Differential responses to mutagens among human lymphocyte subpopulations

Mutation Research 672 (2009) 1–9

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Differential responses to mutagens among human lymphocyte subpopulations Huachun Weng, Kanehisa Morimoto ∗ Department of Social and Environmental Medicine, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Osaka 565-0871, Japan

a r t i c l e

i n f o

Article history: Received 17 July 2008 Received in revised form 24 September 2008 Accepted 26 October 2008 Available online 5 November 2008 Keywords: Lymphocyte subpopulations DNA Micronuclei Sister chromatid exchanges Single cell gel electrophoresis

a b s t r a c t Many previous studies have described differential responses to mutagens among human lymphocyte subpopulations. However, there are some confusing data about the sensitivity to mutagens among lymphocyte subpopulations. In this paper we review the studies published to date reporting differential responses among CD4+ T-cells, CD8+ T-cells, B-cells and NK-cells exposed to different mutagens, using different assay methods, such as micronuclei (MN), sister chromatid exchanges (SCEs), single cell gel electrophoresis (SCGE), and fluorescence-activated cell sorting (FACS). These methods are mostly used for genetic biomonitoring of human populations exposed to potential mutagens. Thus, this review may provide a reference of target cells sensitive to mutagens that will be useful for genetic biomonitoring of human populations. © 2008 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differential responses to mutagens between CD4+ T-cells and CD8+ T-cells (Table 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differential responses to mutagens between B-cells and T-cells (Table 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differential responses to mutagens among NK-cells and other lymphocyte subpopulations (Table 3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differential responses to mutagens among lymphocyte subpopulations in vivo study (Table 4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction DNA damage resulting from spontaneous hydrolytic events, radiation, or chemical mutagens is thought to play a relevant role in degenerative diseases and aging [1]. Further, genetic biomonitoring of human populations exposed to potential mutagens is an early warning system for genetic diseases or cancer. It also allows identification of risk factors at a time when control measures could still be implemented. Human biomonitoring can be performed using different genetic markers [2–4]. In addition, human peripheral blood or freshly isolated lymphocytes are often used to determine the effects of mutagens based on cytogenetic markers, such as chromosomal aberrations (CAs), micronuclei (MN), sister chromatid

∗ Corresponding author. Tel.: +81 6 6879 3920; fax: +81 6 6879 3929. E-mail address: [email protected] (K. Morimoto). 1383-5718/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2008.10.010

exchanges (SCEs), and single cell gel electrophoresis (SCGE). However, there are some confusing data, mainly from the biomonitoring of human populations. In some cases, where DNA damage was detected as a consequence of occupational or environmental exposure, no parallel increases in DNA damage induced by mutagens were found, i.e. the effect of cigarette smoking [5–8]. Although these inconsistencies can be easily associated with the different types of genetic damage examined, the level of exposure to toxicants, the methods used and the target cell type may be important factors. The following review describes the differential responses to mutagens among human lymphocyte subpopulations (CD4+ T-cells, CD8+ T-cells, B-cells and NK-cells). Many previous studies, using different methods, have indicated that the level of DNA damage induced by mutagens may differ among lymphocyte subpopulations, and also that different types of mutagens may act differently on the same subpopulations.

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2. Differential responses to mutagens between CD4+ T-cells and CD8+ T-cells (Table 1)

3. Differential responses to mutagens between B-cells and T-cells (Table 2)

Wilkins et al. used a neutral SCGE assay to characterize the apoptotic responses of various subpopulations of human lymphocytes exposed to radiation [9]. The results indicate that CD8+ T-cells were more radiation-responsive than CD4+ T-cells. Similar result shows that CD8+ T-cells have higher apoptotic responses to 137 Csirradiation compared to CD4+ T-cells [10]. Furthermore, Mori et al. assessed the differential transcriptional responses to the radiation of Cu filter installation among lymphocyte subpopulations [11], finding that the expression levels of the BAX gene (a proapoptotic gene), which were increased by activation with ionizing radiation, are higher in CD8+ T-cells than in CD4+ T-cells. There is a consistent result reported by Wuttke et al. [12]. They found that CD8+ T-cells had higher numbers of micronuclei than CD4+ T-cells when exposed to 2.5 Gy of irradiation (Cu filter installation). Although this difference was not statistically significant, after irradiation with 5 Gy, this tendency increased to a significant difference. However, using the same assay method, an opposite result that CD4+ T-cells had higher numbers of micronuclei than CD8+ T-cells when exposed to 60 Co-irradiation (dose <2.0 Gy) was also reported [13]. Interestingly, CD4+ T-cells were also more prone to apoptosis induced by a 2 Gy dose of 60 Co-irradiation than CD8+ T-cells [14]. Thus, these two different results may be due to the different effect of 60 Co-irradiation compared to other types of irradiation. Our recent study demonstrated that CD4+ T-cells had slightly higher value of tail moment (DNA fragment) compared to CD8+ Tcells exposed to H2 O2 by alkaline SCGE assay [15], consistent with the results of Morllas et al. [16]. However, CD8+ T-cells have higher apoptotic responses to H2 O2 compared with CD4+ T-cells using flow cytometry basing on the altered characteristics displayed by endstage apoptotic cells, were also described in the other report [17]. Whether or not these different results would be due to different endpoint, is not fully clear, and it would be the focus of further investigation. However, the other reports also indicated that CD8+ T-cells have higher apoptotic responses to prednisone or xanthine oxidoreductase compared with CD4+ T-cells [18,19]. Furthermore, CD8+ T-cells had higher rates of cell death induced by sulfamethoxazole hydroxylamine than CD4+ T-cells [20]. Also, two-color annexin V/PI staining showed an increase in the percentage of apoptotic cells among CD8+ T-cells compared with that among CD4+ T-cells by day 5 of cell stimulation with anti-CD3 plus anti-CD28 [21], which may be due to the mitogenic responses of CD4+ T-cells to activation by antiCD3/CD28 antibodies owing to their higher concentrations of surface thiols compared with CD8+ T-cells [22]. Surface thiols in general are believed to play a role in protection against oxidants, signaling associated with cell growth, and apoptosis. However, investigation of the surface thiols on resting lymphocytes revealed that CD8+ T-cells have higher levels than CD4+ T-cells [22,23]. This seems to indicate that CD4+ T-cells are more sensitive to mutagens than CD8+ T-cells in a non-activated cell environment, but not in the activated cell environment. Furthermore, by using MN, CD4+ T-cells are more sensitive than CD8+ T-cells (and B-cells) to the genotoxicity of 3-azido-3-deoxythymidine (AZT), an antiretroviral drug active against human immunodeficiency virus, and mitomycin-C [13,24]. These results seem to indicate that (1) CD8+ T-cells have higher sensitivities compared with CD4+ T-cells when exposure to radiation except for 60 Co-radiation. (2) When exposed to some other mutagens, CD8+ T-cells have higher apoptotic response than CD4+ T-cells, but it appears that higher numbers of micronuclei or higher level of DNA fragments were found in CD4+ T-cells as compared to CD8+ T-cells.

Wuttke et al. [12] found that B-cells show micronucleus expression, the frequency of which was 2.5–4.0 times higher than that in T-cells after irradiation with 0.5 Gy or 1.0 Gy. On the other hand, although T-cells have a significantly high micronucleus frequency after irradiation with 5 Gy, their result also demonstrated that with a higher dose (2.5 Gy or 5 Gy) range, B-cell proliferation was inhibited nearly completely. And in this study, B-cells were not isolated or enriched, as a result of which only a small number of binucleate B-cells could be analyzed. However, similar results showing that B-cells have higher levels of micronuclei induction after exposure to lower doses of irradiation (< 1 Gy) than T-cells [25,26], have been reported, but the MN yields became lower in B-cells than in T-cells when the irradiation was above a dose of 1 Gy [13,25,27]. In addition, in the studies of Högstedt et al. and Holmén et al. [13,27], although lower frequencies of MN were seen in B-cells compared with CD4+ and CD8+ T-cells when exposed to 1–2.0 Gy irradiation, B-cells had a lower mitotic index than CD4+ and CD8+ T-cells. In these two studies both B-cells and T-cells were only stimulated with phytohemagglutinin, which may induce a lower proliferative index in B-cells. It is very important to examine the proliferation of a lymphocyte subpopulation when evaluating its radiation-induced micronucleus frequency. It is well known that mitosis is necessary for the expression of micronuclei. Therefore, an appropriate mitotic index is required to assess micronucleus expression. Differential apoptotic responses to ionizing radiation among subpopulations of human lymphocytes were measured using a modified neutral comet assay [9]. The results indicated that B-cells have the highest background apoptotic fraction (AF), and CD4+ and CD8+ T-cells have a low, stable, spontaneous AF, which gives them the highest response ratio (it is used to assess sensitivity, which was defined as the ratio between AF after exposure to X-rays and the AF of the unirradiated cells.). Although B-cells have the highest radiation-induced apoptotic response to 1 Gy of X-rays, CD4+ and CD8+ T-cells show higher radiation-responsiveness owing to their low spontaneous AF. However, Philippé et al. demonstrated that Bcells were most sensitive to apoptosis, followed by T-cells; NK-cells turned out to be most apoptosis-resistant following irradiation [14]. In this study, annexin V binding to phosphatidylserine was evaluated by flow cytometry to examine apoptosis. These contradictory results may be explained by differences in following factors: (1) Different endpoints: one study used flow cytometry the other one used a neutral comet assay, in which the percentage of apoptotic cells was assessed by distinguishing typical apoptotic morphology with a fragmented nucleus. It may easily affect the veracity of results by man-made. (2) The different methods of calculating sensitivity: one study used response ratios, the other one used different number of apoptotic cells among lymphocyte subsets following irradiation without spontaneous apoptotic cells taken into account (In this study B-cells also have the highest number of spontaneous apoptotic cells). Thus, these two results are difficult to be directly compared. However, the expression level of BAX (a pro-apoptotic gene) was measured in peripheral blood lymphocytes, CD4+ and CD8+ -T-cells, B-cells, and NK-cells 8 h after 1-Gy X-ray irradiation exposure [11]. Although there were no differences in the basal expression level of BAX among these cell types, the levels of activation induced by radiation differed among the cell subpopulations. Moreover, the expression levels of the BAX gene were increased in lymphocytes in the following order: B-cells > T-cells > NK-cells. Rensburg et al. had also provided information relevant to an understanding of the molecular basis for the differential radiosensitivity between enriched B-cells and enriched T-cells. Cell survival, DNA supercoiled domain sizes, superhelical density and

Table 1 The following articles are about differential responses to mutagens between CD4+ T-cells and CD8+ T-cells. Assay method

Results (the extent of response to mutagens)

Cell separating methods

References

Radiation Low energy photons, 0–1.5 Gy

Single cell gel electrophoresis (SCGE) (neutrality)

CD8+ T-cells > CD4 + T-cells

[9]

Agarose gel electrophorsis: DNA fragments, flow cytometry: Apoptosis, Bcl-2 Micronuclei (MN) FACSort (apoptosis, staining with annexin V) MN RT-PCR (BAX gene)

CD8+ T-cells > CD4+ T-cells

Magnetic activated cell sorting (MACS) Epics Elite flow cytometer An immune- magnetic method Immuni-fluorescence staining MACS

[13] [14] [12] [11]

SCGE (alkaline) SCGE (alkaline) Flow cytometry (the altered characteristics displayed by end-stage apoptotic cells) SCGE (alkaline) MN MN EPICS XL flow cytometer (staining with annexin-V) FACScan (apoptosis, staining with annexin-V), Western blotting.

CD4+ T-cells > CD8+ T-cells CD4+ T-cells > CD8+ T-cells CD8+ T-cells > CD4+ T-cells

FACSvantage MACS Flow cytometry

[15] [16] [17]

CD4+ T-cells > CD8+ T-cells CD4+ T-cells > CD8+ T-cells CD4+ T-cell > CD8+ T-cells Apoptosis: CD8+ T-cells > CD4+ T-cells Period of culture: CD4+ T-cells > CD8+ T-cells, Apoptosis: CD8+ T-cells > CD4+ T-cells, Bcl-X: CD8+ T-cells = CD4+ T-cells The number of dead cells: CD8+ T-cells > CD4+ T-cells Apoptosis: CD8+ T-cells > CD4+ T-cells, PDN inhibited the expression of IL-2R: CD8+ T-cells > CD4+ T-cells

FACSvantage An immune- magnetic method Immuno-peroxidase staining An immune-magnetic method

[15] [13] [24] [19] [21]

MACS

[20]

Negative selection with immunomagnetic beads

[18]

137

Cs, 6 Gy/min, 1500 cGy

60

Co, <2 Gy Co, 2.0 Gy Cu filter installation, 2.5 or 5.0 Gy Cu filter installation 1 Gy 60

Other mutagens H2 O2 , 5.0–50.0 ␮M H2 O2 , 10.0 and 20.0 ␮M H2 O2 , 125 ␮M, or mononuclear phagocytes-derived reactive oxygen metabolites Bleomycin, 0.05–0.2 ␮g/ml Mitomycin-C: <250 nmol/l Azidothymidine, 100 ␮g/ml Xanthine oxidoreductase, 30 and 100 mU/ml. Cells stimulated with cd3/cd28 monoclonal antibody

Sulfamethoxazole hydroxylamine (SMX-HA), <400 ␮M Prednisone (PDN), 1.0 × 10−12 –1.0 × 10−3 M

Cell viability assay Epics Elite flow cytometer: apoptosis cytofluorimentric analysis: IL-2R␣-chain expression

CD4+ CD4+ CD8+ CD8+

T-cells > CD8+ T-cells > CD8+ T-cells > CD4+ T-cells > CD4+

T-cells T-cells T-cells T-cell

[10]

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Mutagens

3

4

Table 2 The following articles are about differential responses to mutagens between T-cells and B-cells. Mutagens Radiation 60 Co, 1.2 Gy/min

Co, 0.05–1.00 Gy Co, <2 Gy 60 Co, 1.5 and 2.0 Gy 60 Co, 2 Gy 60 Co, < 6 Gy

Results (the extent of response to mutagens)

Cell separating methods

References

Micronuclei (MN) (PHA, PMW), Western blotting, DNA-PK assay

<0.8 Gy: B-cells > T-cells >0.8 Gy: T-cells > B-cells DNA-PK, DNA DSB induction and rejoining: T-cells = B-cells B-cells > T-cells T-cells > B-cells T-cells > B-cells B-cells > T-cells B-cells > T-cells

Magnetic activated cell sorting (MACS)

[25]

FACSort flow cytometer An immune- magnetic method An immunomagnetic method

[26] [13] [27] [14] [28]

Cu filter installation, 0.5–5.0 Gy

MN (PHA PMW) MN (PHA) MN (PHA) FACSort (Apoptosis, Staining with annexin V) Supercoil domain sizes, super-helical density (A nucleoid sedimentation), cell viability (trypan blue) MN (PHA PMW)

Cu filter installation 1 Gy Low energy photons, 0.5 and 1.0 Gy 60 Co, 4 Gy

RT-PCR Single cell gel electrophoresis (SCGE) (Neutrality) Filter elution technique

60

Other mutagens H2 O2 , 5.0–50.0 ␮M H2 O2 , 0.24 M Nitrogen mustard: 1.0 × 10−4 M, methylmethane sulphonate: 1.0 × 10−3 M Ethylnitrosourea (ENU) Bleomycin (BLM), 0.05–0.2 ␮g/ml BLM, <30 ␮g/ml, cyclophosphamide (CP), <3.0 × 10−5 M, Ethylmethanescllfonate (EMS), < 1.0 × 10−3 M Trenimon: 0.5 × 10−6 M Pelomycin: 1.0 ␮g/ml Mytomycin-C: <100 nmol/l

SCGE (Alkaline) Cell viability H3+ thymidine incorporated (liquid scintillation/autoradiographic determination) Filter elution technique SCGE (alkaline) Sister chromatid exchange (SCE)

0.5 or 1.0 Gy: B-cells > T-cells 5.0 Gy: T-cells > B-cells Bax gene: B-cells > T-cells T-cells > B-cells DNA repair capacity: T-cells = B- cells

SCE MN (PHA, PMW)

B-cells > T-cells The number of dead cells: B-cells > T-cells DNA repair synthesis: T-cells > B-cells Unscheduled DNA synthesis: T-cells = B- cells DNA repair capacity: T-cells > B-cells B-cells > T-cells CP:T-cells > B-cells EMS:T-cells = B-cells BLM: no increase T-cells > B-cells B-cells > T-cells

MN (PHA)

B-cells > T-cells

Discontinuous percoll gradient centrifugation Immuni- fluorescence staining

[12]

MACS MACS E-rossetting

[11] [9] [31]

FACSventage E-rossetting E-rossetting

[15] [35] [29]

E-rossetting FACSventage E-rossetting

[31] [15] [42]

Morphology, Antibody, and Chromosomes method An immunomagnetic method

[44] [36] [27]

H. Weng, K. Morimoto / Mutation Research 672 (2009) 1–9

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Assay method

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supercoiling were measured to assess the radio-sensitivity of lymphocyte subpopulations [28]. During the 4-h incubation period the viability of unirradiated cells remained relatively stable. However, exposure to 2 and 6 Gy of a 60 Co-source at 37 ◦ C for 4 h revealed greater sensitivity of B-cells than T-cells (93% of T-cells survived compared with 84% of B-cells after 2 Gy exposure; and 87% of Tcells survived compared with 39% of B-cells after 6 Gy exopsure). The radio-sensitivity of B-cells could be ascribed to the relatively higher content of larger domains in these cells, but there are no significant differences in the domain sizes of B- and T-cells. Furthermore, nucleoids of unirradiated B-cells sediment at slightly higher rates than those of T lymphocytes. However, the differences in radio-sensitivity of lymphocyte subpopulations cannot entirely be ascribed to differences in DNA superstructures. Radio-sensitivity encompasses not only the damage of cells, but also their capacity to repair the damage. Lewensohn et al. [29] assessed DNA repair synthesis in the presence of hydroxyurea, which can inhibit replicative DNA synthesis effectively, in B-cells and T-cells exposed to nitrogen mustard. They found that DNA repair synthesis, which was measured by H3+ thymidine incorporation, was consistently lower in B-cells than in T-cell-enriched fractions. In order to exclude the possibility that differences in the background incorporation of 3H thymidine in cell populations incubated with hydroxyurea could influence DNA repair synthesis, these authors also measured unscheduled DNA synthesis in B-cells and T-cells exposed to irradiation, nitrogen mustard, or methylmethane sulphonate. They found no statistically significant differences between B-cells and T-cells after exposure. On the other hand, they also found that the background replicative DNA synthesis was statistically significantly higher in B-cells, not only in the presence of hydroxyurea, but also in the absence of hydroxyurea, as compared to the T-cell-enriched fraction. These results indicate that T-cells have a higher capacity for DNA repair, but not DNA replication, compared with B-cells. Furthermore, CD4+ - and CD8+ -T-cells were shown to express higher activities of uracil-DNA glycosylase (a DNA repair enzyme) than B-cells [30]. Moreover, using the alkaline filter elution technique, ethylnitrosourea-induced single-strand breaks (SSBs) were shown to be removed only in T-cells; no significant amount of SSBs disappearance was observed in B-cells [31]. By contrast, when exposed to ␥-irradiation, no significant differences in SSBs repair between B-cells and T-cells, which may be partly due to DNA repair being almost accomplished within 1 h in this study. In addition, it has been identified that DNA-dependent protein kinase (DNA-PK) is a crucial component of the non-homologous end-joining (NHEJ) repair system, which in mammalian cells is the major mechanism for rejoining double-strand breaks (DSBs) induced by ionizing radiation [32]. Muller et al. [33] reported that the radio-sensitivity of B-cells results from a defect in Ku86, a DNA-dependent protein kinase catalytic subunit (DNA PKcs). B-cells express a variant form of the Ku86 protein with an apparent molecular weight of 69 kDa, and not the 86-kDa full-length protein. Although the heterodimer Ku70/variant-Ku86 binds to DNA-ends, this altered form of the Ku heterodimer has a decreased ability to recruit the catalytic component of the complex DNA-PKcs, which contributes to an absence of detectable DNA-PK activity in B-cells. However, this was not supported by Vral et al. [25]. These authors reported no difference between T- and B-cells in the basal expression and activity of DNAPK repair proteins. They separated B-cells and T-cells by positive selection with CD19 and CD3 immunomagnetic beads; however, it remains unclear whether these antibodies can affect the result. On the other hand, Morio et al. showed that resting B-cell lines have low DNA-PK activity due to the absence of nuclear Ku86, and Ku is in the cytoplasm of these cells, resulting in minimal DNA-PK activity, which requires activation of B-cells following incubation with IL-4

5

and anti-CD40mAb to induce translocation of Ku into the nucleus and increase in the activity of DNA-PKcs [34]. These studies indicate that B-cells have lower DNA repair capacity; by contrast, T-cells have higher DNA repair capacity. Enriched B-cells showed lower cell viability than enriched Tcells when exposed to a high dose of H2 O2 (about 0.24 mM) [35]. Our recent study also indicated that B-cells had higher sensitivity to H2 O2 compared with T-cells by using the alkaline comet assay, owing to their lower spontaneous tail moment values [15], partly consistent with previous studies [22,23] of the surface thiols on resting lymphocytes, showing that B-cells had higher levels than CD4+ and CD8+ T-cells. Furthermore, B-cells have higher levels of micronuclei induction after exposure to pelomycin or mitomycin-C than T-cells [27,36]. By using SCE, the levels of DNA damage in B-cells and T-cells were assessed. Spontaneous SCE frequencies were higher in Tcells than in B-cells [37–42]. B-cells have shorter cell-cycle times than T-cells, and this difference in proliferation kinetics is likely to contribute to the observed differences in spontaneous SCE levels [43]. Furthermore, the SCE-inducing effects of bleomycin (BLM), cyclophosphamide (CP) and ethyl methanesulfonate (EMS) were tested in mitogen-stimulated B- and T-cells [42]. The S-phasedependent clastogens CP and EMS significantly induced SCE in B-cells as well as in T-cells. Compared with the monofunctional alkylating agent EMS, the bifunctional alkylating CP had a greater effect in terms of SCE induction and proliferation delay in T-cells than in B-cells. However, the situation is different for the S-phaseindependent clastogen BLM. No or only modest increases in SCE frequencies were induced in B- and T-cells. Even in the case of modest increases in SCE frequencies, BLM had almost the same effect on T-cells and B-cells, which is inconsistent with our recent study that B-cells had higher sensitivity to BLM compared with T-cells by using the alkaline comet assay. These different results may be due to different assay methods used. In addition, Riedel et al. indicated that T-cells are more sensitive than B-cells with respect to the induction of SCE frequency by trenimon [44]. These results seem to indicate that the relative sensitivities between B-cells and T-cells exposed to radiation may depend on doses of radiation by using MN. Furthermore, B-cells appear to be more sensitive to some other mutagens and have lower DNA repair capacity than T-cells. However, some authors also suggested that T-cells likely had higher spontaneous and mutagen-induced SCE frequencies than B-cells. 4. Differential responses to mutagens among NK-cells and other lymphocyte subpopulations (Table 3) There have been some controversial results about the different sensitivities to mutagens among NK-cells and other lymphocyte subpopulations. Typical apoptotic morphology with a fragmented nucleus, condensed chromatin, and DNA laddering, and levels of Bcl-2, were assessed in B-cells, CD4+ T-cells, CD8+ T-cells, and NKcells exposed to 1500 cGy of 137 Cs-irradiation [10]. The results showed that NK-cells were the most radiosensitive, whereas CD8+ Tcells and B-cells showed weaker sensitivities to radiation and CD4+ T-cells were relatively radioresistant. It is consistent with Hansson’s study [45] that NK-cells were shown to be considerably more susceptible to apoptosis induced by 137 Cs -irradiation (the dose of 25 Gy) as compared with T-cells. However, when lymphocyte subpopulations were exposed to 60 Co-radiation, Jan et al. indicated that B-cells had the highest numbers of apoptotic cells, followed by T-cells; NK-cells turned out to be the most apoptosisresistant subpopulation [14]. By using the same method, NK-cells were less prone to apoptosis than T-cells when exposed to 1 and 2 Gy of 60 Co-irradiation [46]. However, these authors also indicated

6

Table 3 The following articles are about differential responses to mutagens among NK-cells and other lymphocyte subpopulations. Assay method

Results (the extent of response to mutagens)

Radiation 60 Co, 1.0 and 2.0 Gy

Micronuclei (MN), FACSort (apoptotic cells stained with annexin V)

MN: NK-cells > T-cells Apoptosis: T-cells > NK-cells B-cells > CD4+ T-cells > CD8+ T-cells > NK-cells NK-cells > T-cells

60

Co, 2.0 Gy

FACSort (apoptotic cells stained with annexin V)

137

Cs, 5–50 Gy

Apoptotic nuclei, TdT assay, agarose gel electrophoresis

137

Cs, 6 Gy/min, 1500 cGy

Low energy photons, 0.5 and 1.0 Gy

Agarose gel electrophoresis: DNA fragments, flow cytometry: apoptosis, Bcl-2 Single cell gel electrophoresis (SCGE) (Neutrality)

Cu filter installation, 1 Gy

RT-PCR

Other mutagens H2 O2 , 5.0–50.0 ␮M H2 O2, 125 ␮M, or monocytes derived reactive oxygen metabolites H2 O2 , 2–8 ␮M, monocytes-derived reactive oxygen metabolites

NK-cells > CD8+ T-cells and B-cells > CD4+ T-cells CD8+ T-cells > CD4+ T-cells and NK-cells > B-cells Bax gene: B-cells > CD8+ T-cells > CD4+ T-cell > NK-cells

SCGE (alkaline) Flow cytometry (apoptotic cells) Apoptotic nuclei, TdT assay, agarose gel electrophoresis

B-cells > NK-cells > T-cells NK-cells > T-cells NK-cells > T-cells NK-cells > T-cells

Etoposide, 0.05–0.2 mg/ml

Flow cytometry (The altered characteristics displayed by apoptotic cells), Annexin-V apoptosis detection kit, the analysis of intra-cellular content of reduced glutathione Flow cytometry (the altered characteristics displayed by apoptotic cells), TUNEL assay (intracellular content of reduced glutathione) Apoptotic nuclei, TdT assay, agarose gel electrophoresis

Bleomycin, 0.05–0.2 ␮g/ml

SCGE (alkaline)

B-cells > NK-cells > T-cells

Granulocyte- or chronic myelogenous leukemia-derived reactive oxygen metabolites Monocytes-derived reactive oxygen metabolities

NK-cells > T-cells NK-cells > T-cells

Cell separating methods

References

[46] [14] Negative selection with special antibody Epics Elite flow cytometer Magnetic activated cell sorting (MACS) MACS

[45] [10] [9] [11]

FACSventage Flow cytometry Negative selection with special antibody Counter-current centrifugal elutriation

[15] [17] [45]

Counter-current centrifugal elutriation Negative selection with special antibody FACSventage

[47]

[48]

[45] [15]

H. Weng, K. Morimoto / Mutation Research 672 (2009) 1–9

Mutagens

[57] Tail moment: B-cells > T-cells

Tail moment: B-cells > T-cells

41 workers from a printing company

Single cell gel electrophoresis (SCGE) (alkaline) SCGE (alkaline) 28 workers from an incineration company

LI Total body irradiation (TBI) TBI and cyclophosphamide treatment Polycyclic aromatic hydrocarbons Benzene

40 patients with breast cancer 30 patients with head and neck squamous carcinoma 15 patients with non-small-cell lung cancer 41 patients with breast cancer, 71 patients with bladder cancer or 13 patients with prostate cancer 8 patients with cervix or endometrium cancer 30 patients with leukemia 8 patients with leukemia LI LI LI and paclitaxel treatment LI

FACS (Cell number) FACS (Cell number) Cytofluorometry (cell number)

E-rossetting

Magnetic activated cell sorting (MACS) MACS

[58]

[55] [49] [50]

[54] [53] [52] [56]

CD4+ T-cells > CD8+ T-cells or NK-cells CD4+ T-cells > CD8+ T-cells or NK-cells CD4+ T-cells or B-cells > CD8+ T-cells or NK-cells Breast cancer: B-cells > T-cells Bladder cancer or prostate cancer: B-cells = T-cells B-cells > CD4+ -T-cells > CD8+ T-cells > NK-cells B-cells > CD4+ T-cells = CD8+ T-cells > NK-cells B-cells = CD4+ T-cells = CD8+ T-cells

Results (the extent of response to mutagens) Assay method

Local irradiation (LI)

Flow Cytometry or Cytofluorometry (Cell number) FACS (Cell number) FACS (Cell number) FACS (Cell number) Cell number

Subjects

37 patients with lung cancer

Mutagens

Some authors have described no differences in radiosensitivity between CD4+ T-cells and CD8+ T-cells in leukemia patients undergoing therapy of total body irradiation [49,50]. It is consistent with Ogawa’s study [51] that CD4+ T-cells and CD8+ T-cells showed similar radiosensitivity in 37 patients with lung cancer after local irradiation therapy. However, Reckzeh et al. indicated that a more pronounced decrease in the number of CD4+ T-cells compared with CD8+ T-cells in 15 patients with non-small cell lung cancer after local irradiation therapy [52]. But in this study a radiosensitizer paclitaxel was also used. Some other authors’ studies also showed that CD4+ T-cells had higher sensitivity to local irradiation therapy than CD8+ T-cells in patients with head and neck squamous carcinoma [53], with breast cancer [54], or with carcinoma of the cervix or endometrium [55]. When comparing the sensitivities of B-cells and T-cells, some studies have shown that B-cells have higher sensitivities than Tcells [49,52,55]. However, one author observed no difference in radiosensitivity between B- and T-cells after total body irradiation of leukemic patients [50]. In this study, the patients also received induction chemotherapy. Thus, the results cannot be compared directly with those of studies in which patients only received radiotherapy. And Blomgren’ study [56] showed that the proportion of B-cells was significantly reduced following local radiation therapy compared to T-cells in patients with breast cancer but not bladder cancer or prostate cancer. It appeared that radiation therapy directed to different anatomic sites might change the distribution of subpopulations of lymphocytes in different ways. Furthermore, most authors have found a relative radio-resistance of NK-cells in patients undergoing radiotherapy compared to B-cells or Tcells [49,52–55]. The relative radio-resistant of NK-cells may be of physiological importance. We know that NK-cells together with monocytes and granulocytes provide a primary immune response at the site of infection and therefore need to survive in these

Table 4 The following articles are about differential responses to mutagens among lymphocyte subpopulations in vivo study.

5. Differential responses to mutagens among lymphocyte subpopulations in vivo study (Table 4)

Cell separating methods

points used. And chromosomal aberrations and apoptosis are not necessarily correlated. It should be remembered that MN only represent the more persistent cytogenetic damage and do not cover the whole range of cytogenetic damage. By using a modified neutral comet assay, Wilkins et al. characterized the differential apoptotic responses to ionizing radiation in lymphocyte subpopulations. Bcells showed significantly lower susceptibility than NK-cells and T-cells, and the sensitivity of CD8+ T-cells was significantly higher than that of NK-cells [9]. In addition, the expression level of the BAX gene in NK-cells was the lowest compared with that in other lymphocyte subpopulations exposed to 1 Gy of irradiation (Cu filter installation [11]). These different results may be due to different types and doses of irradiation or different assay methods used. NK-cells were shown to be considerably more susceptible to apoptosis induced by H2 O2 , monocyte- or granulocyte-derived reactive oxygen metabolites, or a topoisomerase II inhibitor (etoposide), as compared with T-cells [17,45,47,48]. Furthermore, in our recent study, we also found that NK-cells were more highly sensitive to H2 O2 or bleomycin compared with T-cells, but not B-cells, by using an alkaline SCGE assay [15]. These results seem to indicate that the relative sensitivity of NK-cells exposed to radiation may depend on doses and types of radiation. Furthermore, when exposed to reactive oxygen species or bleomycin the sensitivities of NK-cells are higher than T-cells but lower than B-cells.

[51]

that NK-cells had higher numbers of micronuclei than T-cells after 60 Co-irradiation. Contradictory results may be due to different end-

7

CD4+ T-cells = CD8+ T-cells

References

H. Weng, K. Morimoto / Mutation Research 672 (2009) 1–9

8

H. Weng, K. Morimoto / Mutation Research 672 (2009) 1–9

unfavourable conditions in order to resist infection. In a word, these results seem to indicate that the type of cancer, different body regions of radiotherapy and whether or not other methods of therapy used may be important to compare the sensitivities of lymphocyte subpopulations exposed to radiation. In a human population study, the SCGE assay was carried out to evaluate DNA damage in B-cells and T-cells from 41 workers exposed to benzene in a printing company and 41 unexposed donors. The results indicated that B-cells were more sensitive than T-cells to the genotoxicity of benzene [57]. Similar results [58] were also reported by the same authors that sensitivities of B-cells were higher than T-cells from workers exposed to polycyclic aromatic hydrocarbons. 6. Conclusions Previous studies suggest that in vitro study the sensitivity of lymphocyte subpopulations to radiation may depend on doses and types of radiation, and B-cells have the highest sensitivity, followed by NK-cells, T-cells turn out to be the most resistant subpopulation when exposed to other mutagens. However, in vivo study B-cells seem to be more mutagen-sensitive than T-cells, NK-cells would be the most radio-resistant. When comparing different studies, the method used to assess DNA damage and mutagen-source must be also taken into account. Also, in biomonitoring and genotoxicity testing, the analysis of DNA damage using human peripheral blood or freshly isolated lymphocytes has to account for possible changes in cellular composition. Then, appropriate cell subpopulations and detection methods should be selected to assess the effects of different mutagens.

[11]

[12] [13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22] [23]

Conflict of interest statement The authors declare that there are no conflicts of interest.

[24] [25]

Acknowledgements

[26]

We wish to thank Yuquan Lu for his useful discussions and support.

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