cytochalasin B (Cyt-B, 3 μg/ml) on the frequency of micronuclei (MN) harbouring whole chromosomes and acentric fragments was investigated in purified lymphocyte cultures of five nonsmoking male donors aged 41–50 years. Centromere-positive (C +) MN were identified by fluorescence in situ hybridization, using an alphoid DNA oligomer probe (SO- αAllCen) hybridizing to all human centromeres. For each culture time, 2000 cells and 60 MN were scored per donor, both with and without Cyt-B, making a total of 60 000 cells and 1800 MN. The frequency of MN and the proportion of C + MN were higher at 64 h and 76 h than at 52 h, irrespective of Cyt-B. The culture time-dependent increase in MN frequency was mainly due to C + MN which were about 1.5-times more frequent at 64 h and 72 h than at 52 h. The frequencies of C + MN, expressed per 1000 nuclei, were similar with and without Cyt-B, although the prevalence of C + MN was consistently about 10 percent units higher in the former type of culture. This effect was due to a decreased frequency of centromere-negative (C −) MN in the binucleate cells, possibly reflecting, e.g. increased inclusion of acentric chromosomal fragments within the main nuclei of such cells, enhanced expulsion of C − MN, or selection against binucleate cells carrying such MN. In conclusion, the present findings indicate that MN harbouring whole chromosomes become more frequent at long culture times with and without Cyt-B and that Cyt-B-induced binucleate cells show a reduced frequency of MN containing acentric fragments. Due to the high background of whole-chromosome-containing MN (mean C + MN proportions ranged from 42.3% to 62.7%), it may be recommended that centromeric fluorescence in situ hybridization is routinely applied when lymphocyte MN are used as a biomarker of human exposure to clastogens.

Detection of whole chromosomes in micronuclei of cytokinesis-blocked human lymphocytes by antikinetochore staining and in situ hybridization.

The effect of cytochalasin B (Cyt-B; 3 and 6 micrograms/ml; for the last 28 h) on micronuclei (MN) was studied in 72-h purified lymphocyte cultures of three male donors. The frequency of MN was much higher in multinucleate cells (mean 100-204 MN per 1000 cells) than in binucleate cells (mean 8.2-21.0 MN per 1000 cells), tetranucleate cells containing more MN than trinucleate cells. The presence of whole chromosomes in the MN was studied in two separate experiments by immunofluorescence using antikinetochore (CREST) serum and by a centromeric alphoid DNA oligomer probe (in situ hybridization, ISH). In the tri- and tetra-nucleate cells produced by Cyt-B, MN were clearly more often kinetochore-positive (K+) (mean 82-86%) and centromere-positive (C+) (mean 73-83%) than in mononucleate cells of cultures containing no Cyt-B (mean 63% for CREST and 50% for ISH), indicating that most of the excess MN in the multinucleate cells were due to whole chromosomes. The binucleate lymphocytes had about as high prevalence of K+MN (mean 79-84%) as the tri- and tetra-nucleate cells, despite their low MN count. Also in the ISH analysis, the majority of MN in binucleate cells were positively stained (mean 58-62%). If it is assumed that the extra labelled MN are due to Cyt-B, the present findings suggest that Cyt-B could be responsible for approximately 45-57% (CREST data) or approximately 17-23% (ISH data) of MN in binucleate cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Selection and Evaluation of an Appropriate Model for Screening Adenosine Analogs for Chromosome Aberrations

As part of a preclinical safety assessment profile, the adenosine analog SC-46267, a candidate antihypertensive drug, was tested for potential genotoxicity in a series of in vitro and in vivo assays. Negative results were obtained in the Salmonella/microsome and CHO/HGPRT point mutation assays and in an acute mouse micronucleus test. However, in the presence of exogenous metabolism, positive results in the mouse lymphoma L5178Y assay and an in vitro chromosome aberration assay with purified rat lymphocytes indicated that the analog was a potential clastogen. Both adenosine and the analog were subsequently tested in rat lymphocytes using whole-blood versus purified lymphocyte cultures. High concentrations of adenosine and the adenosine analog induced chromosome aberrations in the purified lymphocytes. However, neither compound induced chromosome damage when cultured in the presence of whole blood, regardless of the presence or absence of the metabolic activation system. These results were similar to those reported in the literature that suggested that high concentrations of adenosine and other naturally occurring nucleotides can produce nonphysiological perturbations of nucleotide pools that are clastogenic in purified cultures of lymphocytes but not in whole-blood cultures. Our studies appear to support the hypothesis that adenosine deaminase, present at normal physiological levels in whole blood, is capable of detoxifying excessive levels of adenosine. Both compounds were also negative in a 10-day repeated dose mouse micronucleus test, which further ensures that the positive in vitro findings are not biologically significant. An in vitro clastogenicity assay using whole blood appears to be more appropriate than use of purified lymphocytes for screening adenosine analogs. The results from this series of experiments demonstrate the importance of considering culture conditions when designing in vitro test protocols. Furthermore, this study exemplifies the need to explore the mechanisms of activity as well as in vivo relevance of in vitro results.

Variation in the frequency of sister chromatid exchanges in repeated human lymphocyte cultures.

Repeated blood samples from two healthy donors were taken over a period of about one year to determine the temporal variation in human lymphocyte baseline sister chromatid exchange (SCE)-frequencies. The investigations were performed on whole blood cultures and purified lymphocyte cultures using a standardized protocol for blood collection and cultures. Significant differences in the frequencies of SCEs were found between the two cultivation systems and the two blood donors but also between repeated cultures of the same individual. There was no systematic relationship between the proliferation of the cultures and the basal SCE values. The results indicate the necessity of concurrent controls and repeated blood samples whenever SCEs are used as a test for monitoring human exposure to potential mutagens. Temporal variation in human lymphocyte baseline SCE frequencies is a limiting factor for the detection of minor effects of genotoxic agents.

Differences in the induction of SCEs between human whole blood cultures and purified lymphocyte cultures and the effect of an S9 mix

Studies for SCE induction are frequently performed on human blood cultures. Either whole blood cultures (WBC) or purified lymphocyte cultures (PLC) are employed. However, it has been shown that fundamental differences with respect to metabolic activity exist between these two systems. In order to further characterize the whole blood culture and the purified lymphocyte culture, differently acting substances were studied comparatively with and without an Aroclor-1254-induced S9 mix. Treatment with ethyl methanesulfonate (EMS), a direct mutagen, produced distinct SCE induction in both systems. Cyclophosphamide (CP) and benzo[ a]pyrene (BP), two indirect mutagens, also led to a significant increase of SCEs both in WBC and PLC without S9 mix. Only with CP was this effect more pronounced after addition of S9 mix. Sodium selenite (Na 2SeO 3), which induced SCEs in WBC, did not show this effect in the PLC. After S9 mix was added to purified lymphocytes, an increase of SCEs by sodium selenite was observed as in WBC. H 2O 2, a radical former, led to SCE induction in purified lymphocytes but not in the whole blood culture. By adding S9 mix, a distinct reduction of the SCEs induced by H 2O 2 was established. These results show that human lymphocytes can metabolize indirect mutagens and that it should be kept in mind when using S9 mix that, besides mixed-function oxygenases, it also contains enzymes which influence the SCE-inducing effects of substances.

Ethylene oxide and some factors affecting the mutagen sensitivity of sister chromatid exchange in humans.

In our laboratory, we consider mutagenesis and the factors surrounding a mutagenic event as a series of barrier systems (Fig. 1), any one of which can affect a final biological outcome in humans. For example, a mutagen can only exert an effect if it indeed has access to a portal of entry. Physical parameters such as solubility affect skin absorption (1). Particle size will determine whether a particulate is respirable (2). Taken together, the physical/chemical barriers at the portal of entry limit carcinogen access to an organism. Beyond this point, the extracellular fluid (e.g., blood plasma) may limit or augment a mutagenic effect. In our studies shown here and elsewhere in this volume, we (Wiencke, et al.) demonstrate that sister chromatid exchange (SCE) mutagen effects can be modulated by blood plasma (3). Indeed, Lambert et al. (4) have indicated this possibility earlier. The complex environment of peripheral blood presents a myriad of cell types (erythrocytes, other mononuclear cells, neutrophils, and platelets) which may alter the mutagen response in phytohemagglutinin (PHA)-stimulated lymphocytes. An elegant series of studies (5) shows quite clearly that erythrocytes activate at least 1 carcinogen, styrene, which otherwise is inactive in purified lymphocyte cultures, i.e., the target cell.

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.

Induction by inorganic metal salts of sister chromatid exchanges and chromosome aberrations in human and syrian hamster cell strains

AbstractSister chromatid exchange (SCE) and chromosome aberration induction were determined for several inorganic metal salts. Arsenic, nickel, and beryllium salts at concentrations effective in causing transformation of Syrian hamster cells (HEC) induced SCE and chromosome aberrations of HEC and human lymphocytes, whereas sodium tungstate, a non‐transforming chemical, neither induced SCE nor chromosome aberrations. Normal human and hamster cells exhibited equal sensitivity to SCE induction; nontoxic concentrations of sodium arsenite, beryllium sulfate, and nickel sulfate caused an increase of 8–10 SCE/cell over control values. Sodium arsenite, a trivalent arsenic, and sodium arsenate, a pentavalent arsenic, produced increases in SCE but the former was effective at lower concentrations. Both arsenic salts were less efficient in inducing SCE in human whole blood than in purified lymphocyte cultures. Sodium arsenite, sodium arsenate, nickel sulfate, and beryllium sulfate also caused damage consisting primarily of chromatid type of aberrations. In HEC, with doses most effective in SCE induction, all four metals produced aberrations in 16–21% of cells. In human lymphocytes, 34 and 30% of the cells had chromosome damage after sodium arsenite and sodium arsenate, respectively, whereas beryllium sulfate or nickel sulfate caused damage in about 10% of the cells. The induction of SCE and chromosomal aberrations by metals reemphasizes the sensitivity of cytological assays and their importance for detecting genetic damage caused by carcinogens.

Correlation of the lymphocyte transformation, leucocyte migration inhibition and the delayed cutaneous hypersensitivity tests in chickens sensitised to Mycobacterium tuberculosis

Optimum conditions for non-specific and specific stimulation of avian peripheral blood lymphocytes were established using whole blood and purified lymphocyte cultures from control and Mycobacterium tuberculosis (TB) sensitised chickens. The degree of lymphocyte stimulation was quantitated by measurement of 3H-thymidine incorporation. Non-specific stimulation was observed with purified and reagent grade Phytohamagglutinin. The reagent grade gave higher and more consistent stimulation, peak responses occurring at 250 μg/ml. Peak specific stimulation was obtained in TU sensitised birds with purified protein derivative of tuberculin at 40 μg/ml. Other optimal conditions included incubation at 37 C for 66 h (inclusive of an 18 h incorporation time), and the addition of 5 per cent inactivated fetal calf serum to purified lymphocyte cultures.

Impaired Cell-Mediated Immune Response in Patients with Congenital Rubella: Correlation with Gestational Age at Time of Infection

Lymphocyte transformation, interferon, and leukocyte migration inhibition factor synthesis were studied in purified lymphocyte cultures for 20 children with congenital rubella and 18 healthy children (seven susceptible and 11 immune to rubella). Lymphocyte transformation after phytophemagglutinin stimulation was significantly lower in children with congenital rubella as compared to healthy controls. Responses to purified rubella virus were absent in the susceptible controls and absent or at least two times lower in congenital rubella children than in immune controls. After purified rubella virus stimulation, leukocyte migration inhibition factor production was detected in all immune controls, but in none of the susceptible controls, or the congenital rubella-infected children. The results varied with gestational age of intrauterine infection: the impairment of cellular immune response, both after phytohemagglutinin or rubella virus stimulation, was more severe in the children infected in the first two months than in the latter stages of gestation.

Sister-chromatid exchange induction by sodium selenite: Dependence on the presence of red blood cells or red blood cell lysate

Sodium selenite (Na 2SeO 3) sister-chromatid exchange (SCE) induction was studied in both short-term and long-term cell cultures. The ability of Na 2SeO 3 to induce SCEs was found to depend on the culture conditions employed. Concentrations of Na 2SeO 3 (7.90 × 10 −6 M and greater) that produced elevated SCE frequencies in whole blood cultures resulted in control level SCE frequencies (6–8 SCEs/cell) in Ficoll-Hypaque-purified lymphocyte cultures. However, whole blood and purified lymphocyte cultures were equally sensitive to SCE induction by methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), and N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Analysis of different whole blood components showed that the presence of red blood cells (RBCs), and specifically RBC lysate, was a prerequisite for Na 2SeO 3 SCE induction in purified lymphocyte cultures. The SCE frequencies of xeroderma pigmentosum (XP12RO) and normal human lymphoblastoid cell lines were also found to be unaffected by Na 2SeO 3 concentrations that produced elevated SCE frequencies in whole blood cultures. Incubation of these latter two cell types with Na 2SeO 3 and RBC lysate resulted in SCE frequencines comparable to those in Na 2SeO 3-exposed whole blood cultures.