Effects of polyamines on the thermal stability and formation kinetics of DNA duplexes with abnormal structure.

The effects of ions (i.e. Na+, Mg2+ and polyamines including spermidine and spermine) on the stability of various DNA oligonucleotides in solution were studied. These synthetic DNA molecules contained sequences that mimic various cellular DNA structures, such as duplexes, bulged loops, hairpins and/or mismatched base pairs. Melting temperature curves obtained from the ultraviolet spectroscopic experiments indicated that the effectiveness of the stabilization of cations on the duplex formation follows the order of spermine > spermidine > Mg2+ > Na+ > Tris-HCl buffer alone at pH 7.3. Circular dichroism spectra showed that salts and polyamines did not change the secondary structures of those DNA molecules under study. Surface plasmon resonance (SPR) observations suggested that the rates of duplex formation are independent of the kind of cations used or the structure of the duplexes. However, the rate constants of DNA duplex dissociation decrease in the same order when those cations are involved. The enhancement of the duplex stability by polyamines, especially spermine, can compensate for the instability caused by abnormal structures (e.g. bulged loops, hairpins or mismatches). The effects can be so great as to make the abnormal DNAs as stable as the perfect duplex, both kinetically and thermodynamically. Our results may suggest that the interconversion of various DNA structures can be accomplished readily in the presence of polyamine. This may be relevant in understanding the role of DNA polymorphism in cells.

In vivo effects of ascorbate and glutathione on the uptake of chromium, formation of chromium(V), chromium-DNA binding and 8-hydroxy-2'-deoxyguanosine in liver and kidney of osteogenic disorder shionogi rats following treatment with chromium(VI).

Several previous in vitro studies have indicated that ascorbate and glutathione are the major reductants of Cr(VI) in cells. In order to evaluate the in vivo effects of ascorbate and glutathione on Cr(VI)-induced carcinogenesis, Cr uptake and the formation of Cr(V), Cr-DNA adducts and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) were measured in the liver and kidney of Osteogenic Disorder Shionogi (ODS) rats that lack the ability to synthesize ascorbate. Despite a 10-fold difference in tissue ascorbate levels among different dietary ascorbate groups, the Cr(V) signal intensity, Cr uptake and total Cr-DNA binding were not affected in either organ. Treatment of ODS rats with Cr(VI) (10 mg/kg) had no substantial effect on the levels of ascorbate and glutathione in these tissues. The levels of Cr(V) and Cr-DNA binding were approximately 2-fold higher in the liver than in the kidney, although the levels of total Cr uptake were similar in both tissues. Cr uptake levels were significantly lower in the liver and kidney of ODS rats treated with high levels of ascorbate and a high dose of Cr(VI) (40 mg/kg), suggesting a detoxifying role played by plasma ascorbate. Similarly, modulation of glutathione levels by N-acetyl-L-cysteine, L-buthionine-S, R-sulfoximine or phorone in these animals by up to 2-fold had little or no consistent effect on Cr uptake, Cr-DNA binding, Cr(V) levels or 8-OH-dG formation in either organ. One possible explanation is that reduction of ascorbate and glutathione concentration to <10 and 50%, respectively, of normal in these two organs still provides threshold levels of these two reductants that are in excess of what is needed for significant reductive activation of Cr(VI). Alternatively, it is possible that ascorbate and glutathione do not play a major role in the formation of Cr(V), Cr-DNA binding or 8-OH-dG and that other cellular reductants, such as cysteine or other amino acids, might be more important reductants of Cr(VI) in vivo.

Determination by photoreduction of flip-flop kinetics of spin-labeled stearic acids across phospholipid bilayers.

Spin-labeled stearic acid derivatives (N-DS) can be used to determine the rate at which lipid-derived drugs can cross a phospholipid bilayer (flip-flop). The flip-flop rate of N-DS (where N=5, 6, 7, 9, 10, 12, 16), was measured using vectorial photoreduction of nitroxides to their corresponding hydroxylamine by FMN, a charged, membrane-impermeable flavin, by hydrogen atom transfer from EDTA. From the time difference in the photoreduction rates of N-DS located in the outer and inner half of the bilayer, the flip-flop rate of N-DS across the bilayer can be determined. The results show that at pH 8.0 or lower, the photoreduction of 5-DS on one side of the membrane by FMN is slower than the flip-flop rate of 5-DS across phospholipid bilayers. For 5-DS at pH 7.0, this rate is at least 33.8+/-4.24 s or faster. Stearic acids with the spin label at different positions along the acyl chain (N=5, 6, 7, 9, 10, 12) have similar flip-flop rates in the liposomes at pH 7.0 although 16-DS is slower, probably due to the inaccessibility of the nitroxide moiety to FMN. It is most likely that the fast distribution of 5-DS in cells is due to the fast movement of acidic form, but not the salt form, of 5-DS across membrane bilayers. The oxazolidine (nitroxide moiety) does not seem to affect the pKa ( approximately 8.3) of stearic acid at air-water interface. Thus, N-DS are good probes for studying the distribution kinetics of stearic acid derivatives in biological systems.

Kinetics of distribution of 5-doxylstearic acid in mouse lymphocytes.

The rate at which stearic acid nitroxide spin labels distribute through cells affects the interpretation of data obtained from these nitroxides. We used photoreduction of 5-doxylstearic acid (5DS) to determine the rate at which 5DS arrives at the outer half of the plasma membrane of mouse lymphocytes and macrophages. Our results show that 5DS is in equilibrium with the outer half of the plasma membrane of mouse lymphocytes over a time frame of 2 minutes or less. Thus, spectra data obtained from 5DS-labelled cells clearly reflects the spectrally averaged environment of all the cell membranes in lymphocytes and potentially other cells as well. This clarifies the observation that the spectral information obtained from doxylstearic acid nitroxides is relatively insensitive to environmental changes which would be expected to involve only the plasma membrane of the cell.