Minimizing tocopherol-mediated radical phase transfer in low-density lipoprotein oxidation with an amphiphilic unsymmetrical azo initiator.

The antioxidant alpha-tocopherol (alpha-TOH) has been found to act as a pro-oxidant under many in vitro conditions. The observed tocopoherol-mediated peroxidation (TMP) is dependent on two primary factors. (1) Chain transfer: alpha-TO. radical reacts with lipid to form lipid peroxyl radicals. (2) Phase transfer: alpha-TOH can transport radical character into the lipoprotein. Given the limitations of existing initiators, there is a need for new compounds that avoid the requirement for alpha-TOH to act as a phase-transfer agent. We report here a study showing that the new unsymmetrical azo compound, C-8, initiates LDL lipid peroxidation without requirement for alpha-TOH. This initiator provides a steady source of free amphiphilic peroxyl radicals that efficiently initiates oxidation of alpha-TOH-depleted LDL at a rate comparable to that reported for the very reactive hydroxyl radical (.OH). With other initiators tested, unsymmetrical C-12 and C-16 and symmetrical C-0 and MeOAMVN, alpha-TOH-depleted LDL displayed significant resistance to oxidation. Results indicate that the amphiphilic nature of the unsymmetrical initiators increases their partitioning into lipoprotein depending on the hydrocarbon chain length, and the symmetrical azo initiators C-0 and MeOAMVN primarily remain in the aqueous phase. Evidence suggests that even when the phase-transfer activity of alpha-TOH is limited, with the use of an initiator such as C-8, the mechanism of peroxidation remains controlled by TMP chain-transfer activity.

Design of unsymmetrical azo initiators to increase radical generation efficiency in low-density lipoproteins.

Lipid peroxidation studies often employ the use of azo initiators to produce a slow, steady source of free radicals, but the lack of initiators capable of efficiently generating radicals in lipid regions has created persistent problems in these investigations. For example, experiments with symmetrical lipophilic or symmetical hydrophilic azo initiators increasingly suggest that their initiation mechanisms in low-density lipoproteins (LDL) rely upon the presence of alpha-tocopherol to mediate peroxidation. We report here the synthesis and study of the new unsymmetrical azo compounds SA-1, SA-2, C-16, C-12, and C-8 that decompose over a range of convenient temperatures and improve radical generation efficiency and access to lipid compartments. The half-life for decomposition (tau(1/2)) of the unsymmetrical initiators at 37 degrees C in methanol covered a range of 121 hours for SA-1, 77 hours for SA-2, and approximately 25 hours for the series C-16, C-12, and C-8. Agarose gel electrophoresis of LDL incubated with these unsymmetrical initiators supports the conclusion that the initiators associate with lipoprotein without disrupting integrity of the particle. The unsymmetical initiator C-8 when compared to symmetical hydrophilic initiator C-0 is capable of providing increased peroxidation of LDL, as monitored by formation of cholesteryl linoleate oxidation products and consumption of alpha-tocopherol. Efficiency of radical generation in lipophilic and hydrophilic compartments was found to be represented with the use of the radical scavenger combination alpha-tocopherol and uric acid, but not with the use of N,N'-Diphenyl-p-phenylenediamine (DPPD) and uric acid. These unsymmetrical initiators, when compared to the widely used symmetrical azo initiators, provide an advantage of free radical production, lipophilic access, and constant radical generation in the investigation of lipid peroxidation in low-density lipoproteins.

Comparison of sensitivities of cells to X-ray therapy, chemotherapy, and isotope therapy using a tumor spheroid model.

Numerous treatment modalities have been used in treating microscopic carcinoma confined to the peritoneal cavity. However, there is little information at the cellular level regarding which modality is most effective in eradicating small nests of tumor cells. In order to determine whether chemotherapy, X rays, or isotope therapy is most effective, we have compared in a three-dimensional spheroid model the survival of cells after treatment with cis-platinum, X ray, or the investigational high-energy isotope 212-bismuth (Bi-212). In this study, V-79 cells were grown to 100-microns diameter spheroids. In killing spheroids, high-energy isotopes were at least six times more effective than X rays at fractionated doses of up to 1300 cGy. High-energy isotopes appear also to be as efficient as cis-platinum. Regardless of exposure time to treatment, Bi-212 was more toxic to spheroids than the other treatment modalities. From this study we conclude that high-energy isotope therapy biologically is very effective in eradicating microscopic nests of cells.

Choice of a terminating over a non-terminating signal in free-operant avoidance.

Rats chose between two signalled avoidance schedules. Under one schedule, responses in the presence of the signal terminated it and resulted in avoidance of shock; in the other, responses in the presence of the signal resulted in shock avoidance but signal termination was delayed for 10 sec. Pressing a second (changeover) lever produced change from one schedule to the other for 1 min. Once this 1-min period timed out, subjects could remain under the schedule in effect or could reinstate the other schedule for another 1-min period. All four subjects continuously changed over from the non-terminating to the terminating signal schedule. Changeover responding was not maintained when the termination contingency was removed. When changeover responding resulted in a change from a terminating to a non-terminating signal schedule, changeover responding did not occur.

Choice of longer or stronger signalled shock over shorter or weaker unsignalled shock.

Unsignalled, inescapable shocks were presented to four albino rats in one study and to six rats in a second study. By pressing a lever, subjects could change the condition to signalled shock for 3 min after which unsignalled shock was automatically reinstated. All subjects changed frequently to the signalled shock schedule. After a minimum of three 6-hr sessions or after changeover responding stabilized at the previous values, higher values of signalled shock intensity or duration were introduced. In the first study, the duration of signalled shock was increased in increments of 0.5 sec. In the second study, the intensity of signalled shock was increased in increments of either 0.2 or 0.4 mA. Duration subjects chose signalled shock four (2.0 sec) to nine times (4.5 sec) longer than unsignalled shock (0.5 sec). Intensity subjects chose signalled shock two (2.0 mA) to three times (3.0 mA) more intense than unsignalled shock (1.0 mA).

The relative aversiveness of signalled vs unsignalled escapable and inescapable shock.

In the first study, subjects escaped shock by pressing on a lever under an unsignalled condition. By pressing a different lever they changed the condition to signalled escape for three minute periods. The second study used the changeover procedure to study inescapable-unavoidable shock. Seven rats were used in each study. All subjects in both studies changed over from unsignalled to signalled conditions. Once contact with the signal condition was made, subjects responded to remain in that condition. The three different extinction conditions showed that the correlated stimulus without the signal had greater control over responding than the signal without the correlated stimulus. An analysis based upon shock and shock-free periods was presented.

The relative aversiveness of signalled vs unsignalled avoidance.

Subjects avoided shock by pressing on one lever under an unsignalled condition, but by pressing a separate lever they changed the condition to signalled avoidance for 1-min periods. Signalled avoidance periods were identified by a correlated stimulus. All eight subjects responded to change the unsignalled schedule to a signalled one. Once contact with signalled avoidance was made, subjects continued responding to remain in that condition. Other tests showed that changeover responding was greater when the correlated stimulus was presented without the signal than when the signal was presented without the correlated stimulus. An analysis based upon shock and shock-free periods is presented.