Key words: nitric oxide, mutagenesis
Nitric oxide (NO) is a bioregulator and toxin induced in the immune, cardiovascular, neuronal and other systems. It is the endogenous mediator of toxic shock resulting from certain bacterial infections. However, it has increasing therapeutic uses related to the control of blood pressure (drugs such as nitroglycerine) and alleviation of respiratory insufficiency in newborns (devices instilling NO gas directly into the lungs). Studies in OST, in collaboration with scientists at NCI, have established that NO gas and drugs that generate it, including nitroglycerine, are mutagenic. Therefore, it is necessary to understand the genetic toxicology of NO in order to assess the benefits and risks of NO-based therapies. OST is working to understand the types of damage and the types of mutations generated by NO in vitro, as a fingerprint that can be identified in vivo. Recently we found that E. coli, a likely target of NO in vivo, is immune to mutagenesis by NO, even when a major repair system is inactivated. A close relative, Salmonella typhimurium, is susceptible to mutagenesis. Understanding the reason for this surprising result in these closely related organisms should help to understand how NO functions as a genotoxin in cells. [ProA]
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Key words: biomarkers, polyurethane, cytotoxic
Poly(ester)urethanes are or have been used in a number of medical devices, for example breast implants, gastric bubbles, endotracheal tubes, catheters, blood and plasma bags, etc. In order to understand the impact of this material in human beings, some means to identify and track the degradation of the polyurethane in vivo needs to be developed. OST has identified a possible in vivo biomarker of exposure to polyurethane exposure in humans: 2, 4-TDA-Human Serum Albumin. We are currently examining this as a potential biomarker for women with breast implants. Preliminary results indicate that this may not be a conjugate which would exclusively indicate polyurethane exposure, as with a breast implant. This may be due to a number of factors, not least of which is "environmental" exposure to products which contain TDI (toluene diisocyanate) and TDA, such as furniture refinishing products, paints, etc. Other possible biomarkers selective for polyurethane are currently being explored.
In addition, studies indicate the particles and monomers released from the metabolism of polyurethane can create adverse effects. To assess the cytotoxic potential of a material, simple means to screen for the cytotoxic potential of polyurethane in vivo must be developed. We have used a number of different extraction conditions and solvents to provide a more complete evaluation of the cytotoxic potential of polyurethane to a number of different cell lines in vitro. Some conditions indicate that there is a strong cytotoxic effect of polyurethane extracts over time. OST has just completed a full scale study to characterize this cytotoxic effect and then will apply these new techniques to assess the cytotoxicity of other biomaterial extracts.
The impact of this work would be to provide better methods and strategies to test biomaterials in general. By designing methods to easily screen for cytotoxic effects and strategies for developing and validating biomarkers, the benefit to the regulatory aspect of this work is to provide specific information on polyurethanes. The general benefit to the FDA would be to provide information on strategies to better evaluate and regulate materials for toxic potential. [ProA]
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Key words: immunotoxicity
OST, with ODE, prepared an overview of the current role of immunotoxicity testing in regulatory review of medical device applications. Based on this survey, a joint OST/ODE committee to develop an "Immunotoxicity Testing Framework" that will provide reviewers and manufacturers with a coherent process for optimizing immunotoxicity testing was established. A revised draft framework is being prepared. [ProA]
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Key words: latex, allergenic, protein measurement
Natural latex in medical devices can cause life-threatening Type 1 allergic reactions in individuals sensitized to latex proteins. HSB latex research project was initiated to solve the problem of severe allergic reactions. It included efforts to provide basic data on the nature of allergens and to develop methodology for evaluating the allergenic potential of latex products. The studies are focused on the identification of allergenic proteins in latex in order to reduce or eliminate them from finished latex products. Furthermore, OST is studying the correlation of the extractable protein level and several in vitro tests for latex allergenicity with skin testing in vivo. This initiative resulted in the collaborative project with the Johns Hopkins University and will ultimately define, clinically, the most relevant in vitro test as an alternative to skin testing in humans for the evaluation of the safety of latex products and assessment of latex sensitivity.
Significant effort is focused on the development of methodology for a total protein measurement. In collaboration with ASTM, OST's modification of the Lowry method was developed into a standard protocol. OST scientists are continuing joint efforts with ASTM to further develop a more sensitive method for detecting very small amounts of protein and insure the safety of latex products. A standardized, relevant methodology for the assessment of allergenic potential of finished latex products is critical to manufacturers in their efforts to develop safe, nonallergenic products and help prevent life-threatening reactions. [ProA]
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Key words: gloves, powder, ASTM D11.40
Sensitivity to natural rubber latex and the fact that powder used on latex gloves can carry latex allergens are both well established. Minimizing or eliminating powder on gloves helps address the sensitivity problem. Hence, a need for determining powder residues on "powderless" gloves arose in order to establish operational qualifications for these products as needed by FDA for product review and compliance assessments. ASTM D11.40 task group proposed a draft method for determining powder residues. In-house laboratory efforts showed that the original method for determining powder residues. These included minimization of the number of transfer steps and, most importantly, the inclusion of a glove rinse step. The laboratory efforts also established the validity of the modified method and established that its capabilities are sufficient for regulatory purposes. The results have been communicated to DGRDG and ASTM D11.40. This work has been submitted to the ASTM Journal of Research and Testing for publication. [Stds]
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Key words: mercury, mercury vapor, amalgam
Studies are being conducted in order to evaluate the effects of mercury vapor on gene expression in rat brain. Mercury constitutes 50% of dental amalgam, and amalgam restorations release small amounts of mercury vapor (elemental mercury). This vapor is absorbed and distributed throughout the body, localizing in the kidney and brain. Dental practitioners who use mercury have elevated urinary mercury levels, and in persons who have amalgam restorations, the number of amalgams is directly proportional to the urinary mercury concentration. Since mercury is a known neurotoxicant, exposure to mercury vapor during pregnancy may interfere with brain development. In contrast to animal studies, reports of human reproductive and developmental effects due to occupational vapor exposure have been inconclusive.
Since controlled mercury vapor (the form of mercury that is of concern) exposures require an inhalation facility with analytical capability and safety controls, initial series of experiments will be conducted using methyl mercury (MeHg). Like mercury vapor MeHg is lipophilic, and mercury levels can be easily elevated in brains of rats injected with MeHg. Progress to date has focused on dissecting rat brain into six regions, including targets for mercury (cerebellum, cortex, and hippocampus). Immunoblotting experiments with heat shock protein (hsp) antibodies are planned in order to identify region-specific induction of hsps, as well as radiolabeling studies to asses de novo synthesis and immunohistochemistry to identify cell-specific induction. Future mercury vapor inhalation studies in rats are being coordinated through the NIEHS Inhalation Toxicology Group, RTP, NC.
These studies will help in identifying the responses of target tissues to low-level mercury exposures. This will equip the PHS to better assess the risks associated with exposures to mercury from amalgam restorations. [PreME, PostMS]
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Key words: chemical toxicants, mercury, stress proteins, kidney
Current methods to identify potential hazards of medical device materials are based on limited toxicity information, resemblance to known chemical toxicants, and extensive extrapolation. Perturbation of stress gene expression is being evaluated as a marker of chemical exposure and toxicity. Previous studies in OST labs demonstrated that the expression of inducible gene products, i.e., stress proteins, after exposure to mercury is target tissue-specific. Studies are now being undertaken in order to assess the cell-type specificity of the stress response within a target tissue.
Immunohistochemical localization of stress proteins using monoclonal antibodies specific for various stress proteins was undertaken in kidney. Immunohistochemical staining for stress proteins revealed intense staining primarily in renal tubule cells undamaged by mercury. Induction of stress proteins in kidney may play a role in mediating the nephrotoxicity of mercuty.
Improvements in molecular toxicology methods and their integration into routine testing regimes will enable FDA to enhance identification of toxic materials, respond to materials safety issues, and improve assessments of risk regarding materials used in medical devices. [PreME, PostMS]
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Key words: biomarker, stress protein, gene expression, toxicants
In order to assess the pertubations of gene expression in embryos as a biomarker of developmental toxicity, we are evaluating interspecies comparisons of the effects of metals, known to be developmental toxicants, on stress protein synthesis in developing rat and avian embryos.
Cadmiun, a strong stress protein inducer in many biological systems, does
not induce
stress protein synthesis in cultured rat embryos, even though stress proteins
are easily induced in this organism following heat shock. OST has conducted
experiments to see if this teratogen, along with arsenite and mercury, are
inducers of stress proteins in avian embryos. The metals induced the synthesis
of major proteins (hsp70, hsp90, hsp28) in a dose- and time-dependent manner.
All three metals induced the synthesis of hsp70 and hsp90, with arsenite
being the most efficacious, and arsenite induced a unique protein: hsp27.
Thus, several changes in gene expression appear to be related to specific
metals. Morphologic evaluation of the embryos indicated that the expression
of these gene products occurs prior to gross morphologic changes.
A major uncertainty inherent in toxicology testing is the extrapolation of data across species, e.g., are results from 2-year rodent studies predictive of human health effect? If endogenous molecular mechanisms common to all species appear to be involved with the toxic response, the application of a toxicity screening method for human risk assessments related to medical device materials becomes more plausible. [PreME, PostMS]
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Key words: hormones, estrogenic, women's health
Some chemicals associated with medical devices (as plastics, plasticizers, binders, solvents, monomers, or other components) have been shown to act as hormones. Because of increasing interest in women's health issues, OST has started examining the estrogenic effects of some chemicals associated with medical devices. The first group of chemicals to be examined are the phthalates, used as plasticizers.
A portion of the work this year consisted of developing the mouse uterotrophic assay in the laboratory by working out the details of dose, strain, time, and age. In addition, OST scientists have conducted a survey of nine phthalates. While variability was a problem, OST obtained evidence of oestrogenic activity (either estrogenic or antiestrogenic) for several of these chemicals. Four gave responses that were more equivocal and need further definition. OST scientists are undertaking more definitive assays for each of these responses in ovariectomized mice. They also sought to make the assay more sophisticated by examining the use of heat shock proteins (which are known to be involved in estrogen receptor binding) as markers for estrogenic effect. This work is in the preliminary stages of methods development and is the subject of a pending proposal for next year.
The results already obtained suggest that the initial premise of this research-that materials found as components of medical devices may have hormonal effects-is correct, although these effects are, as yet, poorly defined or quantified. While the levels of these materials released from devices is probably small, hormones are highly potent. In the healthy woman, the amount of estrogenic activity contributed by device materials may be of no significance, but in the compromised patient, or the patient receiving hormonal/antihormonal therapies, the contribution from devices might have medical significance. Knowledge of the hormonal activity from medical device materials might lead to alterations of the therapeutic regime and may significantly affect device evaluation. [PreME, PostMS]
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Key words: inflammatory, macrophage, PMMA, LPS
Any material implanted into the body evokes a biological response. Some responses are simply fibrous capsule formation protecting both the implant and the host. However some responses develop into chronic inflammatory responses with macrophage accumulation and development of giant cells. It is the purpose of these studies to document the in vitro responses of mouse macrophages to particles from biomaterials and to document the in vivo response to these same materials in particle form or as solid materials.
The in vitro studies are being developed, and so far there is much data accumulated on PMMA (polymethyl-methacrylate: bone cement), HA (hydroxyapatite, a calcium phosphate ceramic used to coat dental and orthopedic implants), and the oxides of titanium and cadmium which would be the surface of implants made of these metals. The production of biologically active substances such as nitric oxide and tumor necrosis factor alpha (which are important substances in inflammatory responses and the production of cytokines such as Il-6, Il-4, and Il-10) involved in the immune responses are being assessed. Lipopolysaccharide (LPS), which is a component of the cell wall of Gram negative bacteria and a known stimulant of cells, is added to macrophage cultures in the presence and absence of the particles. It is evident to date that there is strong production of the biologically active substances by the LPS alone. The addition of HA or PMMA, with the LPS to the macrophage cultures, markedly potentiates the production of these substances. Whether it is additive or synergistic is still being assessed. On the other hand, the presence of the metallic oxides depresses the response to LPS.
The in vivo data have indicated the formation of a capsule composed of fibroblasts and macrophages when the material is placed into the peritoneal cavity for several days. These cells can then be grown in culture and their activity assessed. The production of the biologically active substances by these peritoneal cells in the absence of LPS is very low. The addition of LPS stimulates the active response. The in vitro data and the in vivo data seem to be in agreement and giving correlative studies.
These findings on the different responses to the different particles and materials will help delineate the differences in biological responses and nature of the inflammatory responses to devices. It will assist in understanding and predicting tissue responses. The observations on the interaction between LPS and the materials is important in assessing the impact of infection on the tissue response to materials and loss of tissue such as bone resorption. There will be a much better understanding of the role of materials and particles in adverse biological responses to devices and in the development of, or consequences of, infection. [PreME, PostMS]
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Key words: tumors, medical implants, protheses, metallic, mutagenesis
Development of tumors near medical implants raises concerns regarding the safety of certain implant materials. Metal prostheses consist mainly of iron in titanium and cobalt alloys. Copper is the main component of one of the intrauterine contraceptive devices and a component of dental casting alloys and amalgams. All implanted metallic materials corrode and release ions or particulate matter into the surrounding tissue. It has been suggested that long-term use of medical implants, made from either metallic or synthetic materials, may cause mutations or be carcinogenic. Better understanding of the processes and interactions between materials and the biological environment is needed for assessing the risk of a variety of metal-containing medical implants.
In this study, a method was developed for the early detection of DNA base
modifications that are associated with mutagenesis. The mutagenicity of
iron ions alone or combined with a chelator was studied. Genetic modifications
were detected in experimental animals surrounding copper leaching implants.
The data gathered in this project may assist in assessing the potential
health (e.g. carcinogenic) risk of copper-releasing devices.
[PreME, PostMS]
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Key words: prenatal, heat shock proteins (HSP), morphological
This project is designed to examine the association between insults during prenatal organogenesis and the alteration of embryonic and perinatal morphology and the expression of heat shock proteins (HSPs) and other molecular events. The ultimate focus of the project is to determine if HSPs and other molecular endpoints can be used as biological markers of exposure and/or effect of developmental toxicants. Previous work demonstrated that heat exposure at different temperatures and durations of time and, at different critical periods, will alter endpoints of embryo/fetal development.
Our studies have indicated that, both in vivo and in vitro, the temperature and duration of exposure at the target site (i.e., the embryo) is critical. The extent of effect is dependent on the time of insult and may be related to the genetic expression that is occurring at that time. OST studies on HSP expression indicate a strong association with levels of hyperthermia that produce morphological alterations. Future studies in this area are focusing on 1) further definition of the association of morphological development with HSP expression; 2) investigation of the cellular events that are altered by insult; 3) modeling of the relationship of exposure level, duration, and effect for use in biologically-based, dose-response models and risk assessment; and 4) extension of the model to other potential developmental toxicants.
The Center for Devices and Radiological Health, FDA, and the Office of Research
and Development, EPA, are responsible for developing experimental methods
that can assist in the testing and assessment of chemical and physical agents.
Within the field of developmental toxicology, there is an increasing emphasis
on developing biologically-based, dose-response models that will assist
in the incorporation of experimental and test data into the risk assessment
process. Currently, the use of pharmocokinetic and mechanistic data is limited.
The on-going studies noted above are part of a major research effort in
expanding understanding of biologicallybased, dose-response models and developing
more biologically appropriate methods for assessing the human health risk
of real or potential exposures. [ProA]
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