Maternal antioxidant treatment prevents the adverse effects of prenatal stress on the offspring's brain and behavior.

Maternal exposure to stress during pregnancy is associated with an increased risk of psychiatric disorders in the offspring in later life. The mechanisms through which the effects of maternal stress are transmitted to the fetus are unclear, however the placenta, as the interface between mother and fetus, is likely to play a key role. Using a rat model, we investigated a role for placental oxidative stress in conveying the effects of maternal social stress to the fetus and the potential for treatment using a nanoparticle-bound antioxidant to prevent adverse outcomes in the offspring. Maternal psychosocial stress increased circulating corticosterone in the mother, but not in the fetuses. Maternal stress also induced oxidative stress in the placenta, but not in the fetal brain. Blocking oxidative stress using an antioxidant prevented the prenatal stress-induced anxiety phenotype in the male offspring, and prevented sex-specific neurobiological changes, specifically a reduction in dendrite lengths in the hippocampus, as well as reductions in the number of parvalbumin-positive neurons and GABA receptor subunits in the hippocampus and basolateral amygdala of the male offspring. Importantly, many of these effects were mimicked in neuronal cultures by application of placental-conditioned medium or fetal plasma from stressed pregnancies, indicating molecules released from the placenta may mediate the effects of prenatal stress on the fetal brain. Indeed, both placenta-conditioned medium and fetal plasma contained differentially abundant microRNAs following maternal stress, and their predicted targets were enriched for genes relevant to nervous system development and psychiatric disorders. The results highlight placental oxidative stress as a key mediator in transmitting the maternal social stress effects on the offspring's brain and behavior, and offer a potential intervention to prevent stress-induced fetal programming of affective disorders.

Signalling of DNA damage and cytokines across cell barriers exposed to nanoparticles depends on barrier thickness.

The use of nanoparticles in medicine is ever increasing, and it is important to understand their targeted and non-targeted effects. We have previously shown that nanoparticles can cause DNA damage to cells cultured below a cellular barrier without crossing this barrier. Here, we show that this indirect DNA damage depends on the thickness of the cellular barrier, and it is mediated by signalling through gap junction proteins following the generation of mitochondrial free radicals. Indirect damage was seen across both trophoblast and corneal barriers. Signalling, including cytokine release, occurred only across bilayer and multilayer barriers, but not across monolayer barriers. Indirect toxicity was also observed in mice and using ex vivo explants of the human placenta. If the importance of barrier thickness in signalling is a general feature for all types of barriers, our results may offer a principle with which to limit the adverse effects of nanoparticle exposure and offer new therapeutic approaches.

Metal-on-metal bearings: the evidence so far.

Lately, concerns have arisen following the use of large metal-on-metal bearings in hip replacements owing to reports of catastrophic soft-tissue reactions resulting in implant failure and associated complications. This review examines the literature and contemporary presentations on current clinical dilemmas in metal-on-metal hip replacement.

Early failure of the Ultima metal-on-metal total hip replacement in the presence of normal plain radiographs.

Metal-on-metal total hip replacement has been targeted at younger patients with anticipated long-term survival, but the effect of the production of metal ions is a concern because of their possible toxicity to cells. We have reviewed the results of the use of the Ultima hybrid metal-on-metal total hip replacement, with a cemented polished tapered femoral component with a 28 mm diameter and a cobalt-chrome (CoCr) modular head, articulating with a 28 mm CoCr acetabular bearing surface secured in a titanium alloy uncemented shell. Between 1997 and 2004, 545 patients with 652 affected hips underwent replacement using this system. Up to 31 January 2008, 90 (13.8%) hips in 82 patients had been revised. Pain was the sole reason for revision in 44 hips (48.9%) of which 35 had normal plain radiographs. Peri-prosthetic fractures occurred in 17 hips (18.9%) with early dislocation in three (3.3%) and late dislocation in 16 (17.8%). Infection was found in nine hips (10.0%). At operation, a range of changes was noted including cavities containing cloudy fluid under pressure, necrotic soft tissues with avulsed tendons and denuded osteonecrotic upper femora. Corrosion was frequently observed on the retrieved cemented part of the femoral component. Typically, the peri-operative findings confirmed those found on pre-operative metal artefact reduction sequence MRI and histological examination showed severe necrosis. Metal artefact reduction sequence MRI proved to be useful when investigating these patients with pain in the absence of adverse plain radiological features.

Effects of hTERT on genomic instability caused by either metal or radiation or combined exposure.

Genomic instability is considered to be an important component in carcinogenesis. It can be caused by low-dose exposure to agents, which appear to act through induction of stress-response pathways related to oxidative stress. These agents have been studied mostly in the radiation field but evidence is accumulating that chemicals, especially heavy metals such as Cr (VI), can also act in the same manner. Previous work showed that metal ions could initiate long-term genomic instability in human primary fibroblasts and this phenomenon was regulated by telomerase. The aim of this study was to examine the difference in clonogenic survival and cytogenetic damage after exposure to Cr (VI) and radiation both singly and in combination in normal human fibroblasts (hTERT- cells) and engineered human fibroblasts, infected with a retrovirus carrying a cDNA encoding hTERT, which rendered these cells telomerase positive and replicatively immortal (hTERT+ cells). Cr (VI) induced genomic instability in hTERT- cells but not in hTERT+ cells, whereas radiation induced genomic instability in hTERT+ cells and to a lesser extent in hTERT- cells. Combined exposure caused genomic instability in both types of cells. However, this genomic instability was more pronounced in hTERT- cells after radiation followed by Cr (VI) and more pronounced in hTERT+ cells after Cr (VI) followed by radiation. Moreover, the biological effects provoked by combined exposure of Cr (VI) and radiation also led to a synergistic action in both types of cells, compared to either Cr (VI) treatment only or radiation exposure only. This study suggests that telomerase can prevent genomic instability caused by Cr (VI), but not by radiation. Furthermore, genomic instability may be prevented by telomerase when cells are exposed to radiation and then Cr (VI) but not after exposure to Cr (VI) and then radiation.

Macrophages detoxify the genotoxic and cytotoxic effects of surgical cobalt chrome alloy particles but not quartz particles on human cells in vitro.

Particles of surgical cobalt chrome alloy are cytotoxic and genotoxic to human fibroblasts in vitro. In vivo orthopaedic patients are exposed to cobalt chrome particles as a result of wear of a joint replacement. Many of the wear debris particles that are produced are phagocytosed by macrophages that accumulate at the site of the worn implant and are disseminated to local and distant lymph nodes the liver and the spleen. In this study we have tested whether this process of phagocytosis could have altered the cytotoxic and genotoxic properties of the cobalt chrome particles. Quartz particles have been investigated as a control. Micron-sized particles of cobalt chrome alloy were internalised by either white cells of peripheral blood or by THP-1 monocytes for 1 week and 1 day, respectively. The particles were then extracted and presented at different doses to fibroblasts for 1 day. There was a reduction of the cytotoxicity and genotoxicity of the cobalt chrome particles after phagocytosis by white cells or THP-1 cells. Cobalt chrome particles that were internalised by fibroblasts also showed a reduction of their cytotoxicity but not their genotoxicity. In contrast the cytotoxicity and genotoxicity of quartz particles was increased after internalisation by THP-1 cells. The surface morphology of the cobalt chrome particles but not the quartz particles was changed after phagocytosis by THP-1 cells. This study suggests that the genotoxic and cytotoxic properties of particles that fall within the size range for phagocytosis may be highly complex in vivo and depend on the combination of material type and previous phagocytosis. These results may have relevance for particle exposure from orthopaedic implants and from environmental or industrial pollution.

Orthopaedic metals and their potential toxicity in the arthroplasty patient: A review of current knowledge and future strategies.

The long-term effects of metal-on-metal arthroplasty are currently under scrutiny because of the potential biological effects of metal wear debris. This review summarises data describing the release, dissemination, uptake, biological activity, and potential toxicity of metal wear debris released from alloys currently used in modern orthopaedics. The introduction of risk assessment for the evaluation of metal alloys and their use in arthroplasty patients is discussed and this should include potential harmful effects on immunity, reproduction, the kidney, developmental toxicity, the nervous system and carcinogenesis.

The effect of nano- and micron-sized particles of cobalt-chromium alloy on human fibroblasts in vitro.

Wear debris from metal on polyethylene joint replacements causes asceptic loosening as a result of an inflammatory reaction of macrophages to micron-sized particles. Metal on metal implants, which generate nanoparticles, have been reintroduced into surgical practise in order to avoid this problem. There is a current concern about possible long-term effects of exposure to metal particles. In this study, the cytotoxic and genotoxic effects of nanoparticles and micron-sized particles of cobalt chrome alloy have been compared using human fibroblasts in tissue culture. Nanoparticles, which caused more free radicals in an acellular environment, induced more DNA damage than micron-sized particles using the alkaline comet assay. They induced more aneuploidy and more cytotoxicity at equivalent volumetric dose. Nanoparticles appeared to disintegrate within the cells faster than microparticles with the creation of electron dense deposits in the cell, which were enriched in cobalt. The mechanism of cell damage appears to be different after exposure to nanoparticles and microparticles. The concept of nanotoxicology is, therefore, an important consideration in the design of future surgical devices.

Biological effects of metal-on-metal hip replacements.

The advantages seen by patients receiving total hip arthroplasties and the implications of the release of both metal particles and soluble metal ions are discussed. This paper describes some of the early changes that have been observed in metal-on-metal hip arthroplasties, in terms of both changes in metal levels in blood and chromosome changes.

Effects of hTERT on metal ion-induced genomic instability.

There is currently a great interest in delayed chromosomal and other damaging effects of low-dose exposure to a variety of pollutants which appear collectively to act through induction of stress-response pathways related to oxidative stress and ageing. These have been studied mostly in the radiation field but evidence is accumulating that the mechanisms can also be triggered by chemicals, especially heavy metals. Humans are exposed to metals, including chromium (Cr) (VI) and vanadium (V) (V), from the environment, industry and surgical implants. Thus, the impact of low-dose stress responses may be larger than expected from individual toxicity projections. In this study, a short (24 h) exposure of human fibroblasts to low doses of Cr (VI) and V (V) caused both acute chromosome damage and genomic instability in the progeny of exposed cells for at least 30 days after exposure. Acutely, Cr (VI) caused chromatid breaks without aneuploidy while V (V) caused aneuploidy without chromatid breaks. The longer-term genomic instability was similar but depended on hTERT positivity. In telomerase-negative hTERT- cells, Cr (VI) and V (V) caused a long lasting and transmissible induction of dicentric chromosomes, nucleoplasmic bridges, micronuclei and aneuploidy. There was also a long term and transmissible reduction of clonogenic survival, with an increased beta-galactosidase staining and apoptosis. This instability was not present in telomerase-positive hTERT+ cells. In contrast, in hTERT+ cells the metals caused a persistent induction of tetraploidy, which was not noted in hTERT- cells. The growth and survival of both metal-exposed hTERT+ and hTERT- cells differed if they were cultured at subconfluent levels or plated out as colonies. Genomic instability is considered to be a driving force towards cancer. This study suggests that the type of genomic instability in human cells may depend critically on whether they are telomerase-positive or -negative and that their sensitivities to metals could depend on whether they are clustered or diffuse.

Metal-specific differences in levels of DNA damage caused by synovial fluid recovered at revision arthroplasty.

Previous research has shown an increase in chromosomal aberrations in patients with worn implants. The type of aberration depended on the type of metal alloy in the prosthesis. We have investigated the metal-specific difference in the level of DNA damage (DNA stand breaks and alkali labile sites) induced by culturing human fibroblasts in synovial fluid retrieved at revision arthroplasty. All six samples from revision cobalt-chromium metal-on-metal and four of six samples from cobalt-chromium metal-on-polyethylene prostheses caused DNA damage. By contrast, none of six samples from revision stainless-steel metal-on-polyethylene prostheses caused significant damage. Samples of cobalt-chromium alloy left to corrode in phosphate-buffered saline also caused DNA damage and this depended on a synergistic effect between the cobalt and chromium ions. Our results further emphasise that epidemiological studies of orthopaedic implants should take account of the type of metal alloy used.

Effect of synovial fluid, phosphate-buffered saline solution, and water on the dissolution and corrosion properties of CoCrMo alloys as used in orthopedic implants.

The corrosion and dissolution of high- and low-carbon CoCrMo alloys, as used in orthopedic joint replacements, were studied by immersing samples in phosphate-buffered saline (PBS), water, and synovial fluid at 37 degrees C for up to 35 days. Bulk properties were analyzed with a fine ion beam microscope. Surface analyses by X-ray photoelectron spectroscopy and Auger electron spectroscopy showed surprisingly that synovial fluid produced a thin oxide/hydroxide layer. Release of ions into solution from the alloy also followed an unexpected pattern where synovial fluid, of all the samples, had the highest Cr concentration but the lowest Co concentration. The presence of carbide inclusions in the alloy did not affect the corrosion or the dissolution mechanisms, although the carbides were a significant feature on the metal surface. Only one mechanism was recognized as controlling the thickness of the oxide/hydroxide interface. The analysis of the dissolved metal showed two mechanisms at work: (1) a protein film caused ligand-induced dissolution, increasing the Cr concentration in synovial fluid, and was explained by the equilibrium constants; (2) corrosion at the interface increased the Co in PBS. The effect of prepassivating the samples (ASTM F-86-01) did not always have the desired effect of reducing dissolution. The release of Cr into PBS increased after prepassivation. The metal-synovial fluid interface did not contain calcium phosphate as a deposit, typically found where samples are exposed to calcium rich bodily fluids.

An unusual lymphocytic perivascular infiltration in tissues around contemporary metal-on-metal joint replacements.

BACKGROUND: Metal-on-metal bearing surfaces have been reintroduced for use during total hip replacement. To assess tissue reactions to various types of articulations, we studied the histological appearance of periprosthetic tissues retrieved from around metal-on-metal and metal-on-polyethylene total hip replacements and compared these findings with the appearance of control tissues retrieved at the time of primary arthroplasty. METHODS: Periprosthetic tissues were obtained at the time of revision of twenty-five cobalt chromium-on-cobalt chromium, nine cobalt chromium-on-polyethylene, and ten titanium-on-polyethylene total hip arthroplasties. Control tissues were obtained from nine osteoarthritic hips at the time of primary total hip arthroplasty. Each tissue sample was processed for routine histological analysis, and sections were stained with hematoxylin and eosin. Quantitative stereological analysis was performed with use of light microscopy. RESULTS: Tissue samples obtained from hips with metal-on-metal implants displayed a pattern of well-demarcated tissue layers. A prominent feature, seen in seventeen of twenty-five tissue samples, was a pattern of perivascular infiltration of lymphocytes. In ten of the tissue samples obtained from hips with metal-on-metal prostheses, there was also an accumulation of plasma cells in association with macrophages that contained metallic wear-debris particles. The surfaces of tissues obtained from hips with metal-on-metal prostheses were more ulcerated than those obtained from hips with other types of implants, particularly in the region immediately superficial to areas of perivascular lymphocytic infiltration. The lymphocytic infiltration was more pronounced in samples obtained at the time of revision because of aseptic failure than in samples retrieved at the time of autopsy or during arthrotomy for reasons other than aseptic failure. Total-joint-replacement and surface-replacement designs of metal-on-metal prostheses were associated with similar results. Tissue samples obtained from hips with metal-on-polyethylene implants showed far less surface ulceration, much less distinction between tissue layers, no pattern of lymphocytic infiltration, and no plasma cells. The inflammation was predominantly histiocytic. Tissues retrieved from hips undergoing primary joint replacement showed dense scar tissue and minimal inflammation. CONCLUSIONS AND CLINICAL RELEVANCE: The pattern and type of inflammation seen in periprosthetic tissues obtained from hips with metal-on-metal and metal-on-polyethylene implants are very different. At the present time, we do not know the prevalence or clinical implications of these histologic findings, but we suggest that they may represent a novel mode of failure for some metal-on-metal joint replacements.

Wear debris from hip or knee replacements causes chromosomal damage in human cells in tissue culture.

Wear debris was extracted from 21 worn hip and knee replacements. Its mutagenic effects were tested on human cells in tissue culture using the micronucleus assay and fluorescent in situ hybridisation. The extracted wear debris increased the level of micronuclei in a linear dose-dependent manner but with a tenfold difference between samples. The concentration of titanium +/- vanadium and aluminium within the wear debris was linearly related both to the level of centromere-positive micronuclei in tissue culture, indicating an aneuploid event, and to the level of aneuploidy in vivo in peripheral blood lymphocytes. The concentration of cobalt and chromium +/- nickel and molybdenum in the wear debris correlated with the total index of micronuclei in tissue culture, both centromere-positive and centromere-negative i.e. both chromosomal breakage and aneuploidy events. The results show that wear debris can damage chromosomes in a dose-dependent manner which is specific to the type of metal. The results from studies in vitro correlate with those in vivo and suggest that the wear debris from a worn implant is at least partly responsible for the chromosomal damage which is seen in vivo.

Particulate debris from a titanium metal prosthesis induces genomic instability in primary human fibroblast cells.

Previous studies detected both lethal and cumulative chromosomal aberrations in bone marrow and peripheral blood of patients with worn hip and knee replacements. This study shows that wear debris from a worn titanium metal on high-density polyethylene hip replacement also produces chromosomal instability and reproductive failure in cell culture. The progeny of these treated cells also displayed chromosomal instability, mainly consisting of chromatid breaks and minutes, and reproductive failure as determined by clonogenic survival many generations postexposure. These delayed effects are similar to those caused by the heavy metals cadmium and nickel and to those seen for low-dose radiation. These findings may have important implications with regard to the long-term risks of joint replacement surgery. This highlights the need for long-term epidemiological studies of patients with surgical implants.

Increased chromosome translocations and aneuploidy in peripheral blood lymphocytes of patients having revision arthroplasty of the hip.

The long-term biological effects of wear debris are unknown. We have investigated whether there is any evidence of cumulative mutagenic damage in peripheral blood lymphocytes of patients undergoing revision arthroplasty of predominantly metal-on-plastic total hip replacements compared with those at primary arthroplasty. There was a threefold increase in aneuploidy and a twofold increase in chromosomal translocations which could not be explained by the confounding variables of smoking, gender, age and diagnostic radiographs. In the patients with TiVaAl prostheses there was a fivefold increase in aneuploidy but no increase in chromosomal translocations. By contrast, in patients with cobalt-chrome prostheses there was a 2.5-fold increase in aneuploidy and a 3.5-fold increase in chromosomal translocations. In six patients with stainless-steel prostheses there was no increase in either aneuploidy or chromosomal translocations. Our results suggest that future epidemiological studies of the putative long-term risks of joint replacement should take into account the type of alloy used in the prosthesis.

Development of a routine method for the determination of trace metals in whole blood by magnetic sector inductively coupled plasma mass spectrometry with particular relevance to patients with total hip and knee arthroplasty.

BACKGROUND: Joint-replacement surgery has revolutionized the treatment of osteoarthritis and is still the most effective therapy. A recent clinical trend reintroducing metal-on-metal bearing surfaces has in turn stimulated a requirement for accurate measurement of the concentrations of relevant metals in both pre- and postoperative patients. Thus, there is a need for cost-effective, multielement methods for trace metal analysis in whole blood to monitor possible increases in wear metal concentrations. METHODS: A method was developed to allow routine analysis of whole blood samples for molybdenum, cobalt, chromium, and nickel. Sample preparation consisted of a simple 1:10 dilution of whole blood with a solution of 10 mL/L Triton X-100, 0.0002 mol/L EDTA, and 0.01 mol/L ammonium hydroxide. Final determination was performed by a double-focusing magnetic sector inductively coupled plasma mass spectrometer operated in medium-resolution mode (resolution, 3400). Online addition of rhodium was used for internal standardization. RESULTS: Detection limits in whole blood were 0.06 microg/L for chromium, cobalt, and molybdenum and 0.30 microg/L for nickel. Base concentrations of 0.22, 0.17, 0.62, and 0.99 microg/L for chromium, cobalt, molybdenum, and nickel, respectively, in whole blood have been found. Polyatomic interferences on all four elements have been shown to be resolved from the analyte masses by use of a resolution of >3000. CONCLUSIONS: The simple, rapid method of sample preparation is effective in minimizing potential contamination and enables 60 samples (run time, 8 h) to be analyzed before cleaning the instrument is necessary. A resolution >3000 was sufficient to separate polyatomic interferences from the masses of interest. The method was used to analyze a large number of blood samples taken from primary patients awaiting total hip arthroplasty. The method is sensitive enough to provide base concentrations for chromium, cobalt, and molybdenum in whole blood. The results for nickel were compromised by high signals for blank samples.

Nerve injury-associated kinase: a sterile 20-like protein kinase up-regulated in dorsal root ganglia in a rat model of neuropathic pain.

Partial injury of the rat sciatic nerve elicits a variety of characteristic chemical, electrophysical and anatomical changes in primary sensory neurons and constitutes a physiologically relevant model of neuropathic pain. To elucidate molecular mechanisms that underlie the physiology of neuropathic pain, we have used messenger RNA differential display to identify genes that exhibit increased ipsilateral expression in L4/5 dorsal root ganglia, following unilateral partial ligation of the rat sciatic nerve. One set of partial complementary DNA clones identified in this screen was found to encode a protein kinase, nerve injury-associated kinase. Cloning of the full-length human nerve injury-associated kinase complementary DNA, together with recombinant expression analysis, reveal nerve injury-associated kinase to be a functional member of a subgroup of sterile 20-like protein kinases characterised by the presence of a putative carboxy terminal autoregulatory domain. Induction of nerve injury-associated kinase expression in dorsal root ganglia in the rat neuropathic pain model was confirmed by quantitative reverse transcription-polymerase chain reaction, and RNA in situ hybridization analysis revealed enhanced levels of nerve injury-associated kinase within neurons.Together, our data implicate nerve injury-associated kinase as a novel upstream component of an intracellular signalling cascade that is up-regulated in dorsal root ganglia neurons in response to sciatic nerve injury.

Changes in the proportions of peripheral blood lymphocytes in patients with worn implants.

We compared the peripheral blood and periprosthetic tissues of 53 patients at revision arthroplasty with those of 30 patients at primary arthroplasty to determine whether there is a systemic difference in lymphocytes in patients with worn hip implants. The absolute number and relative proportion of lymphocytes bearing CD2, CD3, CD4, CD8, CD16, CD19, HLA-DR, kappa and lambda antigens were compared with the levels of IL-1beta, IL-6 and PGE2 in the pseudosynovial membrane as well as with a semiquantitative estimate of metal and polyethylene particles, necrosis and chronic inflammation and the total concentration of metals within the periprosthetic tissues. There was a significant increase in the relative proportion of CD2-positive T-cells and CD16-positive natural killer cells in the peripheral blood at revision arthroplasty compared with primary arthroplasty and an increased proportion of CD8-positive T-cells and a decreased ratio of CD4 to CD8 (helper inducer/suppressor cytotoxic cells). Three control patients, who went on to have revision surgery, had values at primary arthroplasty which were similar to those of patients at the time of revision surgery. These differences did not correlate with the local concentration of metal, plastic or cement or inflammatory response or the type of prosthesis. An inverse correlation was noted between the necrosis in the periprosthetic tissue and both the local production of IL-6 and the absolute numbers of T-cells in peripheral blood. We conclude that there may be several cell-mediated systemic immune responses to aseptic loosening, at least one of which may be directly related to events in the periprosthetic tissues. We cannot exclude the possibility that the changes in the proportion of CD8-positive cells reflected a predisposition, rather than a reaction, to loosening of the implant.