Combining 3D human in vitro methods for a 3Rs evaluation of novel titanium surfaces in orthopaedic applications.

In this study, we report on a group of complementary human osteoblast in vitro test methods for the preclinical evaluation of 3D porous titanium surfaces. The surfaces were prepared by additive manufacturing (electron beam melting [EBM]) and plasma spraying, allowing the creation of complex lattice surface geometries. Physical properties of the surfaces were characterized by SEM and profilometry and 3D in vitro cell culture using human osteoblasts. Primary human osteoblast cells were found to elicit greater differences between titanium sample surfaces than an MG63 osteoblast-like cell line, particularly in terms of cell survival. Surface morphology was associated with higher osteoblast metabolic activity and mineralization on rougher titanium plasma spray coated surfaces than smoother surfaces. Differences in osteoblast survival and metabolic activity on titanium lattice structures were also found, despite analogous surface morphology at the cellular level. 3D confocal microscopy identified osteoblast organization within complex titanium surface geometries, adhesion, spreading, and alignment to the biomaterial strut geometries. Mineralized nodule formation throughout the lattice structures was also observed, and indicative of early markers of bone in-growth on such materials. Testing methods such as those presented are not traditionally considered by medical device manufacturers, but we suggest have value as an increasingly vital tool in efficiently translating pre-clinical studies, especially in balance with current regulatory practice, commercial demands, the 3Rs, and the relative merits of in vitro and in vivo studies. Biotechnol. Bioeng. 2016;113: 1586-1599. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Amine functionalized nanodiamond promotes cellular adhesion, proliferation and neurite outgrowth.

In this study, we report the production of amine functionalized nanodiamond. The amine functionalized nanodiamond forms a conformal monolayer on a negatively charged surface produced via plasma polymerization of acrylic acid. Nanodiamond terminated surfaces were studied as substrates for neuronal cell culture. NG108-15 neuroblastoma-glioma hybrid cells were successfully cultured upon amine functionalized nanodiamond coated surfaces for between 1 and 7 d. Additionally, primary dorsal root ganglion (DRG) neurons and Schwann cells isolated from Wistar rats were also successfully cultured over a period of 21 d illustrating the potential of the coating for applications in the treatment of peripheral nerve injury.

Direct laser writing of 3D scaffolds for neural tissue engineering applications.

This study reports on the production of high-resolution 3D structures of polylactide-based materials via multi-photon polymerization and explores their use as neural tissue engineering scaffolds. To achieve this, a liquid polylactide resin was synthesized in house and rendered photocurable via attaching methacrylate groups to the hydroxyl end groups of the small molecular weight prepolymer. This resin cures easily under UV irradiation, using a mercury lamp, and under femtosecond IR irradiation. The results showed that the photocurable polylactide (PLA) resin can be readily structured via direct laser write (DLW) with a femtosecond Ti:sapphire laser and submicrometer structures can be produced. The maximum resolution achieved is 800 nm. Neuroblastoma cells were grown on thin films of the cured PLA material, and cell viability and proliferation assays revealed good biocompatibility of the material. Additionally, PC12 and NG108-15 neuroblastoma growth on bespoke scaffolds was studied in more detail to assess potential applications for neuronal implants of this material.

Regionally-selective cell colonization of micropatterned surfaces prepared by plasma polymerization of acrylic acid and 1,7-octadiene.

Micropatterned surfaces have been used as a tool for controlling the extent and strength of cell adhesion, the direction of cell growth and the spatial distribution of cells. In this study, chemically micropatterned surfaces were prepared by successive plasma polymerization of acrylic acid (AA) and 1,7-octadiene (OD) through a mask. Rat vascular smooth muscle cells (VSMC), bovine endothelial cells (EC), porcine mesenchymal stem cells (MSC) or human skeletal muscle cells (HSKMC) were seeded on these surfaces in densities from 9,320 cells/cm(2) to 31,060 cells/cm(2). All cell types adhered and grew preferentially on the strip-like AA domains. Between day 1 and 7 after seeding, the percentage of cells on AA domains ranged from 84.5 to 63.3 % for VSMC, 85.3 to 73.5 % for EC, 98.0 to 90.0 % for MSC, and 93.6 to 55.0 % for HSKMC. The enzyme-linked immunosorbent assay (ELISA) revealed that the concentration of alpha-actin per mg of protein was significantly higher in VSMC on AA. Similarly, immunofluorescence staining of von Willebrand factor showed more apparent Weibel-Palade bodies in EC on AA domains. MSC growing on AA had better developed beta-actin cytoskeleton, although they were less stained for hyaluronan receptor (CD44). In accordance with this, MSC on AA contained a higher concentration of beta-actin, although the concentration of CD44 was lower. HSKMC growing on AA had a better developed alpha-actin cytoskeleton. These results based on four cell types suggest that plasma polymerization is a suitable method for producing spatially defined patterned surfaces for controlled cell adhesion, proliferation and maturation.

Immobilized alpha-melanocyte stimulating hormone 10-13 (GKPV) inhibits tumor necrosis factor-alpha stimulated NF-kappaB activity.

alpha-MSH is an anti-inflammatory peptide which signals by binding to the melanocortin-1 receptor (MC1R) and elevating cyclic AMP in several different cells and tissues. The carboxyl terminal peptides of alpha-MSH (KPV/GKPV) are the smallest minimal sequences that prevent inflammation, but it is not known if they operate via MC1R or cyclic AMP. The aim of this study was to examine the intracellular signaling potential of the GKPV peptide sequence when immobilized to polystyrene beads via a polyethylene glycol moiety. Beads containing an immobilized GKPV peptide were investigated for their ability to inhibit proinflammatory tumor necrosis factor-alpha (TNF-alpha) stimulated activation of NF-kappaB in HBL cells stably transfected with an NF-kappaB-luciferase reporter construct. Peptide functionalized beads were compared with the ability of soluble peptide alone (alpha-MSH or GKPV) or non-functionalized beads to inhibit TNF-alpha stimulated activation of NF-kappaB. GKPV peptide functionalized beads significantly inhibited NF-kappaB-luciferase activity in comparison to beads containing no peptide moiety in one of two growths conditions investigated. Soluble alpha-MSH and GKPV peptides were also confirmed to inhibit NF-kappaB-luciferase. The present study suggests that the carboxyl terminal MSH peptide acts via a cell receptor-based mechanism and furthermore may support the potential use of such immobilized ligands for anti-inflammatory therapeutic use.

Melanocyte stimulating hormone peptides inhibit TNF-alpha signaling in human dermal fibroblast cells.

Alpha-melanocyte stimulating hormone (alpha-MSH) has been identified as a potent anti-inflammatory in various tissues including the skin. It has previously been shown in skin cell keratinocytes and melanocytes/melanoma cells that MSH peptides inhibit TNF-alpha stimulated NF-kappaB activity and intercellular adhesion molecule-1 (ICAM-1) upregulation. However, the precise anti-inflammatory role of MSH peptides in dermal fibroblasts is unclear. Some studies report on pro-inflammatory responses, while others on anti-inflammatory responses. The present study confirms MC1R expression in cultured human dermal fibroblasts and reports that the MSH peptides alpha-MSH and KP(-D-)V inhibit TNF-alpha stimulated NF-kappaB activity and ICAM-1 upregulation, consistent with an anti-inflammatory role. However, involvement of IkappaB-alpha regulation by either peptide was not confirmed, supporting a mechanism independent of the NF-kappaB inhibitor. In conclusion, alpha-MSH and KP(-D-)V peptides have an anti-inflammatory action on dermal fibroblast signaling by inhibiting the pro-inflammatory activity of TNF-alpha in vitro.

Measurement of NF-kappaB in normal and reconstructed human skin in vitro.

The survival of grafted donor skin for the treatment of burn injuries depends on several factors including wound bed vascularisation and the intensity of acute inflammation shortly after injury. However, acceptance rates approximate 50% at best and therefore a clinical need exists for improvement. The aim of the study was to develop a method for assessing the inflammatory response of cells in skin tissue based on activation of the NF-kappa B (NF-kappaB) transcription factor complex, thereby providing a basis for analysing the inflammatory component and anti-inflammatory strategies for tissue-engineered treatments. We have extended a standard method of measuring NF-kappaB in monolayer cultures that relies on determining translocation of the p65 subunit from the cytoplasm to the nucleus. Normal human skin and tissue engineered skin was analysed using an immunofluorescence microscopy technique, that revealed base line NF-kappaB activation in the epidermis and dermis were different. It was possible to determine the activation of NF-kappaB in skin tissue, enabling correlation that NF-kappaB measurement is a sensitive indicator of cellular responses in 3-D tissue. The approach will provide a basis for early responses of skin cells in determining the efficacy of anti-inflammatory delivery via tissue-engineered scaffolds for burn injuries.

Melanoma cell migration is upregulated by tumour necrosis factor-alpha and suppressed by alpha-melanocyte-stimulating hormone.

We reported recently that the inflammatory cytokine tumour necrosis factor alpha (TNF-alpha) can upregulate integrin expression, cell attachment and invasion of cells through fibronectin in a human melanoma cell line (HBL). Furthermore, the actions of TNF-alpha were suppressed by the addition of an anti-inflammatory peptide alpha-melanocyte-stimulating hormone (alpha-MSH). In the current study, we extend this work investigating to what extent TNF-alpha might stimulate melanoma invasion by promoting cell migration and whether alpha-MSH is also inhibitory. Two human melanoma cell lines were examined in vitro (HBL and C8161) using a scratch migration assay. Analysis using either time-lapse video microscopy or imaging software analysis of migrating 'fronts' of cells revealed that C8161 cells migrated more rapidly than HBL cells. However, when cells were stimulated with TNF-alpha both cell types responded with a significant increase in migration distance over a 16-26 h incubation time. alpha-Melanocyte-stimulating hormone had an inhibitory effect on TNF-alpha-stimulated migration for HBL cells, completely blocking migration at 10(-9) M. In contrast, C8161 cells did not respond to alpha-MSH (as these cells have a loss-of-function melanocortin-1 receptor). However, stable transfection of C8161 cells with the wild-type melanocortin-1 receptor produced cells whose migration was significantly inhibited by alpha-MSH. In addition, the use of a neutralising antibody to the beta(1)-integrin subunit significantly reduced migration in both cell types. This data therefore supports an inflammatory environment promoting melanoma cell migration, and in addition shows that alpha-MSH can inhibit inflammatory stimulated migration. The data also support a fundamental role of the beta(1)-integrin receptor in melanoma cell migration.

Depolarization-stimulated catecholamine biosynthesis: involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ.

Depolarizing stimuli increase catecholamine (CA) biosynthesis, tyrosine hydroxylase (TH) activity, and TH phosphorylation at Ser19, Ser31, and Ser40 in a Ca(2+)-dependent manner. However, the identities of the protein kinases that phosphorylate TH under depolarizing conditions are not known. Furthermore, although increases in Ser31 or Ser40 phosphorylation increase TH activity in vitro, the relative influence of phosphorylation at these sites on CA biosynthesis under depolarizing conditions is not known. We investigated the participation of extracellular signal-regulated protein kinase (ERK) and cAMP-dependent protein kinase (PKA) in elevated K(+)-stimulated TH phosphorylation in PC12 cells using an ERK pathway inhibitor, PD98059, and PKA-deficient PC12 cells (A126-B1). In the same paradigm, we measured CA biosynthesis. TH phosphorylation stoichiometry (PS) was determined by quantitative blot-immunolabeling using site- and phosphorylation state-specific antibodies. Treatment with elevated K(+) (+ 58 mM) for 5 min increased TH PS at each site in a Ca(2+)-dependent manner. Pretreatment with PD98059 prevented elevated K(+)-stimulated increases in ERK phosphorylation and Ser31 PS. In A126-B1 cells, Ser40 PS was not significantly increased by forskolin, and elevated K(+)-stimulated Ser40 PS was three- to five-fold less than that in PC12 cells. In both cell lines, CA biosynthesis was increased 1.5-fold after treatment with elevated K(+) and was prevented by pretreatment with PD98059. These results suggest that ERK phosphorylates TH at Ser31 and that PKA phosphorylates TH at Ser40 under depolarizing conditions. They also suggest that the increases in CA biosynthesis under depolarizing conditions are associated with the ERK-mediated increases in Ser31 PS.

Striatal dopaminergic and serotonergic markers in human heroin users.

To establish whether chronic opiate exposure might impair brain dopaminergic or serotonergic function in humans, we assessed biochemical indices of monoaminergic neurotransmitter activity and integrity in post mortem striatum of nine chronic heroin users and 14 control subjects. Striatal levels of the vesicular monoamine transporter were normal, suggesting that the density of dopamine nerve terminals is not reduced in heroin users. In nucleus accumbens, levels of tyrosine hydroxylase protein (-25%) and those of the dopamine metabolite homovanillic acid (-33%) were reduced significantly together with a trend for decreased dopamine (-32%) concentration. These changes could reflect either a compensatory downregulation of dopamine biosynthesis in response to prolonged dopaminergic stimulation caused by heroin, or reduced axoplasmic transport of tyrosine hydroxylase. Striatal levels of serotonin were either normal or elevated whereas concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid were decreased by 27-38%. Our data suggest that chronic heroin exposure might produce a modest reduction in dopaminergic and serotonergic activity that could affect motivational state and impulse control, respectively.

Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size.

While the transporters that accumulate classical neurotransmitters in synaptic vesicles have been identified, little is known about how their expression regulates synaptic transmission. We have used adenoviral-mediated transfection to increase expression of the brain vesicular monoamine transporter VMAT2 and presynaptic amperometric recordings to characterize the effects on quantal release. In presynaptic axonal varicosities of ventral midbrain neurons in postnatal culture, VMAT2 overexpression in small synaptic vesicles increased both quantal size and frequency, consistent with the recruitment of synaptic vesicles that do not normally release dopamine. This was confirmed using noncatecholaminergic AtT-20 cells, in which VMAT2 expression induced the quantal release of dopamine. The ability to increase quantal size in vesicles that were already competent for dopamine release was shown in PC12 cells, in which VMAT2 expression increased the quantal size but not the number of release events. These results demonstrate that vesicle transporters limit the rate of transmitter accumulation and can alter synaptic strength through two distinct mechanisms.

Alpha-melanocyte-stimulating hormone reduces impact of proinflammatory cytokine and peroxide-generated oxidative stress on keratinocyte and melanoma cell lines.

We have previously shown that alpha-melanocyte-stimulating hormone (alpha-MSH) can oppose tumor necrosis factor alpha activation of NF-kappaB (1-2 h) and intercellular adhesion molecule 1 up-regulation (mRNA by 3 h and protein by 24 h) in melanocytes and melanoma cells. The present study reports on the ability of four MSH peptides to control intracellular peroxide levels and glutathione peroxidase (GPx) activity in pigmentary and nonpigmentary cells. In human HBL melanoma and HaCaT keratinocytes tumor necrosis factor alpha and H(2)O(2) both activated GPx in a time- and concentration-dependent manner (by 30-45 min). alpha-MSH peptides were found to inhibit the stimulated GPx activity and had biphasic dose-response curves. MSH 1-13 and MSH [Nle(4)-d-Phe(7)] achieved maximum inhibition at 10(-10) and 10(-12) m, respectively. Higher concentrations (10-100 fold) of MSH 4-10 and MSH 11-13 were required to produce equivalent levels of inhibition. alpha-MSH was also capable of reducing peroxide accumulation within 15 min, and again this inhibition was biphasic. The data support a role of alpha-MSH in acute protection of cells to oxidative/cytokine action that precedes NF-kappaB and GPx activation. The rapidity and potency of the response to alpha-MSH in pigmentary and nonpigmentary cells suggest this to be a central role of this peptide in cutaneous cells.

Alpha-melanocyte-stimulating hormone inhibits NF-kappaB activation in human melanocytes and melanoma cells.

Alpha-melanocyte-stimulating hormone is produced by several different cell types including neural cells, endothelial cells, monocytes, and keratinocytes. A biologic role in melanocyte pigmentation is widely recognized, but more recent studies describe a part in modulating inflammatory and immune responses. The aim of the this study was to investigate the mechanism by which alpha-melanocyte-stimulating hormone antagonizes proinflammatory cytokine action. We report that alpha-melanocyte-stimulating hormone (10-9 M) was effective in opposing a tumor necrosis factor-alpha stimulated increase in NF-kappaB DNA binding activity in: (i) normal ocular melanocytes; (ii) cells cultured from ocular melanoma tumors; and (iii) two cutaneous melanoma cell lines. NF-kappaB is activated by many inflammatory mediators and controls transcription of genes required for immune and inflammatory responses. The transcription factor complex was positively identified as the p50/p65 heterodimer, recognized to have transcriptional activating potential. Maximum reduction of NF-kappaB DNA binding activity with alpha-melanocyte-stimulating hormone was detected 2 h after cellular stimulation and varied from between 53% and 18% depending on cell type. Whereas the acute inhibitory effects could be mimicked by elevating cyclic adenosine monophosphate, alpha-melanocyte-stimulating hormone was not found to have any effect on the relative level of IkappaBalpha protein expression over 24 h. These data show that alpha-melanocyte-stimulating hormone has a pronounced effect on NF-kappaB activity in melanocytes and melanoma cells, identifying a specific dimeric complex, and suggest this to be a key pathway by which immunomodulation/anti-inflammation may operate. The results may also be considered in the broader context of general inflammatory pathologies concerning cells which express alpha-melanocyte-stimulating hormone receptors and utilize the NF-kappaB signaling pathway.

Role of serine-19 phosphorylation in regulating tyrosine hydroxylase studied with site- and phosphospecific antibodies and site-directed mutagenesis.

The effects of depolarization by elevated potassium concentrations were studied in PC12 cells and in stably transfected AtT-20 cells expressing wild-type or [Leu19]-recombinant tyrosine hydroxylase (rTH). Changes in the phosphorylation states of Ser19 and Ser40 in tyrosine hydroxylase (TH) were determined immunochemically using antibodies specific for the phosphorylated state of each site and compared with changes in TH activity in PC12 cell lysates and with changes in L-DOPA biosynthesis rates in intact AtT-20 cells. Treatment of either PC12 cells or AtT-20 cells expressing wild-type rTH with elevated potassium produced a transient increase in the phosphorylation state of Ser19 (up to 0.7 mol of phosphate/mol of subunit) in concert with a more gradual and sustained increase in Ser40 phosphorylation. Elevated potassium treatment also increased TH activity in PC12 cell lysates, but these increases paralleled the temporal course of Ser40, as opposed to Ser19, phosphorylation. Similarly, increases in DOPA accumulation produced by elevated potassium in AtT-20 cells expressing wild-type rTH paralleled the increases in the phosphorylation state of Ser40 but not Ser19. Moreover, elevated potassium produced comparable increases in DOPA accumulation in AtT-20 cells expressing rTH in which Ser19 phosphorylation had been eliminated (by substitution of Leu for Ser19). Thus, depolarization-induced increases in the stoichiometry of Ser19 phosphorylation do not appear to influence directly the activity of TH in situ.

Differential protein oxidation in Duchenne and Becker muscular dystrophy.

We describe the use of an immunoblotting technique to investigate the potential role of reaction oxygen species in the pathogenesis of Duchenne muscular dystrophy. Quadriceps femoris muscle biopsy samples were obtained from six patients with Duchenne and six with Becker muscular dystrophy, and from six control subjects. These were analysed for the presence of protein carbonyl moieties (indicative of oxidation to protein) by SDS-polyacrylamide gel electrophoresis and Western blotting, using a commercially available antibody. In all Duchenne and Becker patient samples analysed, a heavily oxidized protein species was identified migrating at 125 kDa. This oxidized species was not present (or was present at very low levels) in normal control samples. Use of the present technique also identified that the various muscle proteins in Duchenne and Becker muscular dystrophy muscle are oxidized to varying degrees, supporting the hypothesis of a differential susceptibility of proteins to oxidation in these disorders. Work from the present study further supports the hypothesis that reactive oxygen species play a role in dystrophic muscle cell pathogenesis.

A DEVD-inhibited caspase other than CPP32 is involved in the commitment of cerebellar granule neurons to apoptosis induced by K+ deprivation.

Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K+ (25 mM KCl) to medium containing lower levels of K+ (5 mM KCl). We used this paradigm to investigate the role of caspases in the death process. Two broad-spectrum caspase inhibitors, tert-butoxycarbonyl-Asp x (O-methyl) x fluoromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp x fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10-25 microM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N-acetyl Tyr-Val-Ala-Asp x chloromethyl ketone (YVAD x cmk), an interleukin-1beta converting enzyme-preferring inhibitor, and N-acetyl Asp-Glu-Val-Asp x fluoromethyl ketone (DEVD x fmk), a CPP32-preferring inhibitor, were also evaluated. YVAD x cmk provided only modest (<20%) protection and only at the highest concentration (100 microM) tested, suggesting that interleukin-1beta converting enzyme and/or closely related caspases were not involved. In comparison, DEVD x fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP-ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD x fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD-sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K+-deprived granule neurons.

The participation of proliferative keratinocytes in the preimmune response to sensitizing agents.

The aims of this study were to investigate whether keratinocytes are capable of playing a direct preimmune role in the pathophysiology of allergic contact dermatitis (ACD) and to examine to what extent the degree of differentiation might influence this. We measured the ability of sensitizing agents to up-regulate intercellular adhesion molecule 1 (ICAM-1) expression in cultured normal human keratinocytes (NHK) and in the transformed human keratinocyte HaCaT cell line. In proliferative HaCaT cells, following a 24 h exposure, nickel compounds, para-phenylenediamine (pPD) and 1-chloro-2,4-dinitrobenzene produced a concentration-dependent up-regulation of ICAM-1 expression without reducing cell viability, while K2Cr2O7 led to ICAM-1 up-regulation at cytotoxic concentrations, and CrCl3 was without effect. In NHK, NiSO4 and pPD induced ICAM-1 expression to a significantly greater extent in proliferative cells than in differentiated cells, where involucrin expression was measured to assess the differentiation state. NiSO4- or pPD-pretreatment of proliferative HaCaT cells enhanced T-cell binding, which was abolished by neutralizing antibodies to ICAM-1 or CD18. Our investigations concerning the involvement of oxidative stress in the induction of ICAM-1 expression in response to sensitizing agents were inconclusive. The oxidizing agents FeCl3 and H2O2 up-regulated ICAM-1 expression in HaCaT cells but there was no clear relationship between the ability of agents to induce ICAM-1 expression and their ability to alter the levels of reduced glutathione. Although pPD increased interleukin-1 alpha release from NHK, this cytokine was not capable of inducing ICAM-1 expression in NHK. Tumour necrosis factor-alpha, which does induce ICAM-1 expression in NHK, was not detected in response to pPD, arguing against an autocrine pathway of ICAM-1 induction in response to pPD. In summary, we report the direct interaction of sensitizing agents with keratinocytes leading to the generation of immune signals, particularly by proliferative keratinocytes, suggesting an active role for the proliferative keratinocyte in the pathophysiology of ACD.

Short- and long-term regulation of tyrosine hydroxylase in chromaffin cells by VIP and PACAP.

Whereas cholinergic neurotransmitters are responsible for the release of a substantial portion of the CAs released from rat chromaffin cells by activation of the splanchinc nerves, the present data suggest that noncholinergic neurotransmission appears to play a more substantial role in the short- and long-term homeostatic regulation of TH activity, which serves to maintain the stores of CAs for subsequent release. In addition, studies using the PKA-deficient PC12 cells provided the first direct evidence that PKA actually mediates the phosphorylation of Ser40 in situ.

Differential susceptibility of human skeletal muscle proteins to free radical induced oxidative damage: a histochemical, immunocytochemical and electron microscopical study in vitro.

In this study we describe a novel experimental approach to quantify the relative susceptibility of (membrane-associated, contractile and mitochondrial) proteins in normal human muscle tissue sections to oxidative damage by the reactive oxygen species (ROS), hydroxyl (OH.) or superoxide (O2.-) radicals. The latter species were generated under controlled experimental conditions in vitro using a 60Co gamma radiation source, with subsequent analysis of damage to target proteins (dystrophin, beta-dystroglycan, beta-spectrin, fast and slow myosin heavy chain, NADH tetrazolium reductase, succinate dehydrogenase and cytochrome oxidase) via standard histochemistry, immunocytochemistry and electron microscopy of muscle tissue sections. In general terms, each of the proteins listed above was more susceptible to oxidative damage by OH., compared to O2.-. Different proteins (differing in structure, function or intracellular localisation) showed different susceptibility to oxidative damage, with certain mitochondrial proteins (succinate dehydrogenase, cytochrome oxidase) showing particular susceptibility. In addition, the use of monoclonal antibodies to four different regions of dystrophin showed the latter to contain both resistant and susceptible regions to ROS induced oxidative damage. At the ultrastructural level of subcellular organelle damage, mitochondria were identified as being particularly susceptible to ROS induced oxidative damage. We therefore speculate that oxidative damage to mitochondria and/or mitochondrial proteins may represent the principal initial route of free radical-induced damage within skeletal muscle tissue.