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. 2012 Nov 24;65(4):621–628. doi: 10.1007/s10616-012-9516-4

Genotoxicity of pemetrexed in human peripheral blood lymphocytes

Erman Salih İstifli 1,2,✉, Mehmet Topaktaş 2
PMCID: PMC3720975  PMID: 23179091

Abstract

Pemetrexed (PMX) is an antineoplastic antifolate used in the treatment of non-small cell lung cancer, mesothelioma and several types of neoplasms. Its toxicity in tumor cells has been linked with the potent inhibition of thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyl transferase, and subsequent depletion of both purine and pyrimidine nucleotides. However, cytogenetic toxicity of PMX in non-diseased cells has not been adequately studied; despite the increasing data on the DNA-damaging potential of antineoplastic agents on normal cells. In the present study, the genotoxic potential of PMX was evaluated in peripheral blood lymphocytes obtained from healthy human subjects using chromosome aberration (CA), sister chromatid exchange (SCE) and micronucleus (MN) assays as the cytogenetic damage markers. Human peripheral blood lymphocytes were exposed to four different concentrations (25, 50, 75 and 100 μg/mL) of PMX for 24- and 48-h treatment periods. PMX significantly increased the formation of CA in 24-h treatment, but not in 48-h treatment. PMX did not increase the mean SCE frequency in 24- and 48-h treatment periods; however, there was a striking increase (although not statistically significant, p > 0.05) in the number of SCEs at 25 μg/mL (24- and 48-h treatment) and 50 μg/mL (24-h treatment) due to an increase of SCE at the single-cell level. Interestingly, PMX did not induce MN formation in either 24- or 48-h treatment periods. PMX strongly decreased the mitotic index (MI), proliferation index (PI) and nuclear division index (NDI) in 24- and 48-h treatment periods. Our results suggest that PMX has a potent cytotoxic effect against human peripheral blood lymphocytes at concentrations which are reached in vivo in the blood plasma.

Keywords: Chromosome aberration, Micronucleus, Sister chromatid exchange, Cytotoxicity, Pemetrexed

Introduction

Pemetrexed (PMX) is a folic acid analogue with a mode of action responsible for disrupting folate-dependent biosynthetic cycles required for both purine and pyrimidine synthesis. PMX potently exerts clastogenic and cytotoxic effects on tumor cells by inhibiting thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT) (Shih et al. 1998). The inhibition of TS results in depletion of dTTP pools and promotes misincorporation of deoxyuridine triphosphate (dUTP) into newly synthesized DNA, which then leads to the formation of chromosome breakage (Blount et al. 1997; Tonkinson et al. 1997). In antifolate-treated normal cells, the inhibition of the crucial enzymes along the de novo pathway of purine and pyrimidine synthesis have been shown to cause gross chromosomal abnormalities and transmissible chromosomal damage (Choudhury et al. 2001; Chow et al. 1998).

PMX [N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-benzoyl]-l-glutamic acid] is used against a wide variety of neoplasms including lymphoma, pancreatic, liver, lung, breast, ductal, prostate, genital neoplasms, gestational trophoblastic tumour and mesothelioma (Dalkic et al. 2010; Shih et al. 1998). It has been previously argued that polyglutamated derivatives of PMX are selectively accumulated in susceptible tumor cells at substantially higher levels than in non-diseased progenitor cells (Chattopadhyay et al. 2007). However, PMX treatment in cancer patients has been frequently associated with low blood cell counts. Signs of PMX toxicity primarily include myelosupression and neutropenia (Kao et al. 2010; Smit et al. 2003).

The small number of genotoxicity studies on antifolates, especially with methotrexate (MTX) and aminopterin (APT), has indicated that these compounds can induce both short and long term DNA damage in several in vitro and in vivo test systems. The genotoxic damage was associated with chromosome and chromatid aberrations, and micronucleus formation in human peripheral blood, in in vivo animal studies and in vitro cell culture assays (Chow et al. 1998; Hittelman 1973; Jensen and Nyfors 1979; Kasahara et al. 1992a).

Although PMX has been investigated in several in vitro tumor cell lines to gain mechanistic insight into its cytotoxic effect, there still exists a lack of appropriate in vitro model for the evaluation of the genotoxicity of PMX on normal cells. Therefore, the genotoxic potential of PMX was investigated in cultured human peripheral blood lymphocytes. Chromosome aberration (CA), sister chromatid exchange (SCE) and micronucleus (MN) were used as the cytogenetic damage markers. Besides, cytotoxicity of PMX was evaluated using the mitotic index (MI), the proliferation index (PI) and the nuclear division index (NDI).

Materials and methods

Commercially available pemetrexed disodium (PMX) (ALIMTA, manufactured by Eli Lilly, Indianapolis, IN, USA) was used as the test substance for the in vitro tests. The chemical formula (Fig. 1) and properties of PMX are shown as follows:

  • CAS number: 150399-23-8

  • Linear formula: C20H21N5O6

  • Molecular weight: 427.410 g/mol

Fig. 1.

Fig. 1

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Chemical structure of disodium pemetrexed

Concentration selection

The test concentrations were chosen as 25, 50, 75, and 100 μg/mL (roughly 2,25–9 times less than the maximum plasma level (225 μg/mL) achieved in patients receiving this drug) based on the top concentration (100 μg/mL) that resulted a ≥50 % (LD50) reduction in the mitotic index (MI).

In vitro chromosome aberration and sister chromatid exchange assay

The methods of Evans (1984) and Perry and Thomson (1984) were followed for preparation of the CA and the SCE tests with minor modifications. This study was conducted according to the IPCS guidelines (Albertini et al. 2000). Whole blood (0.2 mL) from four healthy donors (two male and two female, nonsmokers, age: 22–25), was added to 2.5 mL PB-MAX (GIBCO—Life Technologies, Carlsbad, CA, USA) chromosome medium, and a final concentration of 10 μg/mL of BrdU was added to the medium just after setting up the blood cultures. Cultures were incubated at 37 °C for 72 h. The cells were treated with 25, 50, 75, and 100 μg/mL concentrations of PMX dissolved in sterile bidistilled water, for 24 h (PMX was added 48 h after initiating the culture) and 48 h (PMX was added 24 h after initiating the culture), respectively. A negative control and a positive control (Mitomycin-C, 0.25 μg/mL, Sigma M-05030 (Sigma, St. Louis, MO, USA) were also used. The cells were exposed to colchicine (0.06 μg/mL, Sigma C9754) 2 h before harvesting. At the end of the incubation, cells were centrifuged at 2,000 rpm for 5 min. Then, the cells were treated with 0.4 % KCl (37 °C) as the hypotonic solution and methanol: glacial acetic acid (3:1) as the fixative (at room temperature 22 °C ± 1 °C, fixative treatments were repeated three times). The cells were centrifuged at 1,200 rpm for 10 min after each fixative treatment. The staining of the air-dried slides was performed following the standard methods using 5 % Giemsa stain for CA and the modified fluorescence plus Giemsa method for SCE (Speit and Haupter 1985). To obtain sister chromatid differentiation (SCD), the slides were irradiated with 30 W, 254 nm UV lamp at 15 cm distance in Sorensen buffer for 30 min, then incubated with 1× SSC (standard saline citrate) at 60 °C for 50 min and stained with 5 % Giemsa prepared with Sorensen buffer. The percentage of cells showing structural and/or numerical chromosome alterations were obtained by calculating the percentage of the aberrant metaphases from each concentration and treatment period. The CA was classified according to the ISCN (International System for Human Cytogenetic Nomenclature) (Paz-y-Miño et al. 2002). CAs were evaluated in 100 well-spread metaphases per donor (totally 400 metaphases per concentration). Gaps were not evaluated as CA (Mace et al. 1978). CAs were classified as structural and numerical aberrations. Structural CAs consisted of the chromatid type (breaks and exchanges) and the chromosome type (breaks, fragments, sister unions, dicentrics and translocations) abnormalities, whereas the numerical CAs consisted of polyploid cells. For the determination of the genotoxicity, only the structural CAs were taken into consideration.

The scoring of SCE was carried out according to the IPCS guidelines (Albertini et al. 2000). To score SCE, a total of 100 division metaphases per concentration (25 cells per donor) were analyzed. The results were used to determine the mean number of SCE (SCE/cell). In addition, a total of 400 cells (100 cells per donor) were scored to obtain the proliferation index (PI) for each treatment concentration. The PI was calculated according to PI = [(M1 × 1) + (M2 × 2) + (M3 × 3)]/total scored cells. M1, M2, and M3 are the fraction of cells undergoing the first, second, and third mitosis during 72-h cell culture period. MI was determined by scoring 3,000 cells from each donor for each concentration.

In vitro cytokinesis-block micronucleus assay

For the analysis of MN in binucleated lymphocytes, 0.2 mL of fresh whole blood (1/10 heparinized) was used to establish the cultures and the cultures were incubated for 68 h. The cells were treated with 25, 50, 75, and 100 μg/mL concentrations of PMX for 24- and 48-h treatment periods. To block cytokinesis, cytochalasin B (Sigma, C6762) was added at 44 h of the incubation at a final concentration of 6 μg/mL. After an additional 24-h incubation at 37 °C, cells were initially harvested by centrifugation at 2,000 rpm for 5 min and further processed identically as described for the preparation of CA and SCE slides, with the exception of a 5 min hypotonic treatment step at 37 °C. Finally, the slides were stained with 5 % Giemsa (Kirsch-Volders et al. 2003; Rothfuss et al. 2000). In all subjects, 1,000 binucleated lymphocytes were scored from each donor (4,000 binucleated cells were scored per concentration). A total of 1,000 cells (4,000 cells for each treatment concentration) were scored to calculate the nuclear division index (NDI) for the cytotoxicity of PMX using the formula: NDI = [(1 × M1) + (2 × M2) + (3 × M3) + (4 × M4)]/N, where M1-M4 represent the number of cells with one to four nuclei and N is the total number of the cells scored (Fenech 2000).

Statistics

Values of the control, positive control and the exposed groups were expressed as the mean (±SE) from four separate experiments. The multiple comparisons between the control, the positive control and the exposed groups were performed using One-Way ANOVA (LSD test) at p ≤ 0.05.

Results

The effects of PMX on CA formation in human peripheral blood lymphocytes are summarized in Table 1. In 24-h treatment, PMX significantly (p < 0.05–0.01) increased the percentage of cells with aberrations at all concentrations in comparison with the control. However, the increase of the CAs at 24-h treatment period were not concentration-dependent. Noticeably, at 24-h treatment, the total number of chromosome type breaks for each concentration was approximately two-fold higher (three-fold difference at 100 μg/mL) as compared to the number of chromatid type breaks. In contrast, treatment with PMX for 48 h did not significantly induce the formation of CA when compared with the control.

Table 1.

Effect of pemetrexed (PMX) on CA in human peripheral lymphocytes for 24- and 48-h

Test substance Treatment Structural CA Polyploid cells Percentage of cells with aberrations ± SE
Time (hr) Conc. (μg/mL) Chromatid type Chromosome type
Control – – 3 5 1 2.00 ± 0.70
MMC 24 0.25 26 33 1 12.75 ± 1.43
PMX 24 25 7 15 1 5.25 ± 0.25 a1b3
PMX 24 50 7 11 0 5.00 ± 1.08 a1b3
PMX 24 75 8 18 0 6.25 ± 0.62 a2b3
PMX 24 100 5 16 0 5.00 ± 1.08 a1b3
MMC 48 0.25 199 61 1 42.75 ± 9.51
PMX 48 25 6 6 0 2.25 ± 1.03 b3
PMX 48 50 3 10 0 3.00 ± 0.70 b3
PMX 48 75 2 7 0 2.50 ± 0.64 b3
PMX 48 100 1 3 0 1.00 ± 0.40 b3
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Data are expressed as the mean ± SE (n = 4)

a Statistically significant decrease vs. control, b Statistically significant vs. positive control

a1b1: p < 0.05; a2b2: p < 0.01; a3b3: p < 0.001

24- and 48-h treatment with PMX did not induce a statistically significant SCE formation when compared to the control group (Table 2). However, some cells of the treated cultures at 25 μg/mL (24- and 48-h treatment) and 50 μg/mL (24-h treatment) gave an unexpected positive SCE response. The positive control MMC significantly induced SCE in comparison with all concentrations of PMX (Table 2).

Table 2.

Mean SCE values in human peripheral lymphocytes treated with PMX for 24- and 48-h

Test substance Treatment Min–Max SCE SCE/cell ± SE
Time (hr) Conc. (μg/mL)
Control – – 0–12 3.79 ± 0.35
MMC 24 0.25 0–67 36.27 ± 4.14
PMX 24 25 0–56 9.52 ± 3.09 b3
PMX 24 50 0–43 4.73 ± 1.29 b3
PMX 24 75 0–9 3.67 ± 0.48 b3
PMX 24 100 0–12 3.42 ± 0.40 b3
MMC 48 0.25 1–140 53.09 ± 6.41
PMX 48 25 0–75 7.14 ± 2.11 b3
PMX 48 50 0–10 2.87 ± 0.20 b3
PMX 48 75 0–10 3.32 ± 0.29 b3
PMX 48 100 0–10 3.05 ± 0.33 b3
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Data are expressed as the mean ± SE (n = 4)

a Statistically significant vs. control, b Statistically significant vs. positive control

a1b1: p < 0.05; a2b2: p < 0.01; a3b3: p < 0.001

Increasing PMX concentrations did not produce a significant increase in the percentage of binuclear cells with micronuclei or %MN for the 24- and 48-h treatment periods (Table 3). In addition, the %MNBN and %MN were significantly reduced at 100 μg/mL when compared with 25 μg/mL (Table 3).

Table 3.

Percentage of micronucleus (MN) and percentage of micronucleated binuclear (MNBN) cells in cultured human peripheral lymphocytes treated with PMX for 24- and 48-h

Test substance Treatment MN ± SE (%) MNBN ± SE (%)
Time (hr) Concentration (μg/mL)
Control – – 0.27 ± 0.09 0.25 ± 0.08
MMC 24 0.25 2.15 ± 0.25 2.02 ± 0.21
PMX 24 25 0.60 ± 0.07 b3 0.60 ± 0.07 b3
PMX 24 50 0.45 ± 0.17 b3 0.42 ± 0.14 b3
PMX 24 75 0.52 ± 0.13 b3 0.50 ± 0.12 b3
PMX 24 100 0.15 ± 0.06* b3 0.15 ± 0.06* b3
MMC 48 0.25 10.32 ± 2.72 10.32 ± 2.72
PMX 48 25 0.55 ± 0.11 b3 0.55 ± 0.11 b3
PMX 48 50 0.27 ± 0.06 b3 0.25 ± 0.06 b3
PMX 48 75 0.05 ± 0.03 b3 0.05 ± 0.03 b3
PMX 48 100 0.12 ± 0.06 b3 0.10 ± 0.04 b3
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Data are expressed as the mean ± SE (n = 4)

* Statistically significant decrease vs.25 μg/mL for 24 h

a Statistically significant vs. control, b Statistically significant vs. positive control

a1b1: p < 0.05; a2b2: p < 0.01; a3b3: p < 0.001; * < 0.05

PMX treatment for 24 h and 48 h significantly (p < 0.01–0.001) decreased the PI and MI in human peripheral blood lymphocytes (Table 4). Furthermore, PMX decreased the PI and MI to the same extent as the positive control MMC for both 24- and 48-h treatment periods. However, the observed reduction was not concentration-dependent. Furthermore, the majority of cells was undergoing the first-mitosis in the cultures treated with PMX for 48-h.

Table 4.

Proliferation index (PI), mitotic index (MI) and nuclear division index (NDI) in human peripheral lymphocytes treated with PMX for 24- and 48-h

Test substance Treatment M1 M2 M3 PI ± SE MI ± SE NDI ± SE (%)
Time (hr) Conc. (μg/mL)
Control – – 57 91 252 2.48 ± 0.78 7.43 ± 0.52 1.50 ± 0.13
MMC 24 0.25 99 228 73 1.93 ± 0.78 3.17 ± 0.15 1.34 ± 0.07
PMX 24 25 177 131 92 1.78 ± 0.15 a2 3.29 ± 0.47 a3 1.26 ± 0.05 a1b1
PMX 24 50 187 125 88 1.72 ± 0.14 a3 3.53 ± 0.62 a3 1.27 ± 0.06 a1b1
PMX 24 75 139 133 124 1.96 ± 0.09 a2 3.25 ± 0.46 a3 1.19 ± 0.02 a2b2
PMX 24 100 119 138 143 2.06 ± 0.05 a2 3.73 ± 0.89 a3 1.21 ± 0.04 a1b1
MMC 48 0.25 298 63 39 1.35 ± 0.09 1.59 ± 0.16 1.18 ± 0.22
PMX 48 25 146 158 96 1.87 ± 0.72 a2 b1 2.20 ± 0.49 a3 1.06 ± 0.21 a3
PMX 48 50 213 114 73 1.65 ± 0.16 a3 2.81 ± 0.62 a3 1.10 ± 0.26 a3
PMX 48 75 204 112 84 1.70 ± 0.19 a2 2.00 ± 0.51 a3 1.11 ± 0.02 a3
PMX 48 100 253 84 63 1.52 ± 0.16 a3 1.61 ± 0.68 a3 1.09 ± 0.24 a3
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Data are expressed as the mean ± SE (n = 4)

a Statistically significant vs. control, b Statistically significant vs. positive control

a1b1: p < 0.05; a2b2: p < 0.01; a3b3: p < 0.001

Finally, PMX caused a statistically significant (p < 0.05–0.001) reduction in the NDI for both 24- and 48-h treatment periods (Table 4). In addition, the decrease in the NDI after the 24-h PMX treatment was significantly (p < 0.05–0.01) greater than that caused by the positive control MMC.

Discussion

In the present study, we evaluated the genotoxic potential of PMX using CA and MN as the genetic endpoints, and the SCE test was used as an indicator of mutagenesis in human peripheral blood lymphocytes. We also evaluated the effect of PMX on the cell division ratio, using MI, PI and NDI. Our results showed that PMX significantly increased the CA for 24-h treatment, but not for 48-h treatment. Interestingly, PMX increased neither %MN nor %MNBN in both 24- and 48-h treatment. PMX did not induce SCEs during both treatment periods. On the other hand, we observed a statistically very significant reduction in the MI, PI and NDI in PMX-treated peripheral blood lymphocytes in 24- and 48-h treatment periods.

We obtained a completely different cytogenetic toxicity profile with PMX compared to the previous reports on MTX that have shown a significant increase in CA both in the short and long term genotoxicity assays in vitro and in vivo. Chow et al. (1998) reported that CAs significantly increased in the NIH 3T3 mouse cell line after treatment with low doses of MTX. Similarly, the formation of CA and MN was found to increase in mice both in acute and chronic treatments with MTX (Choudhury et al. 2000, 2001; Kasahara et al. 1992a, b). Keshava et al. (1997) found a significant increase of CA in V79 cells treated with 5, 10, 25, 50 and 100 μg/mL of MTX after 6-h treatment. In that study, MTX increased the CA while decreasing the MI. However, in our study, we observed that PMX treatment caused a gradual decrease in the MI, which prevented the CA formation from reaching the statistical significance at 48-h treatment.

Hittelman (1973) demonstrated that 10−7 M of APT increased the chromatid type breaks in cultured potorous cells only at the first metaphase following treatment. Our results are in agreement with the results reported by Hittelman. Furthermore, we observed more chromosome type breaks as compared to the chromatid type breaks in peripheral blood lymphocytes treated with PMX for 24-h treatment. It was reported that the depletion of dTTP pools led to the misincorporation of uracil into the newly synthesized DNA, which is associated with the formation of chromosome breakage (Blount et al. 1997; Tonkinson et al. 1997). Our results are in agreement with those obtained earlier. However, the potent inhibition of multiple enzymes in the de novo biosynthetic pathway of thymidine led to a potent decrease of the MI, PI and NDI in 48-h treatment period, a mechanism which is responsible for the prevention of cells to enter mitosis bearing highly damaged DNA.

In this study, the exposure of human peripheral blood lymphocytes to the various concentrations (25, 50, 75 and 100 μg/mL) of PMX did not induce SCE either in 24- or 48-h treatment periods. However, an obviously greater SCE induction was observed in some cells of PMX-treated cultures (Table 2). It has been previously suggested that SCE induction at the single-cell level is a reflection of the differential sensitivity of lymphocyte subpopulations against DNA damage and SCE formation (Bender and Brewen 1969; Lezana et al. 1977). This suggestion had been further confirmed by Kelsey et al. (1988) in which they reported the existence of some lymphocytes cells with a high frequency with regard to SCE induction following ethylene oxide treatment in vivo. Thus, our findings confirm and extend earlier observations on the existence of drug-sensitive lymphocyte subpopulations and show that PMX is able to induce SCE. However, the persistence of cells bearing elevated SCE levels was decreased due to the death of cells with excessive chromosome damage.

Enzyme inhibition studies (Shih et al. 1998) showed that PMX exerts stronger inhibition of TS, DHFR and GARFT when compared to MTX, which inhibits primarily DHFR. Thus, in our study, statistically significant decline of the MI, PI and NDI in both 24- and 48-h treatment times is most probably associated with a strong inhibition of TS, DHFR and GARFT. In contrast, other studies did not report a significant decline in MI in MTX treated cells in vivo. Thus, we suggest that PMX has a novel and potent mechanism of cytotoxicity in comparison with MTX. It has been previously argued that MTX and its polyglutamated derivatives confer selective toxicity on tumor cells, due to an increased polyglutamate synthesis in tumor cells compared to normal proliferative cells (Poser et al. 1981). However, our results are not in agreement with this argument. PMX significantly decreased the mitotic division in normal human peripheral blood lymphocytes as evidenced by the strong decreases in MI, PI and NDI.

Data from unpublished studies (Tweats et al. 2007) showed that PMX significantly increased the number of micronucleated polychromatic erythrocytes in mouse bone marrow cells at the highest dose in 24-h treatment period. In contrast, we did not observe such a significant increase in either the %MN or the %MNBN for both 24- and 48-h treatment periods. Furthermore, %MN and %MNBN were significantly decreased at the highest concentration (100 μg/mL) in the 24-h treatment period. The finding presented here strongly suggests that continuing cell death is responsible for the decrease in the DNA-damaging capacity of PMX. The accumulation of DNA strand breaks have been shown to be a cell-lethal event in MTX treated Ehrlich ascites tumor cells and was associated with a significant cytotoxicity after 10-h treatment (Li and Kaminskas 1984). Our results are in strong agreement with those obtained from the study of Li and Kaminskas. In contrast, the induction of CA without a concomitant decrease of the MI in mice bone marrow cells has been reported previously (Choudhury et al. 2000, 2001). The discrepancy between the in vitro and in vivo findings may be explained by the drug-induced deficiency in the DNA repair activity, which can not be tolerated in vitro.

The results of this study show that PMX causes damage in chromosomes of cultured human peripheral lymphocytes and rapidly decreases the MI, PI and NDI by killing cytogenetically damaged cells. Thus, we propose on the basis of its genotoxicity, that PMX treatment can be a safer medication in cancer treatment in comparison with other antifolates.

Acknowledgments

This work was supported by Cukurova University Research Fund (FEF2011D8).

References

  1. Albertini RJ, Anderson D, Douglas GR, Hagmar L, Hemminki K, Merlo F, Natarajan AT, Norppa H, Shuker DE, Tice R, Waters MD, Aitio A. IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. Mutat Res. 2000;463:111–172. doi: 10.1016/S1383-5742(00)00049-1. [DOI] [PubMed] [Google Scholar]
  2. Bender MA, Brewen JG. Factors influencing chromosome aberration yields in the human peripheral leukocyte system. Mutat Res. 1969;8:383–399. doi: 10.1016/0027-5107(69)90016-5. [DOI] [PubMed] [Google Scholar]
  3. Blount BC, Mack MM, Wehr CM, MacGregor JT, Hiatt RA, Wang G, Wickramasinghe SN, Everson RB, Ames BN. Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage. Proc Natl Acad Sci. 1997;94:3290–3295. doi: 10.1073/pnas.94.7.3290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chattopadhyay S, Moran RG, Goldman ID. Pemetrexed: biochemical and cellular pharmacology, mechanisms, and clinical applications. Mol Cancer Ther. 2007;6:404–417. doi: 10.1158/1535-7163.MCT-06-0343. [DOI] [PubMed] [Google Scholar]
  5. Choudhury R, Ghosh SK, Palo AK. Cytogenetic toxicity of methotrexate in mouse bone marrow. Environ Toxicol Pharmacol. 2000;8:191–196. doi: 10.1016/S1382-6689(00)00041-7. [DOI] [PubMed] [Google Scholar]
  6. Choudhury R, Ghosh SK, Palo AK. Potential transmission of the cytogenetic toxic effects of methotrexate in the male germline cells of Swiss mice. Environ Toxicol Pharmacol. 2001;10:81–88. doi: 10.1016/S1382-6689(01)00073-4. [DOI] [PubMed] [Google Scholar]
  7. Chow M, Koo J, Ng P, Rubin H. Random population-wide genetic damage induced in replicating cells treated with methotrexate. Mutat Res. 1998;413:251–264. doi: 10.1016/S1383-5718(98)00025-4. [DOI] [PubMed] [Google Scholar]
  8. Dalkic E, Wang X, Wright N, Chan C. Cancer-drug associations: a complex system. PLoS ONE. 2010;5:e10031. doi: 10.1371/journal.pone.0010031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Evans HJ. Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. In: Kilbey BJ, Legator M, Nichols W, Ramel C, editors. Handbook of mutagenicity test procedures. 2. Amsterdam: Elsevier; 1984. pp. 405–427. [Google Scholar]
  10. Fenech M. The in vitro micronucleus technique. Mutat Res. 2000;455:81–95. doi: 10.1016/S0027-5107(00)00065-8. [DOI] [PubMed] [Google Scholar]
  11. Hittelman WN. The type and time of occurrence of aminopterin-induced chromosome aberrations in cultured potorous cells. Mutat Res. 1973;18:93–102. doi: 10.1016/0027-5107(73)90024-9. [DOI] [PubMed] [Google Scholar]
  12. Jensen MK, Nyfors A. Cytogenetic effects of methotrexate on human cells in vivo: comparison between results obtained by chromosome studies on bone-marrow cells and blood lymphocytes and by the micronucleus test. Mutat Res. 1979;64:339–343. doi: 10.1016/0165-1161(79)90126-2. [DOI] [PubMed] [Google Scholar]
  13. Kao SCH, Phan VH, Clarke SJ. Predictive markers for haematological toxicity of pemetrexed. Curr Drug Targets. 2010;11:48–57. doi: 10.2174/138945010790031072. [DOI] [PubMed] [Google Scholar]
  14. Kasahara Y, Nakai Y, Miura D, Yagi K, Hirabayashi K, Makita T. Mechanism of induction of micronuclei and chromosome aberrations in mouse bone marrow by multiple treatments of methotrexate. Mutat Res. 1992;278:145–151. doi: 10.1016/0165-1218(92)90225-O. [DOI] [PubMed] [Google Scholar]
  15. Kasahara Y, Wakata A, Nakai Y, Yuno K, Miura D, Yagi K, Hirabayashi K, Makita T. The micronucleus test using peripheral blood reticulocytes from methotrexate-treated mice. Mutat Res. 1992;280:117–128. doi: 10.1016/0165-1218(92)90007-M. [DOI] [PubMed] [Google Scholar]
  16. Kelsey KT, Wiencke JK, Eisen EA, Lynch DW, Lewis TR, Little JB. Persistently elevated sister chromatid exchanges in ethylene oxide-exposed primates: the role of a subpopulation of high frequency cells. Cancer Res. 1988;48:5045–5050. [PubMed] [Google Scholar]
  17. Keshava C, Keshava N, Whong WZ, Nath J, Ong TM. Inhibition of methotrexate-induced chromosomal damage by vanillin and chlorophyllin in V79 cells. Teratog Carcinog Mutagen. 1997;17:313–326. doi: 10.1002/(SICI)1520-6866(1997)17:6&#x0003c;313::AID-TCM2&#x0003e;3.0.CO;2-K. [DOI] [PubMed] [Google Scholar]
  18. Kirsch-Volders M, Sofuni T, Aardema M, Albertini S, Eastmond D, Fenech M, Ishidate M, Jr, Lorge E, Norppa H, Surrallés J, von der Hude W, Wakata A. Report from the in vitro micronucleus assay working group. Mutat Res. 2003;540:153–163. doi: 10.1016/j.mrgentox.2003.07.005. [DOI] [PubMed] [Google Scholar]
  19. Lezana EA, Bianchi NO, Bianchi MS, Zabala-Suarez JE. Sister chromatid exchanges in down syndromes and normal human beings. Mutat Res. 1977;5:85–90. doi: 10.1016/0027-5107(77)90046-x. [DOI] [PubMed] [Google Scholar]
  20. Li JC, Kaminskas E. Accumulation of DNA strand breaks and methotrexate cytotoxicity. Proc Natl Acad Sci. 1984;81:5694–5698. doi: 10.1073/pnas.81.18.5694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mace ML, Daskal Y, Wray W. Scanning electron microscopy of chromosome aberrations. Mutat Res. 1978;52:199–206. doi: 10.1016/0027-5107(78)90141-0. [DOI] [PubMed] [Google Scholar]
  22. Paz-y-Miño C, Bustamante G, Sánchez ME, Leone PE. Cytogenetic monitoring in a population occupationally exposed to pesticide in Ecuador. Environ Health Persp. 2002;110:1077–1080. doi: 10.1289/ehp.021101077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Perry PE, Thomson EJ. The methodology of sister chromatid exchanges. In: Kilbey BJ, Legator M, Nichols W, Ramel C, editors. Handbook of mutagenicity test procedures. 2. Amsterdam: Elsevier; 1984. pp. 459–529. [Google Scholar]
  24. Poser RG, Sirotnak FM, Chello PL. Differential synthesis of methotrexate polyglutamates in normal proliferative and neoplastic mouse tissues in vivo. Cancer Res. 1981;41:4441–4446. [PubMed] [Google Scholar]
  25. Rothfuss A, Schütz P, Bochum S, Volm T, Eberhardt E, Kreienberg R, Vogel W, Speit G. Induced micronucleus frequencies in peripheral lymphocytes as a screening test for carriers of a BRCA1 mutation in breast cancer families. Cancer Res. 2000;60:390–394. [PubMed] [Google Scholar]
  26. Shih C, Habeck LL, Mendelsohn LG, Chen VJ, Schultz RM. Multiple folate enzyme inhibition: mechanism of a novel pyrrolopyrimidine-based antifolate LY231514 (MTA) Advan Enzyme Regul. 1998;38:135–152. doi: 10.1016/S0065-2571(97)00017-4. [DOI] [PubMed] [Google Scholar]
  27. Smit EF, Mattson K, von Pawel J, Manegold C, Clarke S, Postmus PE. ALIMTA (pemetrexed disodium) as second-line treatment of non-small-cell lung cancer: a phase II study. Ann Oncol. 2003;14:455–460. doi: 10.1093/annonc/mdg099. [DOI] [PubMed] [Google Scholar]
  28. Speit G, Haupter S. On the mechanisms of differential giemsa staining of bromodeoxyuridine-substituted chromosomes. II. Differences between the demonstration of sister chromatid differentiation and replication patterns. Hum Genet. 1985;70:126–129. doi: 10.1007/BF00273070. [DOI] [PubMed] [Google Scholar]
  29. Tonkinson JL, Marder P, Andis SL, Schultz RM, Gossett LS, Shih C, Mendelsohn LG. Cell cycle effects of antifolate antimetabolites: implications for cytotoxicity and cytostasis. Cancer Chemoth Pharm. 1997;39:521–531. doi: 10.1007/s002800050608. [DOI] [PubMed] [Google Scholar]
  30. Tweats DJ, Blakey D, Heflich RH, Jacobs A, Jacobsen SD, Morita T, Nohmi T, O’Donovan MR, Sasaki YF, Sofuni T, Tice R. Report of the IWGT working group on strategy/interpretation for regulatory in vivo tests II. Identification of in vivo-only positive compounds in the bone marrow micronucleus test. Mutat Res. 2007;627:92–105. doi: 10.1016/j.mrgentox.2006.10.006. [DOI] [PMC free article] [PubMed] [Google Scholar]

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