In vivo monitoring of hydroxyl radical generation caused by x-ray irradiation of rats using the spin trapping/EPR technique

Measurement of hydroxyl radical (*OH) in living animals irradiated with ionizing radiation should be required to clarify the mechanisms of radiation injury and the in vivo assessment of radiation protectors, because generation of *OH is believed to be one of the major triggers of radiation injury. In this study, *OH generation was monitored by spin trapping the secondary methyl radical formed by the reaction of *OH with dimethyl sulfoxide (DMSO). Rats were injected intraperitoneally with a DMSO solution of alpha-phenyl-N-tert-butylnitrone (PBN). X-irradiation of the rats remarkedly increased the six-line EPR signal in the bile. The strengthened signal was detectable above 40 Gy. Use of 13C-substituted DMSO revealed that the signal included the methyl radical adduct of PBN as a major component. The EPR signal of the PBN-methyl radical adduct was completely suppressed by preadministration of methyl gallate, a scavenger of *OH but not of methyl radical. Methyl gallate did not reduce the spin adducts to EPR-silent forms. These observations indicate that what we were measuring was *OH generated in vivo by x-irradiation. This is the first report of the in vivo monitoring of *OH generation at a radiation dose close to what people might receive in the case of radiological accident or radiation therapy.     

The metabolism of halothane by hepatocytes: a comparison between free radical spin trapping and lipid peroxidation in relation to cell damage

The technique of free radical spin trapping has been applied to demonstrate the formation of free radicals produced during the metabolism of halothane by rat liver hepatocytes under hypoxic conditions. The results obtained support previous findings that reported sex differences in the metabolic activation of halothane by rats in vivo. Cell viability under hypoxic conditions, as judged by trypan blue staining and lactate dehydrogenase release, shows a correlation with the extent of metabolism of halothane as measured by electron spin resonance spectroscopy. The extent of lipid peroxidation was measured by diene conjugation, malondialdehyde production and chemiluminescence. The latter technique allowed the demonstration of lipid peroxidation during incubations of hepatocytes under aerobic conditions. The magnitude of the aerobic chemiluminescence showed a similar sex dependency to the extent of free radical formation under hypoxic conditions. Cell viability measurements show that halothane metabolism in both hypoxic and aerobic conditions can lead to cell death. Consequently, oxidative lipid damage could be a cause of cell damage, as judged by cell viability, additional to covalent binding.     

Iron and dioxygen chemistry is an important route to initiation of biological free radical oxidations: an electron paramagnetic resonance spin trapping study

Iron can be a detrimental catalyst in biological free radical oxidations. Because of the high physiological ratio of [O2]/[H2O2] (> or = 10(3)), we hypothesize that the Fenton reaction with pre-existing H2O2 is only a minor initiator of free radical oxidations and that the major initiators of biological free radical oxidations are the oxidizing species formed by the reaction of Fe2+ with dioxygen. We have employed electron paramagnetic resonance spin trapping to examine this hypothesis. Free radical oxidation of: 1) chemical (ethanol, dimethyl sulfoxide); 2) biochemical (glucose, glyceraldehyde); and 3) cellular (L1210 murine leukemia cells) targets were examined when subjected to an aerobic Fenton (Fe2+ + H2O2 + O2) or an aerobic (Fe2+ + O2) system. As anticipated, the Fenton reaction initiates radical formation in all the above targets. Without pre-existing H2O2, however, Fe2+ and O2 also induce substantial target radical formation. Under various experimental ratios of [O2]/[H2O2] (1-100 with [O2] approximately 250 microM), we compared the radical yield from the Fenton reaction vs. the radical yield from Fe2+ + O2 reactions. When [O2]/[H2O2] < 10, the Fenton reaction dominates target molecule radical formation; however, production of target-molecule radicals via the Fenton reaction is minor when [O2]/[H2O2] > or = 100. Interestingly, when L1210 cells are the oxidation targets, Fe2+ + O2 is observed to be responsible for formation of nearly all of the cell-derived radicals detected, no matter the ratio of [O2]/[H2O2]. Our data demonstrate that when [O2]/[H2O2] > or = 100, Fe2+ + O2 chemistry is an important route to initiation of detrimental biological free radical oxidations.     

A novel protocol to identify and quantify all spin trapped free radicals from in vitro/in vivo interaction of HO(.-) and DMSO: LC/ESR, LC/MS, and dual spin trapping combinations

When dimethyl sulfoxide (DMSO) is oxidized via hydroxyl radical (HO(.-)), it forms methyl radicals ((.-)CH(3)) that can be spin trapped and detected by electron spin resonance (ESR). This ESR spin trapping technique has been widely used in many biological systems to indicate in vivo HO(.-) formation. However, we recently reported that (.-)CH(3) might not be the only carbon-centered radical that was trapped and detected by ESR from in vivo DMSO oxidation. In the present study, newly developed combination techniques consisting of dual spin trapping (free radicals trapped by both regular and deuterated alpha-[4-pyridyl 1]-N-tert-butyl nitrone, d(0)/d(9)-POBN) followed by LC/ESR and LC/MS were used to characterize and quantify all POBN-trapped free radicals from the interaction of HO(.-) and DMSO. In addition to identifying the two well-known free radicals, (.-)CH(3) and (.-)OCH(3), from this interaction, we also characterized two additional free radicals, (.-)CH(2)OH and (.-)CH(2)S(O)CH(3). Unlike ESR, which can measure POBN adducts only in their radical forms, LC/MS identified and quantified all three redox forms, including the ESR-active radical adduct and two ESR-silent forms, the nitrone adduct (oxidized adduct) and the hydroxylamine (reduced adduct). In the bile of rats treated with DMSO and POBN, the ESR-active form of POBN/(.-)CH(3) was not detected. However, with the addition of the LC/MS technique, we found approximately 0.75 microM POBN/(.-)CH(3) hydroxylamine, which represents a great improvement in radical detection sensitivity and reliability. This novel protocol provides a comprehensive way to characterize and quantify in vitro and in vivo free radical formation and will have many applications in biological research.     

Improvement of the method to estimate the relative reaction rate constants of hydroxyl radical with polyphenols using ESR spin trap: X-ray irradiation of water with a flowing system

UV-photolysis of hydrogen peroxide is a useful technique to produce hydroxyl radical. However, it is not an appropriate method to estimate the reactivity of polyphenols with hydroxyl radicals because many of the polyphenol derivatives also absorb the UV-light to generate hydroxyl radicals. In this study, X-ray irradiation of water with a flowing system was applied to estimate the reactivity of hydroxyl radicals with polyphenols using electron spin resonance (ESR) spin trap. The obtained relative reaction rates reasonably agreed with previous data by pulse radiolysis. This method will be a useful technique to estimate the reactivity of antioxidants including polyphenols with hydroxyl radicals.     

In vivo evidence of free radical generation in the mouse lung after exposure to Pseudomonas aeruginosa bacterium: an ESR spin-trapping investigation

In the Pseudomonas aeruginosa-induced rodent pneumonia model, it is thought that free radicals are significantly associated with the disease pathogenesis. However, until now there has been no direct evidence of free radical generation in vivo. Here we used electron spin resonance (ESR) and in vivo spin trapping with α-(4-pyridyl-1-oxide)-N-tert-butylnitrone to investigate free radical production in a murine model. We detected and identified generation of lipid-derived free radicals in vivo (a(N) =14.86 ± 0.03 G and a(H)(β) =2.48 ± 0.09 G). To further investigate the mechanism of lipid radical production, we used modulating agents and knockout mice. We found that with GdCl(3) (phagocytic toxicant), NADPH-oxidase knockout mice (Nox2(-)/(-)), allopurinol (xanthine-oxidase inhibitor) and Desferal (metal chelator), generation of lipid radicals was decreased; histopathological and biological markers of acute lung injury were noticeably improved. Our study demonstrates that lipid-derived free radical formation is mediated by NADPH-oxidase and xanthine-oxidase activation and that metal-catalysed hydroxyl radical-like species play important roles in lung injury caused by Pseudomonas aeruginosa.     

Studies of AIDS vaccination using an ex vivo feline immunodeficiency virus model: protection conferred by a fixed-cell vaccine against cell-free and cell-associated challenge differs in duration and is not easily boosted.

Cats immunized with cells infected with a primary isolate of feline immunodeficiency virus (FIV) and fixed with paraformaldehyde were challenged with cell-free or cell-associated homologous virus obtained ex vivo. Complete protection was observed in animals challenged with cell-free virus 4 months after completion of vaccination (p.v.) or with cell-associated virus 12 months p.v. In contrast, no protection was observed in cats challenged with cell-free virus 12 or 28 months p.v. or with cell-associated virus 37.5 months p.v. Prior to the 28- and 37.5-month challenges, the animals had received a booster dose of vaccine that had elicited a robust anamnestic immune response. These results show that vaccine-induced protection against ex vivo FIV is achievable but is relatively short-lived and can be difficult to boost.     

AIDS vaccination studies using an ex vivo feline immunodeficiency virus model: failure to protect and possible enhancement of challenge infection by four cell-based vaccines prepared with autologous lymphoblasts

Immunogenicity and protective activity of four cell-based feline immunodeficiency virus (FIV) vaccines prepared with autologous lymphoblasts were investigated. One vaccine was composed of FIV-infected cells that were paraformaldehyde fixed at the peak of viral expression. The other vaccines were attempts to maximize the expression of protective epitopes that might become exposed as a result of virion binding to cells and essentially consisted of cells mildly fixed after saturation of their surface with adsorbed, internally inactivated FIV particles. The levels of FIV-specific lymphoproliferation exhibited by the vaccinees were comparable to the ones previously observed in vaccine-protected cats, but antibodies were largely directed to cell-derived constituents rather than to truly viral epitopes and had very poor FIV-neutralizing activity. Moreover, under one condition of testing, some vaccine sera enhanced FIV replication in vitro. As a further limit, the vaccines proved inefficient at priming animals for anamnestic immune responses. Two months after completion of primary immunization, the animals were challenged with a low dose of homologous ex vivo FIV. Collectively, 8 of 20 vaccinees developed infection versus one of nine animals mock immunized with fixed uninfected autologous lymphoblasts. After a boosting and rechallenge with a higher virus dose, all remaining animals became infected, thus confirming their lack of protection.     

Transforming growth factor alpha (TGFalpha)-stimulated secretion of HSP90alpha: using the receptor LRP-1/CD91 to promote human skin cell migration against a TGFbeta-rich environment during wound healing

Jump-starting and subsequently maintaining epidermal and dermal cell migration are essential processes for skin wound healing. These events are often disrupted in nonhealing wounds, causing patient morbidity and even fatality. Currently available treatments are unsatisfactory. To identify novel wound-healing targets, we investigated secreted molecules from transforming growth factor alpha (TGFalpha)-stimulated human keratinoytes, which contained strong motogenic, but not mitogenic, activity. Protein purification allowed us to identify the heat shock protein 90alpha (hsp90alpha) as the factor fully responsible for the motogenic activity in keratinocyte secretion. TGFalpha causes rapid membrane translocation and subsequent secretion of hsp90alpha via the unconventional exosome pathway in the cells. Secreted hsp90alpha promotes both epidermal and dermal cell migration through the surface receptor LRP-1 (LDL receptor-related protein 1)/CD91. The promotility activity resides in the middle domain plus the charged sequence of hsp90alpha but is independent of the ATPase activity. Neutralizing the extracellular function of hsp90alpha blocks TGFalpha-induced keratinicyte migration. Most intriguingly, unlike the effects of canonical growth factors, the hsp90alpha signaling overrides the inhibition of TGFbeta, an abundant inhibitor of dermal cell migration in skin wounds. This finding provides a long-sought answer to the question of how dermal cells migrate into the wound environment to build new connective tissues and blood vessels. Thus, secreted hsp90alpha is potentially a new agent for wound healing.