Partial purification of linoleic acid isomerase enzyme from Lactobacillus paracasei bacteria isolated from milk

Abstract Conjugated Linoleic Acid (CLA) has attracted the attention of many researchers, especially that of microbial origin due to its biological importance to the consumer. The current study aims to extract LA Isomerase enzyme from Lactobacillus paracasei bacteria from milk and to use the enzyme in the production of CLA. Selective media, including MRS and MRS-Dagatose, were used in isolating local strains. The selected bacterial isolates were tested for their ability to produce LA-Isomerase enzyme. The isolate with high enzymatic activity was selected. After extraction and partial purification of the enzyme, the optimal conditions for the production of conjugated fatty acid were studied, and the reaction products were diagnosed using GC-MS technology. It was found that 11 isolates have the ability to produce CLA at different concentrations, H1 isolate showed the highest production of conjugated fatty acid at a concentration of 120.45 g.ml-1, this isolate was selected as the source for enzyme extraction. The enzymatic activity of the crude extract and partially purified with ammonium sulfate was estimated using color methods at wavelength of 233 nm. The effect of the optimum conditions (pH, temperature, linoleic acid concentration and enzyme concentration) on the CLA product was studied using the partially purified LA Isomerase enzyme, the optimum conditions for production were 6.5, 45 °C, 100 g.ml-1 and 0.7 ml, respectively. The GC-MS technique showed the presence of a number of reaction products that are isomers of conjugated linoleic acid (C9T11, T9T12, T10C12) with different concentrations.

Resumo O Ácido Linoleico Conjugado (CLA) tem chamado a atenção de diversos pesquisadores, principalmente aquele de origem microbiana, devido à sua importância biológica para o consumidor. O presente estudo visa extrair a enzima LA Isomerase da bactéria Lactobacillus paracasei do leite e usar essa enzima na produção de CLA. Meios seletivos, incluindo MRS e MRS-Dagatose, foram usados no isolamento de cepas locais. Os isolados bacterianos selecionados foram testados quanto à sua capacidade de produzir a enzima LA-Isomerase. Foi selecionado o isolado com alta atividade enzimática. Após a extração e purificação parcial da enzima, as condições ideais para a produção de ácido graxo conjugado foram estudadas e os produtos da reação foram identificados usando a tecnologia GC-MS. Verificou-se que 11 isolados possuem capacidade de produzir CLA em diferentes concentrações. O isolado H1 apresentou a maior produção de ácido graxo conjugado, na concentração de 120,45 g.ml-1, e este isolado foi selecionado como fonte para extração enzimática. A atividade enzimática do extrato bruto e parcialmente purificado com sulfato de amônio foi estimada por métodos de coloração em comprimento de onda de 233 nm. O efeito das condições ótimas (pH, temperatura, concentração de ácido linoleico e concentração de enzima) no produto CLA foi estudado usando a enzima LA Isomerase parcialmente purificada e as condições ótimas para produção foram 6,5, 45 °C, 100 g.ml-1 e 0,7 mL, respectivamente. A técnica de GC-MS mostrou a presença de uma série de produtos de reação que são isômeros do ácido linoleico conjugado (C9T11, T9T12, T10C12) com diferentes concentrações.

Partial purification of linoleic acid isomerase enzyme from Lactobacillus paracasei bacteria isolated from milk / Purificação parcial da enzima isomerase do ácido linoleico da bactéria Lactobacillus paracasei isolada do leite

Conjugated Linoleic Acid (CLA) has attracted the attention of many researchers, especially that of microbial origin due to its biological importance to the consumer. The current study aims to extract LA Isomerase enzyme from Lactobacillus paracasei bacteria from milk and to use the enzyme in the production of CLA. Selective media, including MRS and MRS-Dagatose, were used in isolating local strains. The selected bacterial isolates were tested for their ability to produce LA-Isomerase enzyme. The isolate with high enzymatic activity was selected. After extraction and partial purification of the enzyme, the optimal conditions for the production of conjugated fatty acid were studied, and the reaction products were diagnosed using GC-MS technology. It was found that 11 isolates have the ability to produce CLA at different concentrations, H1 isolate showed the highest production of conjugated fatty acid at a concentration of 120.45 g.ml-1, this isolate was selected as the source for enzyme extraction. The enzymatic activity of the crude extract and partially purified with ammonium sulfate was estimated using color methods at wavelength of 233 nm. The effect of the optimum conditions (pH, temperature, linoleic acid concentration and enzyme concentration) on the CLA product was studied using the partially purified LA Isomerase enzyme, the optimum conditions for production were 6.5, 45 °C, 100 µg.ml-1 and 0.7 ml, respectively. The GC-MS technique showed the presence of a number of reaction products that are isomers of conjugated linoleic acid (C9T11, T9T12, T10C12) with different concentrations.

O Ácido Linoleico Conjugado (CLA) tem chamado a atenção de diversos pesquisadores, principalmente aquele de origem microbiana, devido à sua importância biológica para o consumidor. O presente estudo visa extrair a enzima LA Isomerase da bactéria Lactobacillus paracasei do leite e usar essa enzima na produção de CLA. Meios seletivos, incluindo MRS e MRS-Dagatose, foram usados no isolamento de cepas locais. Os isolados bacterianos selecionados foram testados quanto à sua capacidade de produzir a enzima LA-Isomerase. Foi selecionado o isolado com alta atividade enzimática. Após a extração e purificação parcial da enzima, as condições ideais para a produção de ácido graxo conjugado foram estudadas e os produtos da reação foram identificados usando a tecnologia GC-MS. Verificou-se que 11 isolados possuem capacidade de produzir CLA em diferentes concentrações. O isolado H1 apresentou a maior produção de ácido graxo conjugado, na concentração de 120,45 g.ml-1, e este isolado foi selecionado como fonte para extração enzimática. A atividade enzimática do extrato bruto e parcialmente purificado com sulfato de amônio foi estimada por métodos de coloração em comprimento de onda de 233 nm. O efeito das condições ótimas (pH, temperatura, concentração de ácido linoleico e concentração de enzima) no produto CLA foi estudado usando a enzima LA Isomerase parcialmente purificada e as condições ótimas para produção foram 6,5, 45 °C, 100 µg.ml-1 e 0,7 mL, respectivamente. A técnica de GC-MS mostrou a presença de uma série de produtos de reação que são isômeros do ácido linoleico conjugado (C9T11, T9T12, T10C12) com diferentes concentrações.

Partial purification of linoleic acid isomerase enzyme from Lactobacillus paracasei bacteria isolated from milk.

Conjugated Linoleic Acid (CLA) has attracted the attention of many researchers, especially that of microbial origin due to its biological importance to the consumer. The current study aims to extract LA Isomerase enzyme from Lactobacillus paracasei bacteria from milk and to use the enzyme in the production of CLA. Selective media, including MRS and MRS-Dagatose, were used in isolating local strains. The selected bacterial isolates were tested for their ability to produce LA-Isomerase enzyme. The isolate with high enzymatic activity was selected. After extraction and partial purification of the enzyme, the optimal conditions for the production of conjugated fatty acid were studied, and the reaction products were diagnosed using GC-MS technology. It was found that 11 isolates have the ability to produce CLA at different concentrations, H1 isolate showed the highest production of conjugated fatty acid at a concentration of 120.45 g.ml-1, this isolate was selected as the source for enzyme extraction. The enzymatic activity of the crude extract and partially purified with ammonium sulfate was estimated using color methods at wavelength of 233 nm. The effect of the optimum conditions (pH, temperature, linoleic acid concentration and enzyme concentration) on the CLA product was studied using the partially purified LA Isomerase enzyme, the optimum conditions for production were 6.5, 45 °C, 100 µg.ml-1 and 0.7 ml, respectively. The GC-MS technique showed the presence of a number of reaction products that are isomers of conjugated linoleic acid (C9T11, T9T12, T10C12) with different concentrations.

Non-targeted effects of radiation exposure: recent advances and implications.

The target theory of radiation-induced effects has been challenged by numerous studies, which indicate that in addition to biological effects resulting from direct DNA damage within the cell, a variety of non-DNA targeted effects (NTE) may make important contributions to the overall outcome. Ionising radiation induces complex, global cellular responses, such as genomic instability (GI) in both irradiated and never-irradiated 'bystander' cells that receive molecular signals produced by irradiated cells. GI is a well-known feature of many cancers, increasing the probability of cells to acquire the 'hallmarks of cancer' during the development of tumours. Although epidemiological data include contributions of both direct and NTE, they lack (i) statistical power at low dose where differences in dose response for NTE and direct effects are likely to be more important and (ii) heterogeneity of non-targeted responses due to genetic variability between individuals. In this article, NTE focussing on GI and bystander effects were critically examined, the specific principles of NTE were discussed and the potential influence on human health risk assessment from low-dose radiation was considered.

Oxidative stress signalling: a potential mediator of tumour necrosis factor alpha-induced genomic instability in primary vascular endothelial cells.

Studying the potential role of tumour necrosis factor (TNF)alpha in the initiation of genomic instability is necessary to understand whether TNFalpha can serve as a signalling mediator of radiation-induced genomic instability in non-irradiated bystander cells. In this study, we examined whether TNFalpha could initiate processes through oxidative stress signalling that lead to DNA damage and genomic instability in primary vascular endothelium. In these cells, low linear energy transfer (LET) radiation (0.1-2 Gy) induced the secretion of TNFalpha into the culture medium. When added ectopically, TNFalpha at concentrations ranging from 0.1 ng ml(-1) to 10 ng ml(-1) increased (twofold to threefold) intracellular oxidative stress. Next, to examine whether TNFalpha induces genetic damage, cells were treated with TNFalpha for 5 h and analysed immediately using the single cell gel electrophoresis assay or after 3 days, 12 days and 20 days using solid stain chromosomal analysis. Cells exposed to 0.1 Gy, 1 Gy or 2 Gy or treated with 100 microM H2O2 were used as positive controls. The results showed that TNFalpha as low as 0.1 ng ml(-1) could initiate increased DNA damage compared with untreated controls. When examined in the progeny cells after several generations, the chromosomal instability appeared to be carried over even after day 12 and day 20. The increased genetic damage is inhibited in cells that are pre-incubated with the antioxidant enzyme catalase, the antioxidant N-acetyl-L-cysteine or the metal chelator pyrrolidine dithiocarbamate. These results clearly indicate that TNFalpha at concentrations at which no cytotoxicity is observed could induce genetic damage through free radical generation, which could, in turn, lead to the delayed events associated with genomic instability.

Genomic instability and the role of radiation quality.

Genomic instability (GI) is a hallmark of tumorigenic progression and is observed as delayed genetic damage in the progeny of irradiated and unirradiated bystander cells. The expression of GI can be influenced by genotype, cell type and radiation quality. While several studies have demonstrated the induction of GI by high and low-linear energy transfer (LET) radiation, our work on human and mouse primary cell systems has shown LET-dependent differences in the induction and expression of GI. These differences might be attributed to differences in radiation track structure, dose rate, contribution of bystander cells and radiation dose. This paper reviews the role of radiation quality in the induction of GI and describe the possible mechanisms underlining the observed differences between radiation types on its induction. The experimental results presented suggest that dose might be the most significant factor in determining induction of GI after low-LET radiation.

Particulate debris from a titanium metal prosthesis induces genomic instability in primary human fibroblast cells.

Previous studies detected both lethal and cumulative chromosomal aberrations in bone marrow and peripheral blood of patients with worn hip and knee replacements. This study shows that wear debris from a worn titanium metal on high-density polyethylene hip replacement also produces chromosomal instability and reproductive failure in cell culture. The progeny of these treated cells also displayed chromosomal instability, mainly consisting of chromatid breaks and minutes, and reproductive failure as determined by clonogenic survival many generations postexposure. These delayed effects are similar to those caused by the heavy metals cadmium and nickel and to those seen for low-dose radiation. These findings may have important implications with regard to the long-term risks of joint replacement surgery. This highlights the need for long-term epidemiological studies of patients with surgical implants.

Long-term genomic instability in human lymphocytes induced by single-particle irradiation.

Recent evidence suggests that genomic instability, which is an important step in carcinogenesis, may be important in the effectiveness of radiation as a carcinogen, particularly for high-LET radiations. Understanding the biological effects underpinning the risks associated with low doses of densely ionizing radiations is complicated in experimental systems by the Poisson distribution of particles that can be delivered. In this study, we report an approach to determine the effect of the lowest possible cellular radiation dose of densely ionizing alpha particles, that of a single particle traversal. Using microbeam technology and an approach for immobilizing human T-lymphocytes, we have measured the effects of single alpha-particle traversals on the surviving progeny of cells. A significant increase in the proportion of aberrant cells is observed 12-13 population doublings after exposure, with a high level of chromatid-type aberrations, indicative of an instability phenotype. These data suggest that instability may be important in situations where even a single particle traverses human cells.

Dose- and time-response relationships for lethal mutations and chromosomal instability induced by ionizing radiation in an immortalized human keratinocyte cell line.

PURPOSE: To investigate the relationship between two well-established delayed effects of ionizing radiation, experiments were conducted to determine the induction and expression of lethal mutations (delayed reproductive death) and chromosomal instability with respect to dose and time in a human immortalized keratinocyte cell line. METHODS: HPV-G cells were gamma- or alpha-irradiated and maintained in culture for up to 72 population doublings. At intervals, measurements were made of cloning efficiency and the cells examined for apoptosis and cytogenetic aberrations. RESULTS: The descendants of cells surviving 1 or 3 Gy gamma-irradiation, but not 0.5 Gy gamma-irradiation, exhibited a reduced colony-forming efficiency. The reduction persisted at a constant rate of 15-20% clonogenic cell loss per population doubling for up to 72 population doublings. Apoptosis was demonstrated in all colonies in the 1 and 3 Gy groups at 30 and 72 population doublings post-irradiation but not in the 0.5 Gy group. A significant persistent reduction in colony-forming ability (approximately 80%) was demonstrated in the progeny of cells irradiated with 0.5 Gy alpha-particles. After 30 population doublings, the proportion of chromosomally aberrant cells was significantly greater than control values for all doses of both high- and low-LET radiations. The major cytogenetic aberrations (chromatid breaks, chromosome fragments and minutes) were consistent with the transmission of chromosomal instability. The expression of instability declined between 30 and 72 population doublings in the 0.5 Gy and 3 Gy gamma-irradiation groups, but persisted up to 72 population doublings in the 1 Gy group. The expression of chromosomal instability was greater in the descendants of alpha-irradiated cells and showed little evidence of reduction with time. CONCLUSIONS: Unstable aberrations characteristic of radiation-induced chromosomal instability may commonly result in apoptosis and account for a component of the delayed reproductive death/lethal mutation phenotype in HPV-G cells. However, the absence of lethal mutations in the descendants of 0.5 Gy gamma-irradiated cells indicates a low-LET threshold effect for this particular endpoint. Overall, and particularly at low doses, there is no direct correlation between the two endpoints, indicating the absence of a simple relationship between these manifestations of radiation-induced genomic instability.

Complex chromosome aberrations in peripheral blood lymphocytes as a potential biomarker of exposure to high-LET alpha-particles.

PURPOSE: To determine the induction and transmission, to second and third division cells, of complex chromosome aberrations in peripheral blood lymphocytes after exposure to high-LET alpha-particles in vitro. MATERIALS AND METHODS: Separated peripheral blood lymphocytes collected from four healthy donors were irradiated in vitro with either high-LET alpha-particles (121 keV/microm; 0.5 Gy) or low-LET X-rays (250kV constant potential; 3 Gy). Cells were harvested in first, second and third division post-irradiation and chromosome aberrations observed at each cell division were analysed by combining the techniques of FISH and DAPI/Hoechst 33258 harlequin staining. Whole chromosome probes were used for chromosomes 1, 2 and 5, together with a pan-centromeric probe and the resulting chromosome 'painting' patterns were classified according to the Savage and Simpson (S & S) scheme (Savage and Simpson 1994a, Savage and Tucker 1996). RESULTS: A greater proportion of complex chromosome aberrations was observed, defined as involving three or more breaks in two or more chromosomes, relative to total exchanges, after exposure to 0.5 Gy alpha-particles (mean 1 track/cell) than after the high reference dose of 3 Gy X-rays (49-56% and 20-22%, respectively). Qualitatively, alpha-particles induced chromosome aberrations of far greater complexity than those observed after X-rays. This was reflected by both the rapid reduction in the percentage of damaged cells between first and second division indicative of cell death, and the spectrum of aberration types observed in second and third division cells post-irradiation. Regardless of this complexity, 15% of the complexes induced by alpha-particles at first division were potentially transmissible and by third division, transmissible-type complexes, specifically insertions, represented the predominant complex type (65%). CONCLUSION: Transmissible-type complexes were observed, specifically insertions, in both second and third division cells after exposure to high-LET alpha-particles (0.5 Gy) in vitro. The authors predict these cells to be stable and to be capable of persisting through many cell generations. Considering that the induction of complex chromosome aberrations by low-LET radiation is strongly dependent on dose, so that they are expected to be undetectable at environmental exposures, insertions are much more likely to be a characteristic feature of high-LET radiation at all doses. From this the usefulness of insertions as biomarkers of past exposure to environmentally relevant doses of high-LET alpha-particles is supported.

An improved micro-method for obtaining chromosome preparations from individual haemopoietic colonies.

A robust method for obtaining chromosome preparations from individual haemopoietic colonies in semi-solid media is described. The accumulation of metaphases and the hypotonic treatment of cells were carried out in the culture dish and individual colonies were transferred onto poly-L-lysine-treated slides and fixed stepwise prior to staining. The technique produced high yields of well-spread metaphases facilitating clonal cytogenetic analysis.

Chromosomal instability in the descendants of unirradiated surviving cells after alpha-particle irradiation.

We have demonstrated chromosomal instability in the clonal descendants of hemopoietic stem cells after irradiating murine bone marrow with alpha-particles. However, because cells that are irradiated by alpha-particles are defined by a Poisson distribution of individual particle traversals, there is an inevitable proportion of unirradiated cells in the surviving population. The calculated expected proportions of irradiated and nonirradiated cells indicate that the number of clonogenic cells transmitting chromosomal instability is greater than the number expected to be hit and survive. To investigate further this discrepancy, we studied the effects of interposing a grid between the cells and the alpha-particle source so that the surviving population consists predominantly of untraversed stem cells. Comparison with the same irradiation conditions without the grid reveals that the same level of instability is induced. The data confirm that alpha-particles induce chromosomal instability but instability is demonstrated in the progeny of nonirradiated stem cells and must be due to unexpected interactions between irradiated and nonirradiated cells. This untargeted effect has important implications for mechanistic studies of radiation action and for assessment of radiation risk.

Radiation-induced chromosomal instability in human fibroblasts: temporal effects and the influence of radiation quality.

PURPOSE: To determine whether chromosomal instability is induced in human diploid fibroblasts by ionizing radiation and to investigate the effects of radiation quality by comparing X-rays, neutrons and alpha-particles. MATERIALS AND METHODS: Cells from two human diploid fibroblast lines, HF12 and HF19, were irradiated and analysed cytogenetically at 3, 20 and 35 population doublings post-irradiation. RESULTS: Exposure of HF19 cells to neutrons and alpha-particles resulted in a consistently increased frequency of unstable aberrations, particularly chromatid-type aberrations, compared to control cultures. Aberration frequency after X-irradiation was not significantly greater than controls at 20 population doublings but was significantly increased after 35 population doublings, although not to the same level as that following neutron or alpha-irradiation. No chromosomal instability was demonstrated in the progeny of HF12 cells after X-, neutron or alpha-particle irradiation. CONCLUSIONS: The data are consistent with the progeny of irradiated HF19 cells expressing chromosomal instability. All three radiations are effective in inducing instability, but the expression of the phenotype is influenced by radiation quality. The absence of radiation-induced chromosomal instability in HF12 cells may reflect the influence of genetic factors.

Radiation-induced transmissable chromosomal instability in haemopoietic stem cells.

Heritable radiation-induced genetic alterations have long been assumed to be "fixed" within the first cell division. However, there is a growing body of evidence that a considerable fraction of cells surviving radiation exposure appear normal, but a variety of mutational changes arise in their progeny due to a transmissible genomic instability. In our investigations of G-banded metaphases, non-clonal cytogenetic aberrations, predominantly chromatid-type aberrations, have been observed in the clonal descendants of murine and human haemopoietic stem cells surviving low doses (approximately l track per cell) of alpha-particle irradiations. The data are consistent with a transmissible genetic instability induced in a stem cell resulting in a diversity of chromosomal aberrations in its clonal progeny many cell divisions later. Recent studies have demonstrated that the instability phenotype persists in vivo and that the expression of chromosomal instability has a strong dependence on the genetic characteristics of the irradiated cell. At the time when cytogenetic aberrations are detected, an increased incidence of hprt mutations and apoptotic cells have been observed in the clonal descendants of (alpha-irradiated murine haemopoietic stem cells. Thus, delayed chromosomal abnormalities, delayed cell death by apoptosis and late-arising specific gene mutations may reflect diverse consequences of radiation-induced genomic instability. The relationship, if any, between these effects is not established. Current studies suggest that expression of these delayed heritable effects is determined by the type of radiation exposure, type of cell and a variety of genetic factors.

Genetic factors influencing alpha-particle-induced chromosomal instability.

Alpha-particle-induced chromosomal instability in haemopoietic cells obtained from the CBA/H, DBA/2 and C57BL/6 inbred strains of mouse has been demonstrated at frequencies dependent on genotype. The CBA/H and DBA/2 strains may be regarded as 'sensitive' and the C57BL/6 strain as 'resistant'; resistance was dominant in cells from F1 hybrids. Previously, in cultures where we demonstrated radiation-induced chromosomal instability we also demonstrated an enhanced and persisting oxyradical activity. Quantitative differences in superoxide generation have now been correlated with genetically determined differences in the expression of chromosomal instability. Our findings demonstrate an important influence of genetic factors in alpha-particle-induced chromosomal instability.

No association between p53 status and alpha-particle-induced chromosomal instability in human lymphoblastoid cells.

Previous work has demonstrated that alpha-particle irradiation of primary human bone marrow cells leads to the transmission of chromosomal instability in the descendants of the irradiated cell, although there is some interindividual variation. We have extended these studies to human EBV-transformed lymphoblastoid cell lines in order to establish an in vitro model system. The five cell lines analyzed, including one from a Fanconi anaemia patient, exhibited high levels of persistent chromatid aberrations up to approximately 40 cell generations after alpha-irradiation. The p53 status of the cell lines was defined according to whether cellular p53 levels were induced by irradiation, translocated to the nucleus and were able to bind a p53 DNA consensus recognition sequence in vitro. Together with the primary bone marrow cell studies, we conclude that alpha-particle induced chromosomal instability is independent of the p53 status of the cell as defined in these studies.

Radiation-induced genomic instability: delayed cytogenetic aberrations and apoptosis in primary human bone marrow cells.

Transmissible chromosomal instability, characterized by non-clonal cytogenetic aberrations with a high frequency of chromatid-type aberrations together with a lower frequency of chromosome-type aberrations, has been demonstrated in the clonal descendants of human haemopoietic stem cells after alpha- but not X-irradiation. Comparable cytogenetic abnormalities have also been demonstrated in non-clonal cultures of alpha-irradiated primary human bone marrow, but a different pattern of delayed aberrations, mainly of chromosome-type, was found after X-irradiation in non-clonal cultures. In clonal analyses, delayed apoptotic cell death was evident after both X- and alpha-irradiation. It is suggested that the type of radiation exposure, the type of cell and its genetically determined susceptibility are factors that may influence the expression of delayed effects of radiation.

Alpha-particle-induced chromosomal instability in human bone marrow cells.

alpha-particles, which are ionising radiation of high linear-energy-transfer emitted, for example, from radon or plutonium, pass through tissue as highly structured tracks. Single target cells in the path of the tracks might be damaged by even low-dose alpha-irradiation. We found non-clonal cytogenetic aberrations, characterised by a high frequency of chromatid aberrations with chromosome aberrations, in clonal descendants of haemopoietic stem cells after exposure to alpha-particles of bone marrow cells from two of four haematologically normal individuals (up to 25% abnormal metaphases). The data are consistent with a transmissible genetic instability induced in a stem cell resulting in a diversity of aberrations in its clonal progeny many cell divisions later.

Transmission of chromosomal instability after plutonium alpha-particle irradiation.

When investigating the biological effects of ionizing radiation on the haemopoietic system, a confounding problem lies in possible differences between the biological effects of sparsely ionizing, low linear energy transfer radiation such as X-, beta- or gamma-rays, and densely ionizing, high linear energy transfer radiation such as alpha-particles. To address this problem we have developed novel techniques for studying haemopoietic cells irradiated with environmentally relevant doses of alpha-particles from a plutonium-238 source. Using a clonogenic culture system, cytogenetic aberrations in individual colonies of haemopoietic cells derived from irradiated stem cells have been studied. Exposure to alpha-particles (but not X-rays) produced a high frequency of non-clonal aberrations in the clonal descendants, compatible with alpha-emitters inducing lesions in stem cells that result in the transmission of chromosomal instability to their progeny. Such unexpected instability may have important implications for radiation leukaemogenesis.

32P-postlabelling analysis and micronuclei induction in primary Chinese hamster lung cells exposed to tobacco particulate matter.

The genotoxicity of tobacco particulate matter (TPM) derived from a low-tar, low-nicotine cigarette has been examined by measuring micronucleus induction in a primary pulmonary cell line, both in the absence and presence of an exogenous source of metabolic activation. In an attempt to correlate the cytogenetic damage observed with DNA adduct formation, DNA extracted from TPM-treated cells has been analysed with two different modifications of the 32P-postlabelling assay. The results from the 32P-postlabelling analysis taken together with the pattern of micronucleus induction provide strong evidence that bioreactivated aromatic carcinogens, such as benzo[a]pyrene, are unlikely to be responsible for the TPM-induced cytogenetic damage observed in cultured mammalian cells.