Leigh Henderson

A further assessment of factors influencing measurements of thioguanine-resistant mutant frequency in circulating T-lymphocytes

We have used the T-Lymphocyte cloning technique as a method of monitoring the human population for somatic cell mutant frequency. We present a statistical analysis of the experimental factors which may influence the observed mutant frequency. We have obtained consistently high plating efficiencies of T-cells from the mononuclear cell fraction from donor blood samples (mean of 56%, based on 123 observations from 70 individuals). Nevertheless, an inverse correlation of mutant frequency with plating efficiency was observed, and some experimental factors (serum and interleukin-2 batch, and worker) may have a significant effect on the observed mutant frequency. We discuss the difficulties that these possible effects present in establishment of a reference database and design of long-term studies. No significant effect of donor sex on mutant frequency was observed, but age (1.3% increase per year for normal adults) and smoking (56% increase over normal non-smokers) both significantly increased the mutant frequency. We discuss the utility of the assay for the monitoring of populations for heritable DNA damage, and we compare the results to those obtained with lymphocytes using other endpoints, e.g. chromosome aberrations, micronuclei and sister-chromatid exchange.

Comparative Human Cellular Radiosensitivity: II. The Survival Following Gamma-irradiation of Unstimulated (G0) T-lymphocytes, T-lymphocyte Lines, Lymphoblastoid Cell Lines and Fibroblasts from Normal Donors, from Ataxia-telangiectasia Patients and from Ataxia-telangiectasia Heterozygotes

We have measured clonal survival following gamma-irradiation of unstimulated (G0) T-lymphocytes from 35 donors, of 11 T-lymphocyte cell lines, of six lymphoblastoid cell lines, and of nine primary fibroblast strains for which we have G0 T-lymphocyte material from the same donor. Amongst the G0 lymphocytes we have results from nine normal donors, from eight cord bloods, from seven ataxia-telangiectasia (A-T) patients and from nine A-T heterozygotes. Although there is some variation between samples, G0 T-lymphocytes from normal donors appear to be slightly more radioresistant than T-lymphocyte lines, with a more shouldered survival curve. From our limited sample, lymphoblastoid cell lines appear to be slightly more radiosensitive than T-lymphocytes. The overall radiosensitivity of primary fibroblasts appears to be broadly similar to that of G0 T-lymphocytes. In nine instances, five A-Ts and four A-T heterozygotes, both G0 T-lymphocytes and primary fibroblasts from the same donor were tested. In five cases there was closely similar radiosensitivity in the two cell types, but in four cases there was some discrepancy. Further work, especially with normal donors, will be required in order to establish how reliably radiosensitivity in other cell types can be predicted from that of G0 T-lymphocytes. In all cell types the hypersensitivity of A-T cells was confirmed. Furthermore, the marginally greater sensitivity of A-T heterozygotes, when compared as a group with normals, was confirmed with G0 T-lymphocytes. Our results also suggest a slightly increased radiosensitivity in G0 T-lymphocytes from some, but not all, cord blood samples.

The human lymphocyte micronucleus assay: Response of cord blood lymphocytes to γ-irradiation and bleomycin

The induction of micronuclei in human cord blood lymphocytes by treatment with gamma-irradiation and bleomycin has been measured. Culture durations which gave peak MN frequencies were determined. The lowest tested doses, 0.1 Gy irradiation and 1.25 micrograms/ml bleomycin, produced significant increases in the frequency of micronuclei. The spontaneous frequency of micronucleated lymphocytes in 28 cord blood samples ranged between 0.5 and 9.5 per thousand lymphocytes, with a modal value of 2.5. The method is evaluated for its potential usefulness in monitoring populations for chromosome breakage.

Short-term tests for transplacentally active carcinogens Sensitivity of the transplacental micronucleus test to diethylnitrosamine

DEN is an established animal carcinogen, and is effective transplacentally; but like other nitrosamines, its genotoxicity is difficult to assess in established shortterm cytogenetic tests. The mouse transplacental micronucleus test demonstrates the ability of DEN to cause chromosome breakage in utero. Positive results were obtained from the 15th day of gestation and a linear dose-response relationship is expressed when micronucleated polychromatic erythrocytes are measured in neonatal blood, after transplacental exposure late in gestation. 25mg/kg (the lowest dose tested) caused a significant increase in MNPCEs (p less than 0.05). A micronucleus test based on polychromatic erythrocytes in neonatal blood permits maximal exploitation of the metabolic capacity of the prenatal liver and is therefore sensitive to clastogenic chemicals not activated by adult bone marrow.

Sister-chromatid exchange and micronucleus induction as indicators of genetic damage in maternal and foetal cells

The effectiveness of 3 compounds, procarbazine, mitomycin C and cyclophosphamide as inducers of sister-chromatid exchanges (SCEs) in granulocyte-macrophage progenitor cells, in foetal liver and bone marrow from pregnant mice at day 17 of gestation were determined. Cyclophosphamide and procarbazine induced similar SCE frequencies in maternal and foetal cells. Mitomycin C was slightly less effective in foetal liver than in maternal bone marrow. In contrast to the results of SCE induction, cyclophosphamide produced more micronucleated polychromatic erythrocytes in foetal liver than in bone marrow. The SCE results for mitomycin C and procarbazine are compared with results obtained previously for micronuclei induction in 15-day pregnant animals.

Short-term tests for transplacentally active carcinogens A comparison of sister-chromatid exchange and the micronucleus test in mouse foetal liver erythroblasts

The effectiveness of 6 chemicals (benzo[a]pyrene, (BaP), cyclophosphamide (CP), diethylnitrosamine (DEN), methyl methanesulphonate (MMS), mitomycin C (MC) and procarbazine (PC) ) as inducers of micronuclei in foetal liver and maternal bone marrow erythroblasts has been determined, and related to that of gamma-radiation. CP, DEN, MMS and PC were all more effective in the foetal liver. The induction of micronuclei and SCEs by each chemical in foetal erythroblasts after in vivo exposure was measured. When expressed as induction of sister-chromatid exchanges (SCEs) per erythroblast/induction of micronuclei per erythroblast (/microM/kg), the ratios obtained were MC 580, BaP 470, DEN 430, CP 258, MMS 140 and PC 13. The lowest doses detected as potentially genotoxic by each test in foetal liver erythroblasts are (with the exception of PC which is a relatively ineffective inducer of SCEs) similar. When isolated foetal livers were exposed in vitro, SCE dose responses to BaP, MC, MMS and PC could be directly related to those from in vivo exposure, indicating the role of the foetal liver in metabolic activation, but CP was considerably more cytotoxic. The transplacental micronucleus test, and in vivo/in vitro method for SCEs in foetal liver erythroblasts, provide sensitive, complementary assays for genotoxic effects of chemicals during prenatal life. Since foetal liver possesses greater metabolic potential than adult bone marrow, the transplacental tests respond to genotoxic agents not detected by bone-marrow systems.

Effects of diethylstilboestrol-dipropionate on SCEs, micronuclei, cytotoxicity, aneuploidy and cell proliferation in maternal and foetal mouse cells treated in vivo.

The effect of diethylstilboestrol-dipropionate on the frequency of SCEs and micronuclei, cytotoxicity, aneuploidy and cell proliferation rates of foetal liver and maternal bone marrow cells following exposure of pregnant mice was measured. An increase in the number of aneuploid and polyploid cells was observed in both tissues. There was no effect on micronuclei frequency, SCE frequency, or cell proliferation rate.

SISTER-CHROMATID EXCHANGE FREQUENCIES IN FIBROBLASTS AND LYMPHOCYTES OF PATIENTS WITH SYSTEMIC LUPUS-ERYTHEMATOSUS

Sister-chromatid exchange (SCE) frequencies have been measured in lymphocytes and fibroblasts of patients with systemic lupus erythematous (SLE) and healthy controls, and in lymphocytes of control patients with serum anti-nuclear antibodies (ANA) but no other disease manifestations of SLE. The SCEs of SLE lymphocytes were higher than those of the controls but the SCEs of the SLE fibroblasts did not differ from those of the controls. The SCEs of the controls with positive ANA did not differ significantly from those of the healthy controls. There was no correlation between SCE frequencies of the SLE lymphocytes and disease activity determined by many clinical and laboratory measurements. Primary and secondary DNA-repair defects in SLE cells are considered.

Short-term tests for transplacentally active carcinogens: Induction of sister-chromatid exchanges in foetal brain, lung and blood-forming cells by procarbazine and cyclophosphamide

The induction of sister-chromatid exchanges (SCEs) by cyclophosphamide (CP) and procarbazine (PC) in mouse granulocyte-macrophage precursor cells (GM cells) and erythroblasts from foetal liver, and cells from foetal brain and foetal lung has been measured. Agents were administered in vivo, and cells explanted into BrdUrd-containing medium for 2 cell cycles in vitro (using specific growth-promoting substances where necessary) to determine SCE frequency. Tissue and cell-type differences in responses were observed, and it is concluded that the in vivo/in vitro transplacental SCE technique is a useful indicator of genotoxic effects of agents which are potential transplacental carcinogens.