Anomalous genotoxic responses induced in mouse lymphoma L5178Y cells by potassium bromate

Potassium bromate (KBrO₃) is a well-established rodent kidney carcinogen and its oxidising activity is considered to be a significant factor in its mechanism of action. Although it has also been shown to be clearly genotoxic in a range of in vivo and in vitro test systems, surprisingly, it is not readily detected in several cell lines using the standard alkaline Comet assay. However, previous results from this laboratory demonstrated huge increases in tail intensity by modifying the method to include incubation with either human 8-oxodeoxyguanosine DNA glycosylase-1 (hOGG1) or bacterial formamidopyrimidine DNA glycosylase (FPG) indicating that, as expected, significant amounts of 8-oxodeoxyguanosine (8-OHdG) were induced. The purpose of this work, therefore, was to investigate why KBrO₃, in contrast to other oxidising agents, gives a relatively poor response in the standard Comet assay. Results confirmed that it is a potent genotoxin in mouse lymphoma L5178Y cells inducing micronuclei and mutation at the tk and hprt loci at relatively non-cytotoxic concentrations. Subsequent time-course studies demonstrated that substantial amounts of 8-OHdG appear to remain in cells 24 h after treatment with KBrO₃ but result in no increase in frank stand breaks (FSB) even though phosphorylated histone H2AX (γ-H2AX) antibody labelling confirmed the presence of double-strand breaks. Using bromodeoxyuracil (BrdU) incorporation together with measured increases in cell numbers, L5178Y cells also appeared to go through the cell cycle with unrepaired hOGG1-recognisable damage. Since unrepaired 8-OHdG can give rise to point mutations through G:C → T:A transversions, it was also surprising that mutation could not be detected at the Na⁺/K⁺ ATPase locus as determined by ouabain resistance. Some increases in strand breakage could be seen in the Comet assay by increasing the unwinding time, but only at highly toxic concentrations and to a much smaller extent than would be expected from the magnitude of the other genotoxic responses. It was considered unlikely that these anomalous observations were due to the inability of L5178Y cells to recognise 8-OHdG because these cells were shown to express mOGG1 and have functional cleavage activity at the adducted site. It appears that the responses of L5178Y cells to KBrO₃ are complex and differ from those induced by other oxidising agents.     

Effect of 4-chlorobiphenyl on substrate transport and phospholipid metabolism in mouse L5178Y lymphoma cells

The toxicity of 4-chlorobiphenyl, a constituent of Aroclor 1221, was studied in mouse L5178Y cells, in vitro. 4-Chlorobiphenyl had a varied effect on the uptake of small precursor molecules. Uptake of [³H]l-leucine, [³H]l-serine, [³H]uridine and [³H]thymidine was inhibited, while that of [³H]inositol was stimulated. There was no significant effect on either [¹⁴C]ethanolamine or [¹⁴C]choline uptake. However 4-chlorobiphenyl significantly inhibited incorporation of [¹⁴C]ethanolamine into phosphatidylethanolamine and caused a 2- to 3-fold stimulation in the incorporation of [¹⁴C]choline into phosphatidylcholine. This effect on phosphatidylcholine metabolism depended on the adsorption and continued presence of 4-chlorobiphenyl on the cell plasma membrane. The stimulation of [¹⁴C]choline incorporation was reversed when treated cells were placed in fresh growth medium under conditions where 95 per cent of the 4-chlorobiphenyl was desorbed from the cell surface. The effect of 4-chlorobiphenyl on substrate uptake and phospholipid metabolism appears to depend upon the interaction of the agent with the cell membrane surface.     

马兜铃酸诱导小鼠淋巴瘤细胞突变的机制

目的为研究马兜铃酸(Aristolochic Acid,AA)的致突变机制提供实验依据。方法挑取对照组和100μg/ml AA处理小鼠淋巴瘤L5178Y tk /--3.7.2-细胞得到的胸苷激酶(Thymidine kinase,tk)基因突变克隆,提取DNA,用基于PCR的杂合性缺失(Loss of heterozygosity,LOH)的分析方法检测含功能性tk基因(tk )的11号染色体(11 b)上的断裂情况。结果100μg/ml AA诱导的突变体中tk 基因缺失率为99.01%,其中所有的小克隆(Small Clone,SC)都失去了tk 基因,而大克隆(Large Clone,LC)的LOH发生率为96.6%。进一步分析11号染色体上的分布的3个微卫星位点(D11Mit42,D11Mit29,D11Mit74)的LOH状况如下:6 cm长度的DNA断裂频率为76%,38 cm长度DNA即超过染色体一半长度的DNA断裂频率为34%,整条染色体的缺失率为16%。另外LC在3个微卫星位点(D11Mit42,D11Mit29,D11Mit74)上的LOH发生率分别是:88%,60%和24%;SC的分别为:64%,8%和8%。结论AA是一个强染色体断裂剂。在11b染色体上LC的染色体断裂情况比LC严重。     

Synergistic enhancement of the bleomycin and peplomycin induced mitotic index reduction by verapamil

The cytostatics bleomycin and peplomycin are known to reduce the mitotic index. It is shown that in cultured human lymphocytes the heart drug verapamil (Isoptin) increases this effect synergistically. This seems to be for the first time that synergism in the reduction of the mitotic index by chemical agents has been demonstrated.     

Cytostatic and cytotoxic response of Ehrlich ascites tumor cells in vivo on chronic treatment with cytarabine, bleomycin, and peplomycin

The cytokinetic response of Ehrlich ascites tumor (EAT) cells in vivo upon chronic treatment at low dosage levels with cytarabine (1-beta-D-arabinofuranosylcytosine, ara-c) bleomycin (BLM) and peplomycin (PEP) was estimated. Bivariate DNA histograms allow the simultaneous evaluation of the cell cycle status of living and killed cells. It could be confirmed that ara-C is cytostatic on cells in S phase. Pronounced cytotoxicity was observed in G1 and G2+M phase. BLM and PEP showed no (or neglectable ) accumulation of vital cells in any cycle phase. Both drugs, however, are cytotoxic on cells, regardless their position within the cell cycle. A successive application of ara-C and BLM (or PEP) in a cell kinetics-directed therapy schedule may be taken into account.     

Mutagenicity of chromium picolinate and its components in Salmonella typhimurium and L5178Y mouse lymphoma cells.

Chromium picolinate is one of the most commonly used chromium dietary supplements available in the United States, and it has been marketed to consumers for use in weight loss, increasing muscle mass, and lowering serum cholesterol. Chromium picolinate is a synthetic compound that provides a bioavailable form of Cr(III) that is absorbed better than dietary chromium. However, there are several reports that it can have adverse effects. In order to study the mechanism of observed cellular toxicity and mutagenicity, chromium picolinate and its component compounds, chromium (III) chloride and picolinic acid, were evaluated in Salmonella typhimurium and L5178Y mouse lymphoma cells. Neither chromium picolinate nor chromium chloride induced a mutagenic response in S. typhimurium. However, in the L5178Y mouse lymphoma mutation assay, chromium picolinate induced mutagenic responses without and with the addition of S9.     

Studies on cytotoxic and genotoxic effects of N-hydroxypyridine-2-thione (Omadine) in L5178Y mouse lymphoma cells

The cytotoxicity and genotoxicity of the antifungal and antimicrobial agent Omadine, i.e. N-hydroxypyridine-2-thione (HOPT), has been investigated in L5178Y mouse lymphoma cells in the dark and under UVA irradiation. Omadine inhibits cell growth and induces micronuclei at concentrations >0.5 microM in the absence of light. At a 0.5-microM concentration, an UVA-dose-dependent induction of micronuclei is observed, conditions at which the cytotoxicity and genotoxicity in the dark is negligible. The photogenotoxicity is not accompanied by cytotoxicity. Control experiments with the radical scavengers GSH and GSHOEt implicate the involvement of hydroxyl radicals in the photogenotoxicity of Omadine.     

秋水仙碱和长春新碱诱导L5178Y小鼠淋巴瘤细胞tk基因杂合性缺失分析

目的探讨秋水仙碱(Col)和长春新碱(V in)诱导tk基因分子突变类型及机制。方法挑选经Col,V in诱导的tk基因突变子(tk-/-)和自发突变体,提取基因组DNA,经等位基因特异性PCR扩增,杂合性缺失(LOH)分析等技术,分析其tk基因杂合性缺失。结果Col和V in诱导突变体的tk基因LOH发生率分别为91.5%和92.1%。由LOH分析结果显示,诱导突变体的LOH发生率在自发突变与诱导突变之间,大集落与小集落之间差异均没有显著性。结论Col和V in诱导的tk基因是以功能性等位基因缺失为主的分子突变。     

Mode of action of bredinin with guanylic acid on L5178Y mouse leukemia cells.

Moderate concentrations of bredinin (1.2 X 10(-5) M) strongly inhibited growth of L5178Y cells, with the effect being reversed by guanylic acid (GMP). However, at higher concentrations of breeding the inhibition was not reversed completely by GMP added in excess. Bredinin was cytocidal at concentrations above 2 X 10(-5) M, but 5 X 10(-5) M bredinin in the presence of excess GMP, bredinin was cytostatic. Bredinin inhibited nucleic acid synthesis of L5178Y cells, but bredinin itself was not incorporated into the nucleic acid. Inhibition of nucleic acid synthesis was clearly reversed by GMP. Similarly chromosomal aberrations in L5178Y cells caused by bredinin were reversed by GMP. In contrast, the effect of ahigh concentration of bredinin on cell multiplication was not reversed by GMP. The modal volume of L5178Y cells increased during incubation in the presence of bredinin and GMP for 24 hours, 5 X 10(-5) M bredinin with GMP causing a 70% increase in cell volume. This increase in cell volume was mainly due to an increase in the protein content of the cells. The cytostatic effect of bredinin with GMP was reversed completely by adenosine-3',5'-cyclic monophosphate (cyclic AMP). Other cyclic nucleotides and nucleotides were ineffective. The reversing effect of cyclic AMP on cell survival depended upon the concentration of GMP, and was not seen in the absence of GMP. It was concluded that cyclic AMP influences the secondary cytostatic effect of bredinin, and not the primary cytotoxic effect reversed by GMP.     

Mechanism of adriamycin resistance in a subline of mouse lymphoblastoma L5178Y cells

The biochemical mechanism of anthracycline resistance was studied with an adriamycin-resistant subline of mouse lymphoblastoma L5178Y cells. Both uridine and thymidine uptakes in the resistant cells were observed more resistant to adriamycin and daunorubicin than those in the parental cells. Aclacinomycin A exhibited the same degree of inhibition of nucleic acid syntheses in the sensitive cells and in the resistant cells. The resistance pattern observed by the inhibition of RNA and DNA syntheses seemed to parallel that by growth inhibition. No significant difference was demonstrated between the parental and resistant cells in the inhibition of RNA and DNA polymerase reactions with isolated nuclei. The uptake and retention of [3H]adriamycin was observed significantly less in the resistant cells than in the sensitive cells. The results suggested that the adriamycin resistance may be due to alteration of the cytoplasmic membrane and/or cytoplasm, resulting in decreased uptake and retention of the antibiotic in the resistant cells.     

Alteration of membrane-associated enzymes in drug-resistant sublines of mouse lymphoblastoma L5178Y cells

Activities of marker enzymes for various cell components were studied with extracts of adriamycin-, aclacinomycin A- and bleomycin-resistant cells and with partially purified plasma membrane fraction of aclacinomycin A-resistant cells, in comparison with those of the parental cells. Alkaline phosphodiesterase and Na+-K+-ATPase activities were observed to alter in the drug-resistant sublines, but other enzymes showed similar activities in the resistant cells to those in the parental cells. Alkaline phosphodiesterase activities in all the resistant sublines were higher than that in the parental cells. Na+K+-ATPase activities of anthracycline-resistant sublines were lower and that in bleomycin-resistant cell line was higher than that of the parental cells. The adriamycin-resistant cells exhibited the same level of alkaline phosphodiesterase activity with the aclacinomycin A-resistant cells: Vmax was the same with, and the affinity was twice stronger than the parental cells. The bleomycin-resistant cells showed ca. 30% Vmax in comparison with the sensitive cells, and 17 fold higher affinity than the parental cells. The current results, concerning changes of membrane-associated enzymes in drug-resistant sublines of L5178Y cells, support the assumption that the resistance is due to alteration of plasma membrane transport systems.     

Different roles of Fpg and Endo III on catechol-induced DNA damage in extended-term cultures of human lymphocytes and L5178Y mouse lymphoma cells.

Catechol is a genotoxic agent assumed to induce DNA damage via the oxidative pathway. Using the comet assay and the repair-specific enzymes formamido pyrimidine glycosylase (Fpg) and endonuclease III (Endo III), we examined the ability of catechol to induce DNA damage in extended-term cultures of human lymphocytes and mouse lymphoma cells. Our results suggest that mouse lymphoma cells are somewhat more sensitive towards catechol-induced DNA damage than the extended-term cultures of human lymphocytes. At high concentrations, the catechol-induced damage seemed to be independent of both Fpg and Endo III, possibly indicating a non-oxidative pathway for the DNA damage (involving, for example, a bulky adduct). The fact that Endo III, but not Fpg, enhanced the DNA damaging effect of catechol, suggests that this metabolite of benzene either mediates oxidation of pyrimidines rather than purines, or that oxidised purines are repaired more efficiently, at least in human lymphocytes. In the latter cells, low concentrations of catechol were found to reduce the DNA migration. Considering the role of Fpg and it's adduct specific detection of 8-oxoguanine, this suggests that a low concentration of catechol has an antioxidative effect reducing the background levels of oxidized purines.     

Synergistic cytotoxicity of ex vivo expanded natural killer cells in combination with monoclonal antibody drugs against cancer cells

The adoptive transfer of highly cytotoxic natural killer (NK) cells is an emerging tool for cancer immunotherapy. Antibody-dependent cellular cytotoxicity (ADCC) has recently been identified as one of the critical factors for the clinical efficacy of anticancer antibodies, in which NK cells are the major effectors of ADCC. NK cells were expanded from PBMC by a feeder-cell-free expansion method. NK cell expansion efficiency was evaluated within a period of 21 days. The kinetics of NK cell expansion and the expression of activating and inhibitory receptors on NK cells were monitored. NK cells producing IFN-γ and TNF-α were detected by intracellular cytokine staining. The cytotoxicity of expanded NK cells against various cancer cells was compared with that of freshly isolated NK cells. The ADCC functions of expanded NK cells in combination with rituximab against CD20 + lymphoma cell lines were evaluated. Our method efficiently expanded NK cells ex vivo, which showed a much higher activity to induce the expression of activating receptors and to produce IFN-γ and TNF-α as well as cytotoxicity against various cancer cell lines including CD133 + primary cancer cells than freshly isolated NK cells. We observed a synergistic cytotoxicity of our expanded NK cells against CD20 + B lymphoma cell lines as well as higher IFN-γ and TNF-α production when combined with rituximab. Our results suggest that the adoptive transfer of a large number of ex vivo expanded NK cells, particularly in combination with monoclonal antibody drugs, is a useful tool for cancer immunotherapy.     

Uptake and metabolism of mizoribine,an immunosuppressant,in L5178Y-R mouse lymphoma cells in vitro and peripheral blood mononuclear cells of rats and kidney transplant recipients in vivo

The cellular uptake of mizoribine (MZR), an immunosuppressant, and metabolism of MZR to MZR-5′- monophosphate (MZRP), an active metabolite, were evaluated in L5178Y-R mouse lymphoma cells and peripheral blood mononuclear cells (PBMCs) of rats and kidney transplant recipients (KTRs, n = 22). Real-time PCR analysis revealed the expression of ENT1 and ENT2 mRNAs, but not of CNTs, in L5178Y-R cells and rat's PBMCs. In L5178Y-R cells, the uptake of MZR was suppressed by adenosine, a substrate for ENT1 and ENT2, but not by 5-(4-nitrobenzyl)-6-thioinosine (0.1 μM), an ENT1 inhibitor. Saturable metabolism of MZR to MZRP was observed. In rats, peak plasma concentrations of MZR and peak concentrations of MZR and MZRP in PBMCs were observed 3 h after oral administration. MZR disappeared from PBMCs in parallel with plasma MZR, but the disappearance of MZRP from PBMCs appeared to be slow. In KTRs, the mean plasma concentration of MZR 3–4 h after ingestion was 3.14 μg/ml and the mean MZRP concentration in PBMCs was 16.8% of MZR, reflecting the involvement of ENT in the uptake of MZR. A linear relationship was observed between plasma MZR concentrations ranging from 1 to 6 μg/ml and PBMC's MZRP concentrations ranging from 90 to 200 ng/ml.