Mark J. Poznansky

DUAL ROLE OF MELANINS AND MELANIN PRECURSORS AS PHOTOPROTECTIVE AND PHOTOTOXIC AGENTS: INHIBITION OF ULTRAVIOLET RADIATION‐INDUCED LIPID PEROXIDATION

Ultraviolet radiation (UVR) is one of the risk factors for skin cancer and the main inducer of melanin pigmentation, the major protective mechanism of mammalian skin against radiation damage. The melanin pigments, eumelanin and pheomelanin, are likely to be important in protection against UVR, but their precursors are generally considered as phototoxic. The available data suggest DNA damage as the mechanism of phototoxicity. However, the effect of melanin precursors on membrane damage through lipid peroxidation, another important and probably more relevant (from the point‐of‐view of the melanosomal confinement of these molecules) mechanism of phototoxicity, is not known. As a model system for UVR–melanin–membrane interactions, we irradiated liposomes in the presence of eumelanin, pheomelanin and two of their major precursors, 5,6‐dihydroxyindole (DHI) and 5‐S‐cysteinyldopa (SCD). The presence of the two melanin precursors substantially reduced the formation of lipid peroxidation products resulting from UVR exposure. The antioxidant activity of the melanin precursors was diminished under strong prooxidant conditions (presence of Fe3+). These results suggest that melanin precursors may have an important role in the protection of skin against the harmful effects of UVR including photocarcinogenesis.

Electron spin resonance study on the permeability of superoxide radicals in lipid bilayers and biological membranes.

The permeability of lipid bilayers and biological membranes to superoxide free radicals was examined by using superoxide dismutase (SOD)‐loaded lipid vesicles and SOD‐loaded erythrocyte ghosts. After exposing SOD lipid vesicles and SOD ghosts to enzymatically produced superoxide radicals and using spin‐trapping and electron spin resonance (ESR) techniques, we found that SOD entrapped within erythrocyte ghosts effectively scavenges external O.− 2 while SOD inside the lipid bilayers has no effect. These results confirm that O.− 2 is able to cross through a biological plasma membrane but not across a pure lipid bilayer. The data provide instruction as to how and where anti‐oxidant therapy is to be approached relative to the site of oxygen free radical production.

Effect of intrathecal superoxide dismutase and catalase on oxyhemoglobin-induced vasospasm in monkeys.

A gel consisting of agarose and oxyhemoglobin (OxyHb) was developed so that, when placed in the subarachnoid space, OxyHb would be slowly released, simulating lysis of erythocytes after subarachnoid hemorrhage. The system was used to investigate the importance of reactions mediated by free radicals in the genesis of OxyHb-induced vasospasm in monkeys. Seventeen monkeys were randomly assigned to have subarachnoid placement, on Day 0, of one of the following: 1) agarose gel alone (n = 2); 2) agarose plus OxyHb (n = 3); 3) agarose plus OxyHb plus intrathecal administration of superoxide dismutase and catalase (n = 6); and 4) agarose plus OxyHb plus intrathecal administration of placebo (n = 6). Vasospasm was assessed by comparison of angiograms performed on Day 0 and 7 days after subarachnoid placement of compounds, and by electron microscopy. OxyHb alone caused significant reduction in the diameter of the middle cerebral artery (40 +/- 8%, P less than 0.005, paired t test), which was associated with ultrastructural damage to smooth muscle. Treatment with superoxide dismutase plus catalase or with placebo attenuated vasospasm of the middle cerebral artery, although significant narrowing persisted in both groups (27 +/- 12% and 26 +/- 13%, respectively, P less than 0.05, paired t test). Analysis of variance showed no difference in the degree of vasospasm between groups exposed to subarachnoid placement of OxyHb. Cerebrospinal fluid aspirated from the cisterna magna on Day 7 contained elevated activity of superoxide dismutase in animals that received treatment. Malondialdehyde was undetectable in cerebrospinal fluid after subarachnoid placement of agarose alone, although it was present in similar amounts in all groups that received subarachnoid placement of OxyHb. Since intrathecal superoxide dismutase and catalase failed to protect against OxyHb-induced vasospasm, mechanisms mediated by free radicals may not be important in its genesis. As only one combination of doses of superoxide dismutase and catalase was administered, however, it may be that other dosage schedules might be efficacious.

Bilirubin toxicity in neural cell lines N115 and NBR10A.

ABSTRACT: The toxicity of bilirubin was investigated in 2 neural cell lines NBR10A and N115 using a quantitative dye assay 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium biomide (MTT) as a measure of cell viability and [3H]thymidine incorporation as a measure of DNA synthesis. Short exposures (up to 2 h) to bilirubin, even up to a bilirubin-albumin molar ratio of 1.5, yielded no evidence of toxicity using these assays. At longer exposure times (24 h) a decrease in cell viability and [3H]thymidine incorporation was detected at a molar ratio of 0.8 when the bilirubin concentration was 0.1 mM or higher, whereas lower bilirubin levels at this molar ratio showed no deleterous effect. The effect of bilirubin is more pronounced at a molar ratio of 1.5 with longer incubation periods. The MTT assay showed the N115 cells appeared to be more resistant to bilirubin cytotoxicity than NBR10A cells, a finding which was not obtained from [3H]thymidine incorporation studies. This discrepancy can be explained by the fact that we are measuring two different variables; the MTT assay estimates the number of viable cells at the end of the experiment by measuring mitochondrial function whereas the [3H]thymidine assay measures the rate of DNA synthesis during the last 2 h of the experiment. The concentration effect of bilirubin is evident from the [3H]- thymidine studies in that at a molar ratio of 1.5 and bilirubin concentration of 0.075 mM or higher, there is both cell kill (decrease in DNA) and inhibition of [3H]- thymidine incorporation (decrease in specific activity). When the bilirubin concentration is reduced to 0.03 mM, there is little or no cell death (no change DNA) but inhibition still exists (42% decrease in specific activity). Thus, cell viability and function of these two neural lines is dependent not only on the bilirubin albumin molar ratio, but also on the absolute concentration of bilirubin and albumin as well as the time of exposure.

Growth hormone-albumin conjugates Reduced renal toxicity and altered plasma clearance

The effective therapeutic use of many small peptides such as growth hormone has been limited by their small molecular masses and rapid clearance by the kidneys. Moreover, various degrees of nephrotoxicity have been reported for small proteins which are readily filtered at the level of the glomerulus. We have attempted to circumvent this drawback by conjugating growth hormone (somatotropin) to serum albumin in an effort to alter the peptides pharmacokinetics while retaining its biological activity.

Inhibition of superoxide-generating NADPH oxidase of human neutrophils by lazaroids (21-aminosteroids and 2-methylaminochromans)

Lazaroids (21-aminosteroids and 2-methylaminochromans) are a new series of drugs designed and demonstrated to protect against tissue damage after trauma and/or ischemia. It has been suggested that the protective effects of lazaroids are derived from their potent actions to inhibit iron-dependent lipid peroxidation, but whether this is sufficient to explain their therapeutic effects is unknown. In an effort to better understand their mechanism of action, these drugs were tested for other modes of antioxidant activity such as scavenging superoxide and hydroxyl radicals and inhibition of production of oxygen free radicals by human neutrophils stimulated with phorbol myristate acetate. Using an ESR spin-trapping technique, with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for superoxide and hydroxyl radicals, we found that the lazaroids U74500A and U78518F are, at best, weak scavengers of superoxide radicals whereas U78518F is a strong scavenger of hydroxyl radicals. In addition, lazaroids were found to be strong inhibitors (60-80% inhibition at 50 microM) of the superoxide-generating NADPH oxidase of human neutrophils. Inhibition of NADPH oxidase by lazaroids in cell-free systems suggested the action to be on the activated enzyme rather than on the process of activation. This may represent an important mode action of lazaroids and suggests their potential use in ischemic/inflammatory conditions involving oxygen free radical production by activated phagocytic cells.

A modified tetramethylbenzidine method for measuring lipid hydroperoxides

A simple and sensitive spectrophotometric method for measuring lipid peroxides and peroxides in general is described. The method was developed by modifying an existing method based on the peroxidase activity of hemoglobin with tetramethylbenzidine as the electron donor. The modifications resulted in much improved sensitivity and reproducibility. With the modified method lipid peroxides as low as 2 nmol can be measured, a high sensitivity compared with other spectrophotometric methods. The absorbance is linear over a wide range of concentrations. It is suggested that this modified method in combination with the commonly used thiobarbituric acid method will give a better quantitation of lipid peroxidation.

Bilirubin Toxicity in a Neuroblastoma Cell Line N-115:1. Effects on Na+K+ ATPase, [3H]- Thymidine Uptake, L-[35S]-Methionine Incorporation, and Mitochondrial Function

ABSTRACT: Though bilirubin is reported to affect a variety of cellular functions, the primary target of its toxic effect is still not known. A major problem in understanding this is the wide variation in results reported by different groups. This is probably due to the differences in stability of bilirubin solutions arising from large differences in bilirubin:albumin molar ratios used in experiments. Hence in studying the toxic effects of bilirubin in tissue culture systems, it is important to be certain that the bilirubin is maintained in solution throughout the time of the exposure to bilirubin. Spectrophotometric measurements have shown that bilirubin is stable in Dulbeccos modified Eagle medium solution at bilirubin:albumin molar ratios up to 3. Under these defined conditions, bilirubin was found to affect Na+K+ ATPase, [3H]-thymidine uptake, L-[35S]methionine incorporation into protein and mitochondrial function at bilirubin concentrations up to 125 pM and bilirubin: albumin molar ratio of 1.5. Toxic effects on all parameters measured were evident at bilirubin:albumin molar ratio of 1.5 after a minimum of 2 h of exposure. No effect was evident at a bilirubin:albumin molar ratio below 1. Although it is not possible to identify with certainty the primary target, the effect on mitochondrial function appeared earlier and was more profound than that seen with the other assessed functions.

The Effects Of Ischemia And Ischemia-reperfusion On Bacterial Translocation, Lipid Peroxidation, And Gut Histology: Studies On Hemorrhagic Shock In Pigs

The bacterial translocation hypothesis was tested in two studies (acute and subacute) in a porcine model of hemorrhagic shock. Male pigs (30-40 kg each) under general anesthesia had their femoral vein, femoral artery, and portal vein catheterized. After stabilization (1 hour) they were bled (40% of blood volume) over 30 minutes, then maintained in the hypotensive state (MAP = 30-40 mm Hg) for 2 hours, following which, according to randomization, they entered the control group or were resuscitated with whole blood (WB group) or with lactated Ringers solution (LR group). In the acute study, the mesenteric efferent lymphatic was also cannulated, the control group was not resuscitated, and the animals remained under general anesthesia to the end of the experiment (8.5 hours), when gut tissue was obtained for histologic study and measurement of lipid peroxidation. In the subacute study, the control group was not bled, the animals were awakened at 6.5 hours, and the portal vein catheter remained in situ until 48 hours. In both studies, samples of portal blood were obtained for culture at regular intervals and on completion, samples from mesenteric lymph nodes (MLNs) for culture were taken in the acute study, and in the subacute study samples from MLNs, spleen, and liver were obtained. In the acute study significant bacterial translocation to the MLNs and portal blood did not occur among the controls (n = 3), the LR group (n = 5), and the WB group (n = 6). Significant evidence of lipid peroxidation was found in both the LR and WB groups. Histologic assessment showed no difference among the groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of microwave radiation (2450 MHz) on the active and passive components of 24Na+ efflux from human erythrocytes.

We report an effect of low-level 2450-MHz microwaves on total and ouabain-sensitive