Mitochondrial function, apoptosis and cell cycle delay in the WEHI-3B leukaemia cell line and its variant Ciprofloxacin-resistant WEHI-3B/CPX.

OBJECTIVE: The susceptibility of two cell lines, WEHI-3B myelomonocytic leukaemia and its variant Ciprofloxacin-resistant WEHI-3B/CPX to undergo apoptosis induced by Ciprofloxacin was studied and compared. MATERIALS AND METHODS: Apoptosis was checked by measuring the DNA fragmentation and determining the ratio of apoptotic/necrotic cells. The relationship between the induction of apoptosis and G(1), S or G(2) block in the cell cycle has also been investigated and cytogenetical evaluation of chromosomal aberrations in both cell lines has been carried out. The regulation of expression of Bax and Bcl-2 was also checked by western blotting after Ciprofloxacin treatment. RESULTS: We observed that the resistance of the subline was caused by a small percentage of cells that underwent apoptosis during continuous exposure to Ciprofloxacin in comparison with the parental cell line, whereas the percentage of necrotic cells remained unchanged. The WEHI-3B cells showed a G(2) block and a higher degree of cytogenetic damage after drug exposure. The two cell lines expressed the same level of Bax and Bcl-2 following stimulation by Ciprofloxacin. Only in the resistant subclone, the ratio Bcl-2/Bax reversed in the anti-apoptotic gene expression. CONCLUSION: The resistance to ciprofloxacin observed is not related to mitochondrial function and although Bcl-2/Bax ratio behaviour does not fully explain the resistance of the WEHI3B/CPX subclone it is consistent with phenotypic character of resistance to CPX. The toxic effect on sensitive cells could be mediated by the cell cycle arrest whereas in the resistant clone, the prolonged G(2) phase could play a key role to favour cell cycle progression and proliferation.

Role of SR-4987 stromal cells in the modulation of doxorubicin toxicity to in vitro granulocyte-macrophage progenitors (CFU-GM).

Bone marrow stromal microenvironment is essential for the maintenance of the hematopoietic stem cell renewal both by cell-cell interaction and cytokine production. However, stromal cells also exhibit drug metabolizing activities and they may accumulate the drug and successively affect hematopoietic progenitors by a retarded release. Our study investigated the role of both primary culture of murine bone marrow stroma and established stromal cells (SR-4987) in modulating the "in vitro" toxic activity of Doxorubicin (DXR) against murine granulocyte-macrophage progenitors (CFU-GM). The main part of the study has been performed by a "in vitro" agar bilayer technique based on the CFU-GM assay performed over a feederlayer of stromal cells. The results suggest that bone marrow stromal cells play also an important role in decreasing the toxicity of Doxorubicin. Further SR-4987 stromal cells produce a Doxorubicin metabolite (not belonging to the series of metabolites described in literature) which is completely ineffective in inhibiting the growth of CFU-GM and the activity of topoisomerase I. Our data suggest that bone marrow stromal cells must be considered as a cell population having opposite pharmacological roles in modulating the drug toxicity on hematopoietic progenitors. In our model a mechanism of detoxification concerns the capacity of SR-4987 stromal cells to inactivate the drug. For a better prediction of drug hematotoxicity, it is very important to develop "in vitro" cell models able to discriminate between positive and negative modulation of drug toxicity that stromal cells can exert in the bone marrow microenvironment.

Modulation of proto-oncogene expression by polychlorinated biphenyls in 3T3-L1 cell line.

The effects of two substituted polychlorinated biphenyls, the 3,4,5,3',4,5' (PCB-169) and the 2,3,4,2',4',5' (PCB-138) forms, were examined on the expression of c-myc, c-jun, c-ras, and jun-b in 3T3-L1 cells. Northern blot analysis demonstrated that the two PCBs, which exhibit a coplanar and di-ortho-substituted configuration, activated these oncogenes differently. PCB-138 markedly induced overexpression of ras, jun, and myc, whereas PCB-169 led to the overexpression of jun-b. High-performance liquid chromatography analysis of the cell samples treated in medium without serum revealed a higher intracellular concentration of the 2,3,4,2',4',5'-hexachlorobiphenyl (hexaCB), whereas the 3,4,5,3',4'5'-hexaCB reached the same concentration in the sonicated samples of cells with or without serum. These results indicated that there was a relationship between PCB structure, bioavailability, and the capacity to stimulate oncogene expression.

Establishment and characterization of a new mammary adenocarcinoma cell line derived from MMTV neu transgenic mice.

A new murine cell line, named MG1361, was established from mammary adenocarcinomas arising in a MMTV-neu transgenic mouse lineage where breast tumors develop in 100% of females, due to the overexpression of the activated rat neu oncogene in the mammary gland. The MG1361 cell line shows an epithelial-like morphology, has a poor plating efficiency, low clonogenic capacity, and a doubling time of 23.8 hours. Karyotype and flow cytometry analysis revealed a hypotetraploid number of chromosomes, whereas cell cycle analysis showed 31.2% of cells to be in the G1 phase, 21.4% in S and 47.4% in G2 + M. This cell line maintains a high level of neu expression in vitro. The MG1361 cell line was tumorigenic when inoculated in immunodeficient (nude) mice and the derived tumors showed the same histological features as the primary tumors from which they were isolated. MG1361 cells were positive for specific ER and PgR binding which was competed by tamoxifen, making this cell line useful for the evaluation of endocrine therapy. Moreover, they were sensitive to etoposide treatment, suggesting that they could be a model for the study of chemotherapy-induced apoptosis. As the tumors arising in MMTV-neu transgenic mice have many features in common with human mammary adenocarcinomas (Sacco et al., Gene Therapy 1995; 2: 493-497), this cell line can be utilized to perform basic studies on the role of the neu oncogene in the maintenance of the transformed phenotype, and to test novel protocols of therapeutic strategies.

DNA damage induced by the environmental carcinogen butadiene: identification of a diepoxybutane-adenine adduct and its detection by 32P-postlabelling.

To date only a few studies have been undertaken on DNA adducts formed by epoxybutene (EB) and diepoxybutane (DEB), the two active metabolites of 1,3-butadiene. Our interests have focused on further investigating DNA alkylation by the two epoxides, especially in relation to the development of a method for human biomonitoring. Here, following the reaction of deoxyadenosine monophosphate and poly(dA-dT)(dA-dT) with DEB and subsequent HPLC, we have identified an adenine adduct. MS analyses indicate the structure of an adenine adducted by DEB at the N6 position. A HPLC/32P-postlabelling method was developed for its measurement in DNA samples and the adduct was detected in calf thymus DNA and DNA from Chinese hamster ovary cells exposed to DEB. The 100% labelling efficiency during postlabelling, the amount of the adduct and its elution before the normal nucleotides during HPLC suggest it could be a suitable indicator of BUT exposure.

Detection by HPLC-32P-postlabelling of DNA adducts formed after exposure to epoxybutene and diepoxybutane.

We have developed an HPLC-32P-postlabelling procedure to detect DNA adducts formed by epoxybutene and diepoxybutane. The method exploits the interaction of the two epoxides with deoxynucleotides and polydeoxynucleotides to optimize the HPLC enrichment of adducted nucleotides before 32P-postlabelling. Using this approach, a number of guanine adducts were identified after the exposure of dGMP, poly(dG-dC) or calf thymus DNA to epoxybutene and diepoxybutane, and a major adenine adduct was identified in poly(dA-dT) and calf thymus DNA exposed to diepoxybutane.

Biomonitoring of exposure to 1,3-butadiene: detection by high-performance liquid chromatography and 32P-postlabelling of an adenine adduct formed by diepoxybutane.

1,2-Epoxy-3-butene and 1,2:3,4-diepoxybutane, the two oxidative metabolites of 1,3-butadiene, are considered to be involved in some of the carcinogenicity of the parent compound. Diepoxybutane is a bifunctional alkylating agent and reacts with DNA to form monoadducts and cross-links. We investigated DNA alkylation after exposure to diepoxybutane in order to develop a method for human biomonitoring. After reacting dAMP and then poly(dA-dT) (dA-dT) with diepoxybutane, we identified a major adenine adduct. Preliminary mass spectrometry indicated an adenine adducted by diepoxybutane at the N6 position. A high-performance liquid chromatography/32P-postlabelling method was developed, and the adduct was detected in calf thymus DNA and in DNA from Chinese hamster cells after exposure to diepoxybutane. The labelling efficiency, the amount of the adduct and its stability suggest that it could be a suitable indicator of exposure to butadiene.

Intracellular distribution and chemical forms of arsenic in rabbits exposed to arsenate.

Inhibition of the methylation of arsenic in rabbits by ip injection of periodate-oxidized adenosine (PAD) prior to an iv injection of 74As-arsenate (AsV; 0.4 mg As/kg body wt) caused a marked increase in the retention of 74As in both the cellular organelles and the soluble fractions of liver and kidney. One day after exposure, almost 30% of the arsenic in the liver and about 40% of the arsenic in the kidney was recovered in the nuclear fraction. In the liver nuclei, the inhibition of the methylation increased the 74As content of the insoluble fraction and most of this arsenic was protein-bound. The major part of the soluble intranuclear 74As was in the form of AsIII, formed by reduction of the administered AsV. In the liver, PAD also caused a pronounced increase in the 74As content of the microsomal fraction. In the kidneys, where most of the arsenic was present as AsV, there was a marked accumulation of arsenic in the mitochondria.

Dissolution of two arsenic compounds by rabbit alveolar macrophages in vitro.

The ability of rabbit alveolar macrophages to dissolve two arsenic compounds, 74As-labeled lead arsenate and arsenic trisulfide, was studied in vitro. The solubilities in water of these two compounds are related differently to pH. The solubility of lead arsenate increases and that of arsenic trisulfide decreases with decreasing pH. The radiolabeled particles were incubated with and without macrophages for up to 3 days, whereafter the amount of 74As in soluble form and the amount in particle form and/or bound to macrophages were determined. The results strongly support the hypothesis that the dissolution of particles by macrophages is influenced by the acid milieu in the phagosomes. About 14% of the 74As-labeled lead arsenate particles incubated for 3 days with the macrophages was released into the culture medium, compared with about 2% of the particles incubated with the culture medium without macrophages. With the arsenic trisulfide particles, less soluble 74As was released into the medium in samples with macrophages than in samples without macrophages, although the solubility in all incubations was considerably greater than that for lead arsenate. The results indicate that dissolution in the phagosomes of the macrophages may be of great importance for the clearance of particles such as lead arsenate, which are more soluble at pH 4 than at pH 7.

The role of the methylation in the detoxication of arsenate in the rabbit.

The biotransformation, tissue retention, intracellular binding and biokinetics of arsenic were studied in rabbits exposed to [74As]arsenate (0.4 mg As/kg body wt., i.v.). Inhibition of the methyltransferase activity by injection of periodate-oxidized adenosine (PAD) caused a marked decrease of the formation of [74As]dimethylarsinic acid (DMA), which gave rise to 1.5-4 times increased tissue levels of 74As. This is almost the same as reported for rabbits given arsenite in combination with PAD and was due to a rapid reduction of the arsenate to arsenite which bound to the tissues. Only about 30% of the arsenate given was excreted unchanged in the urine, indicating that a large part was reduced to AsIII. Thus the methylation to DMA seems to be almost as important for the detoxication following exposure to arsenate as that following exposure to arsenite. In the rabbits with normal methylating capacity 50-70% of the produced AsIII was methylated to DMA. The liver was the only organ in which DMA was present 1 h after the administration, indicating that this is the main site of the methylation. The DMA was rapidly cleared from all tissues except the thyroid.

Metabolism in the rat of cadmium biocomplexes from edible mussel exposed to 109CdCl2.

The metabolism in the rat of 109Cd biocomplexes present in the tissues of the edible mussel Mytilus galloprovincialis, previously exposed to 109CdCl2, was studied. The tissue distribution and binding of 109Cd were compared to those caused by an equal dose of 109Cd as CdCl2 or rat liver Cd-metallothionein. Administration of mussel 109Cd to rats resulted in an initial accumulation of 109Cd in the kidneys due to the presence of 109Cd-metallothionein, which constituted 25% of the 109Cd in the tissues of the mussels. Other 109Cd biocomplexes present in the mussel tissues were metabolized in the rat in a way similar to that of inorganic cadmium, i.e., initial accumulation in the liver. These findings indicate that the ingestion of seafood rich in metallothionein may give rise to a faster increase of renal cadmium levels than the consumption of a similar amount of inorganic cadmium.

The effect of methyltransferase inhibition on the metabolism of [74As]arsenite in mice and rabbits.

The effect of periodate-oxidized adenosine (PAD), an inhibitor of certain methyltransferases, on the biotransformation and tissue retention of [74As]arsenite in mice and rabbits was studied. Injection of PAD (100 mumol/kg body wt.), 15-min prior to the injection of [74As]arsenite (0.4 mg As/kg body wt.), resulted in a 25-70% decrease in the production of [74As]dimethylarsinic acid ( [74As]DMA). This implies that S-adenosylmethionine is the methyl-donor in the methylation of inorganic arsenic in vivo. Due to interaction of the unmethylated arsenite with tissue constituents the PAD-treated animals had significantly higher (2-6 times) tissue concentrations of 74As than did the controls. This effect was first observed in the liver, indicating that this organ is the main site of the methylation of arsenic. The increase in the tissue retention due to the PAD-treatment remained also after cessation of the inhibition of methylation. The results can be seen as confirmation that alkylation of inorganic arsenic acts as a detoxification mechanism in mammals.

Relations between iron and vanadium metabolism: in vivo incorporation of vanadium into iron proteins of the rat.

In vivo experiments with 48V and 59Fe radiotracers were performed to study the association of V with Fe proteins. Each male rat was injected ip with 10 micrograms 48VO2+ and then with 1 microgram 59Fe3+ to label Fe-containing proteins. The radioactivities incorporated were measured in plasma transferrin, red blood cell hemoglobin, liver ferritin, partially purified heart myoglobin, and liver mitochondrial and microsomal cytochromes b and c and ferriporphyrin. Liver ferritin can bind V in vivo similarly to plasma transferrin, as shown by gel filtration and immunoprecipitation. Negligible amounts of 48 V were incorporated into hemoglobin, partially purified myoglobin, and cytochromes b and c. These findings suggest that the nonenzymatic Fe-containing proteins may be involved in the V metabolism.

Cadmium toxicity studies under long term-low level exposure (LLE) conditions. I. Metabolic patterns in rats exposed to present environmental dietary levels of Cd for two years.

A long term-low level exposure (LLE) experiment was conducted on rats to determine the metabolic patterns for realistic environmental dietary levels of cadmium. Male rats fed with 61 ppb of cadmium ad libitum, 50 labelled with 109 Cd radiotracer as cadmium chloride via drinking mineral water and 11 unlabelled via food for 2 years. The diet was characterized in its metal content by neutron activation analysis to obtain the total dietary intake of different elements. The kidney was found to be the tissue with the major concentration of cadmium which accumulated continuously during the experiment. The variation of the accumulation pattern of Cd concentration in the liver and intestine indicated an itiial rapid increase of Cd during the first 100 days. After this period an apparent equilibrium was attained in both these tissues until the end of the study. The intracellular distribution of cadmium in kidneys, liver, intestine and pancreas were similar, the cytosol fractions containing about 80% of the cellular cadmium. Dialysis experiments indicated that significant amounts of cadmium were able to be associated with cellular organelles, the mitochondria representing the most important organelle capable of binding cadmium. The cytoplasmatic Cd-profiles obtained at various stages of the experiment showed that the metal was only bound to a low-molecular-weight component, cadmium-binding protein (CdBP), which represents the specific cellular-binding component for cadmium under the long term-low level exposure (LLE) conditions. No significant variations in the concentrations of the elements in different organs were observed in animals supplemented with 109Cd untreated controls.

Accumulation of cadmium in rat liver cadmium binding protein following single and repeated cadmium administration.

The accumulation of cadmium in rat liver cadmium binding protein induced by single and repeated intraperitoneal injections of CdCl2 and the de novo biosynthesis of CdBP were studied by using 109Cd to measure cadmium binding in the CdBP and 35S incorporation as indicator of protein synthesis. The biosynthesis of CdBP is controlled by the cadmium concentrations. For single doses up to 1 mg Cd2+/Kg b.w. about 50% of the cadmium is present in the soluble fraction of liver bound to CdBP and the incorporation of 35S-cysteine is linear with the cadmium concentration. When single doses ranging from 1 to 3 mg Cd2+/Kg b.w. are administered the fractions of both 35S-cysteine and cadmium incorporated into de novo synthesized CdBP gradually decrease. For single doses higher than 3 mg Cd2+/Kg b.w. the biosynthesis capability is maximum and 20 mug Cd/g liver can be incorporated into the de novo biosynthesized CdBP. When rats are treated every day with amounts of cadmium of about 0.8 mg Cd2+/Kg b.w. for up to 8 days a dose-proportional increase in both Cd incorporation and CdBP biosynthesis are observed. This shows a cumulative incorporation of cadmium in the de novo biosynthesized CdBP. Experiments carried out by injecting 65ZnCl2 and 203HgCl2 every day showed that they are not accumulated like cadmium and do not induce the biosynthesis of rat liver CdBP after repeated administration over 7 days.

Heavy metals in rat liver cadmium binding protein.

The simultaneous determination of heavy metals in microsamples of chromatographically isolated cadmium-binding protein (Cd-BP) from rat liver was performed by neutron activation analysis. The results suggested that metals other than those already reported (Cd, Zn, Cu, and Hg) can bind the protein. These observations were confirmed by in vivo radiotracer experiments by injecting i.p. 21 labelled metal ions in cadmium-treated rats. Of the metals tested, 109Cd, 65Zn, 64Cu, 203Hg, 106Ag and 113Sn were found incorporated in the Cd-BP. The incorporation of 35S-cysteine, used as an indicator of Cd-BP biosynthesis, was increased in rats exposed to cadmium as compared to untreated animals. In order to establish the influence of other metal ions on the biosynthesis of Cd-BP and the incorporation of cadmium in the protein, in vivo experiments were carried out by i.p. injection of 109Cd and 35S-cysteine. In the presence of 42 metal ions no influence was observed on the incorporation of the two radioisotopes in the Cd-BP. These observations tend to support the hypothesis that cadmium can act as a highly specific inducer of Cd-BP and that this protein might be involved in the metabolism of several heavy metals.