Effects of harman and norharman on the mutagenicity and binding to DNA of benzo[a]pyrene metabolites in vitro and on aryl hydrocarbon hydroxylase induction in cell culture

Harman and norharman, two β-carboline derivatives known to exist in certain foods and to be formed during pyrolysis of tobacco and meat, were tested for mutagenic activity in the presence of benzo[a]pyrene, mouse liver enzymes, and Salmonella typhimurium TA98 in vitro. Both harman and norharman inhibit benzo[a]pyrene mutagenicity, benzo[a]pyrene metabolism (as measured by aryl hydrocarbon hydroxylase activity), and the binding of all benzo[a]pyrene metabolites to DNA in vitro. Moreover, harman and norharman are quite toxic to cultures of hepatoma-derived H-4-II-E and Hepa-1 established cell lines and therefore were found to be very weak inducers of aryl hydrocarbon hydroxylase activity.     

Effects of 50 Hz magnetic field on cell cycle kinetics and the colony forming ability of budding yeast exposed to ultraviolet radiation.

To investigate the effects of extremely low frequency magnetic fields on ultraviolet radiation (UV) exposed budding yeast, haploid yeast (Saccharomyces cerevisiae) cells of the strain SEy2101a were exposed to 50 Hz sine wave magnetic field (MF) of 120 microT with simultaneous exposure to UV radiation. Most of the UV energy was in the UVB range (280-320 nm). The biologically weighted (CIE action spectrum) dose level for the UV radiation was 175 J/m2. We examined whether 50 Hz MF affected the ability of UV irradiated yeast cells to form colonies (Colony Forming Units, CFUs). In addition, the effect of coexposure on cell cycle kinetics was investigated. Although the significant effect of MF on the cell cycle phases of UV exposed yeast cells was seen only at one time point, the overall results showed that MF exposure may influence the cell cycle kinetics at the first cycle after UV irradiation. The effect of our particular MF exposure on the colony forming ability of the UV irradiated yeast cells was statistically significant 420 min after UV irradiation. Moreover, at 240, 360, and 420 min after UV irradiation, there were fewer CFUs in every experiment in (UV+MF) exposed populations than in only UV exposed yeast populations. These results could indicate that MF exposure in conjunction with UV may have some effects on yeast cell survival or growth.     

Temperature effect on the immune defense functions of Arctic charr Salvelinus alpinus

The Arctic charr Salvelinus alpinus is an endangered fish species in Finland, and thus farming is carried out mainly for stocking purposes. Farmed charr are susceptible to infection with atypical Aeromonas salmonicida (aAS). Losses of valuable brood stock will severely reduce the genetic diversity of stocked charr. No commercial vaccines are available to prevent aAS infection, and vaccines against furunculosis (caused by typical A. salmonicida, tAS) do not protect the charr against aAS infection. The effects of a metabolizable oil-adjuvanted, bivalent vaccine (containing killed aAS and A. salmonicida salmonicida bacteria) on the immune system of 1 yr old hatchery-reared charr originating from Lake Inari in Northern Finland were examined. Fish vaccination in Finland generally takes place either from October to November or from February to April, when the water temperature is low (1 to 3degrees C). The water temperature starts to increase in mid-May. Therefore, we also investigated whether post-vaccination (p.v.) temperature had an influence on the immune system of this cold-water fish species. The fish were immunized intraperitoneally at 2.9 degrees C at the end of April. After 52 d, during which the water temperature increased from 2.9 to 10.0 degrees C, the charr were exposed to 1 of 3 test temperatures: 10.3, 14.1 or 18.1 degrees C. Prior to vaccination, and 49, 75 and 103 d p.v., several immune parameters were measured in both unvaccinated and vaccinated charr. Vaccination induced a significant anti-aAS-specific antibody response, and increased plasma lysozyme activity at all p.v. temperatures. The haemolytic activity of the complement system was unaffected either by vaccination or p.v. temperatures. There was a slight positive correlation between p.v. temperature and lysozyme activity of the charr. The significant increase in lysozyme activity took place in vaccinated charr in the first 49 d p.v. as water temperatures increased from 2.9 to 10 degrees C. Furthermore, the highest activity of lysozyme in the plasma was observed 49 d p.v. Our results indicate that a rise in water temperature above 10 degrees C does not significantly enhance the vaccination response of charr. This could be one reason why farmed Arctic charr, which are well adapted to a cold climate, are highly susceptible to aAS infection in the summer.     

Apoptosis induced by ultraviolet radiation is enhanced by amplitude modulated radiofrequency radiation in mutant yeast cells

The aim of this study was to investigate whether radiofrequency (RF) electromagnetic field (EMF) exposure affects cell death processes of yeast cells. Saccharomyces cerevisiae yeast cells of the strains KFy417 (wild-type) and KFy437 (cdc48-mutant) were exposed to 900 or 872 MHz RF fields, with or without exposure to ultraviolet (UV) radiation, and incubated simultaneously with elevated temperature (+37 degrees C) to induce apoptosis in the cdc48-mutated strain. The RF exposure was carried out in a special waveguide exposure chamber where the temperature of the cell cultures can be precisely controlled. Apoptosis was analyzed using the annexin V-FITC method utilizing flow cytometry. Amplitude modulated (217 pulses per second) RF exposure significantly enhanced UV induced apoptosis in cdc48-mutated cells, but no effect was observed in cells exposed to unmodulated fields at identical time-average specfic absorption rates (SAR, 0.4 or 3.0 W/kg). The findings suggest that amplitude modulated RF fields, together with known damaging agents, can affect the cell death process in mutated yeast cells. Bioelectromagnetics 25:127-133, 2004.     

cAMP mediated upregulation of CYP2A5 in mouse hepatocytes

CYP2A5 is induced by a large number of chemicals including some cAMP modifiers. In a primary hepatocyte model, stimulation of the cAMP signal transduction pathway by glucagon and isoproterenol, acting via specific G-protein coupled plasma membrane receptors, produced up to 17-fold increases in the marker activity of CYP2A5, coumarin 7-hydroxylase. In contrast, glucagon and isoproterenol caused no significant effects on two other major CYP forms, CYP2B10 and CYP1A1/2. Phenobarbital (PB) elicited a 3-fold increase in CYP2A5 expression (catalytic activity and mRNA), while the cAMP and protein kinase A (PKA) stimulators dibutyryl-cAMP, forskolin and Sp-cAMPs caused up to 18-fold increases in the amount of CYP2A5 mRNA. Coadministration of PB and cAMP/PKA stimulating agents produced an additive inducing effect. The expression of CYP2A5, but not CYP2B10 or CYP1A1/2, in DBA/2 mice displayed a marked circadian rhythm, the level of expression being highest in the evening. These results suggest that among xenobiotic metabolizing CYP enzymes, CYP2A5 is uniquely upregulated by cAMP, possibly having the physiological function of priming the olfactory and digestive systems for nocturnal feeding.     

Towards an understanding of apoptosis detection by SYTO dyes.

BACKGROUND: SYTO probes are gaining momentum as reliable and easy to use markers of apoptotic cell death, but the phenomenon underlying reduced SYTO fluorescence in apoptotic cells as compared with normal cells is still not fully elucidated. Herein, we attempt to provide further insights into mechanisms of reduced SYTO16 fluorescence during apoptosis. METHODS: Human follicular lymphoma cell lines were subjected to diverse apoptotic and oncotic stimuli with subsequent multiparametric flow cytometric and fluorescence imaging analysis. SYTO green (SYTO11-16), TMRM, PI, 7AAD, and Hoechst 33342 probes were applied for multivariate analysis of temporal sequence of apoptotic events. Sorting of cells differing in the level of SYTO16 fluorescence and subsequent characterization of obtained subpopulations were also performed. RESULTS: Loss of SYTO16 fluorescence (SYTOlow/PI+ events) has been observed in cells exposed to oncotic stimuli, whereas SYTOhigh/PI+ events did not prevail at any treatment scenario. We tracked similarities and discrepancies between SYTO16 and TMRM probes. Often, SYTO16 and TMRM exhibited the same staining profiles, as loss of their fluorescence was detected in a single cell population. However, both mitochondrial uncoupler FCCP and a small-molecule Bcl-2 inhibitor, HA14-1, appeared to induce distinct staining profiles of SYTO16 and TMRM, with the decrease in TMRM fluorescence preceding the loss of SYTO16 fluorescence. Importantly, in both cases (FCCP and HA14-1) the decrease of SYTO16 fluorescence was blocked by pharmacological inhibition of caspases (with z-VAD-fmk). CONCLUSIONS: The data demonstrate that loss of SYTO16 is caspase-dependent, as is not a mere indicator of Deltapsim dissipation. Commonly observed similarities between SYTO and TMRM may stem from the fast kinetics of apoptotic events once cell death is initiated.     

Nonrandom rearrangement of T cell receptor J alpha genes in bone marrow T cell differentiation cultures

TCR J alpha genes span a distance of approximately 65 kb on mouse chromosome 14. Due to the existence of 50 to 100 discrete J genes, a potential for great diversity exists within the V-J-C alpha gene products and within the ultimate repertoire of alpha beta TCR. We have prepared hybridomas from an in vitro system that supports T cell differentiation among bone marrow cells. We have examined the J alpha genes among these cells and categorized rearrangements according to their location within the J alpha locus. It was found that alpha rearrangements were always present among the hybridomas bearing beta gene rearrangements. When two bone marrow-derived alpha-bearing chromosomes could be demonstrated in these hybridomas, both were always rearranged and rearrangements on homologous chromosomes were shown to reside in similar regions of the J alpha locus. Most surprisingly, when hybridomas were categorized by the culture from which they derived, cells from the same culture (designated as a set) demonstrated a skewing of alpha rearrangements to restricted segments of J alpha genes. In one hybridoma, rearrangements on homologous chromosomes involved J alpha genes that were either identical or situated within a 1-kb segment of DNA. The skewing within sets could not be due to clonal identity between hybridomas as the beta and gamma rearrangements in all hybridomas were different. Results suggested that skewing of J alpha gene rearrangements occurred during the course of T cell development in vitro. Should the same situation occur in vivo, the number of distinct TCR J alpha sequences available for expression in early development may be far less than that predicted by gene number alone.