CaNa2EDTA chelation attenuates cell damage in workers exposed to lead--a pilot study.

Lead induced oxidative cellular damage and long-term persistence of associated adverse effects increases risk of late-onset diseases. CaNa2EDTA chelation is known to remove contaminating metals and to reduce free radical production. The objective was to investigate the impact of chelation therapy on modulation of lead induced cellular damage, restoration of altered enzyme activities and lipid homeostasis in peripheral blood of workers exposed to lead, by comparing the selected biomarkers obtained prior and after five-day CaNa2EDTA chelation intervention. The group of smelting factory workers diagnosed with lead intoxication and current lead exposure 5.8 ± 1.2 years were administered five-day CaNa2EDTA chelation. Elevated baseline activity of antioxidant enzymes Cu, Zn-SOD and CAT as well as depleted thiols and increased protein degradation products-carbonyl groups and nitrites, pointing to Pb induced oxidative damage, were restored toward normal values following the treatment. Lead showed inhibitor potency on both RBC AChE and BChE in exposed workers, and chelation re-established the activity of BChE, while RBC AChE remained unaffected. Also, genotoxic effect of lead detected in peripheral blood lymphocytes was significantly decreased after therapy, exhibiting 18.9% DNA damage reduction. Administration of chelation reversed the depressed activity of serum PON 1 and significantly decreased lipid peroxidation detected by the post-chelation reduction of MDA levels. Lactate dehydrogenase LDH1-5 isoenzymes levels showed evident but no significant trend of restoring toward normal control values following chelation. CaNa2EDTA chelation ameliorates the alterations linked with Pb mediated oxidative stress, indicating possible benefits in reducing health risks associated with increased oxidative damage in lead exposed populations.

Review: cell cycle aberrations and neurodegeneration.

The cell cycle is a highly regulated and fundamental cellular process that involves complex feedback regulation of many proteins, and any compromise to its integrity elicits dire consequences for the cell. For example, in neurodegenerative diseases such as Alzheimer disease (AD), evidence for abnormal cell cycle re-entry precedes other hallmarks of disease and as such, implicates cell cycle aberrations in the aetiology of AD. The mechanism(s) for cell cycle re-entry in AD, however, remain unclear. Current theory suggests it to be part of a combination of early events that together elicit the degenerative pathology and cognitive phenotype consistent with the disease. We propose a 'Two-Hit Hypothesis' that highlights the concerted interaction between cell cycle alterations and oxidative stress that combine to produce neurodegeneration. Here, we review the evidence implicating cell cycle mechanisms in AD and how such changes, especially in combination with oxidative stress, would lead to a cascade of events leading to disease. Based on this concept, we propose new opportunities for disease treatment.

Cytogenetic effects of 8-Cl-cAMP on human and animal chromosomes.

PURPOSE: To assess the cytogenetic effects in vitro and in vivo of a non-cytotoxic antitumor agent with biomodulator activity, 8-chloro-3',5' cyclic adenosine monophosphate (8-ClcAMP). MATERIALS AND METHODS: Cytogenetic effects of 8-Cl-cAMP where evaluated using the in vitro chromosome cytogenetic assay (CA) on human peripheral blood lymphocytes of healthy individuals and by bone marrow micronucleus assay in adult BALB/c mice. RESULTS: In the in vitro chromosome CA, 8-Cl-cAMP (in all respective doses; 1.5 and 15 microm) induced mitotic inhibition and premature centromere separation (PCS) but no chromosomal damage in cultured human peripheral blood lymphocytes. In the in vivo test, single intraperitoneal (i.p.) injection of 8-Cl-cAMP in doses of 10, 80 and 150 mg/kg showed a dose-related effect on the frequency of micronuclei, detected in murine polychromatic erythrocytes (PCE). CONCLUSION: The results of the present study show that genotoxicity of 8-Cl-cAMP has a different matrix of response when comparing results in vitro and in vivo, suggesting that high metabolic activity in vivo is responsible for the clastogenic potential of 8-Cl-cAMP. These comparative results indicate a need of having an available battery of genotoxic tests in order to evaluate possible cytogenetic effects of novel antitumor agents.

A study on the genotoxic effects of 8-Cl-cAMP on human lymphocytes in vitro.

8-chloro-cyclic adenosine 3',5'-monophosphate (8-Cl-cAMP) is the most potent cAMP analogue that selectively inhibits a variety of cancer cell lines in vitro and tumors in vivo. Its action toward a variety of tumors, especially when coupled with other antitumor agents, have lead to phase I clinical investigations and recently phase II clinical investigations. Until today very little was done to evaluate its genotoxic potential. In order to evaluate its genotoxic potential we used the cytogenetic and cytokinesis block micronucleus assay in vitro on peripheral blood lymphocytes of healthy individuals. Using three concentrations (1 microM, 5 microM and 15 microM), 8-Cl-cAMP in normal human peripheral blood lymphocytes did not induce any cytogenetic aberrations of the structural type [chromatid breakage, isochromatid breakage and gaps], but did induce premature centromere separation (PCS) in all respective doses and increased the frequency of micronuclei (p <0.05) only in the highest dose (15 microM). Antiproliferative action of 8-Cl-cAMP was estimated by using the cytokinesis block nuclear division index (NDI). The results showed a decrease in the NDI of cells exposed to all doses of 8-Cl-cAMP when compared to control. Therefore, the overall results show a genotoxic potential of 8-Cl-cAMP in peripheral blood lymphocytes in vitro.

Deregulated sequential motion of centromeres induced by antitumor agents may lead to genome instability in human peripheral blood lymphocytes.

PURPOSE: Segregation of chromosomes in anaphase is preceded by a sequential order of centromere separation. Alteration of the sequence of centromere separation or premature centromere division (PCD) has been found to be significantly higher in populations exposed to various xenobiotics. The purpose of this study was to investigate if PCD induced by various cytostatics can alter the stability of chromosomes and lead to aneuploidy. MATERIALS AND METHODS: Peripheral blood lymphocytes of 10 healthy, non smoking subjects were exposed to 8-Cl-cAMP at a dose of 1, 5 and 15 microM, paclitaxel at a dose of 0.01, 0.05 and 0.2 microM, and cycloheximide (CX) at a dose of 5, 10 and 25 microg/ml. By using the cytohalasin B (CB)-micronucleus (MN) test in vitro, in combination with fluorescent in situ hybridization (FISH), the presence of MN was analyzed in 1000 binuclear cells for each experimental and negative control group. For analysis of MN content we used the alpha-centromeric probe for chromosome 18. RESULTS: 8-Cl-cAMP and paclitaxel induced an increase in the frequency of MN in peripheral blood lymphocytes. 8-Cl-cAMP and paclitaxel proved clastogenic, i.e. they increased the frequency of MN and induced PCD in all respective doses. CX proved not clastogenic in the respected doses when using the CB-MN test in vitro, although CX is a specific PCD inducer. No correlation of PCD and aneuploidy of chromosome 18 was found in cells exposed to 8-Cl-cAMP and paclitaxel by using FISH. In cells exposed to CX we found PCD of chromosome 18 in binuclear cells and single signals in scarce MN. These findings were not statistically significant compared to the negative control group. CONCLUSION: Our results show that the properties of the investigated antitumor agents to induce PCD in peripheral blood lymphocytes and, therefore, aneuploidy and genome instability, is highly based on the nature of the alteration of centromere function, i.e. the temporal order of centromere kinetics are more regulated through the sequence of centromere separation than by the segregation processes. We suggest that PCD induced by novel antitumor agents could be included in preclinical and clinical genetic risk assessment analysis.

A negative adaptive response is expressed in peripheral blood lymphocytes that are exposed to mitomycin C and cycloheximide.

PURPOSE: Mitomycin C (MMC) and cycloheximide (CHX) are known for their apoptotic and antitumor activity. CHX is also known for its property to inhibit protein synthesis and to reduce cytotoxicity of various antitumor drugs, i.e. inducing an adaptive survival response (ASR). The purpose of this study was to evaluate the effect of ASR induced by CHX in cells exposed to clastogenic doses of MMC. MATERIALS AND METHODS: In all experiments we used human peripheral blood lymphocytes of 10 healthy male non-smokers, 25-35 years of age. Three groups were established. One control group of PBS-treated group. Two distinctive experimental groups were based on the induction or non-induction of ASR by CHX, i.e. one with MMC alone and a second one with CHX and MMC. The effect of ASR was induced by CHX at a dose of 10 mug/ml. MMC was used in 3 dose levels: 0.05 muM, 0.15 muM and 0.6 muM. To evaluate ASR induced by CHX in cells exposed to MMC we used the cytokinesis-blocked micronucleus test (CBMN) in vitro. RESULTS: CHX at a dose of 10 mg/ml induced an ASR in human peripheral blood lymphocytes of healthy subjects exposed to increasing doses of MMC. CHX induced statistically highly significant difference (p < 0.001) in the nuclear division index (NDI) compared to cells exposed to MMC alone. Genotoxicity of MMC measured by the percentage of micronuclei in binuclear (BN) cells was not elevated in the presence of CHX. Also, the increase in the NDI was correlated with the decrease in nuclear fragmentation (NF). CONCLUSION: The observed differences in NF and the NDI between the two groups showed that ASR to MMC induced by CHX could be a consequence of inhibition of apoptosis. We argue that adaptation (pro-life processes) can overwhelm its positive aspects (antimutagenic and anticarcinogenic) by increasing the population of cells with chromosome aberrations (chromosome instability) by apoptotic inhibition. CHX disturbs the apoptotic signal. Understanding that ASR can act as a pro-survival process leading to inhibition of apoptosis shall enhance in the future our knowledge of anticarcinogenesis, thus utilizing new paths for better treatment of cancer.