Utility of in vivo metabolomics to support read-across for UVCB substances under REACH.

Structure-based grouping of chemicals for targeted testing and read-across is an efficient way to reduce resources and animal usage. For substances of unknown or variable composition, complex reaction products, or biological materials (UVCBs), structure-based grouping is virtually impossible. Biology-based approaches such as metabolomics could provide a solution. Here, 15 steam-cracked distillates, registered in the EU through the Lower Olefins Aromatics Reach Consortium (LOA), as well as six of the major substance constituents, were tested in a 14-day rat oral gavage study, in line with the fundamental elements of the OECD 407 guideline, in combination with plasma metabolomics. Beyond signs of clinical toxicity, reduced body weight (gain), and food consumption, pathological investigations demonstrated the liver, thyroid, kidneys (males only), and hematological system to be the target organs. These targets were confirmed by metabolome pattern recognition, with no additional targets being identified. While classical toxicological parameters did not allow for a clear distinction between the substances, univariate and multivariate statistical analysis of the respective metabolomes allowed for the identification of several subclusters of biologically most similar substances. These groups were partly associated with the dominant (> 50%) constituents of these UVCBs, i.e., indene and dicyclopentadiene. Despite minor differences in clustering results based on the two statistical analyses, a proposal can be made for the grouping of these UVCBs. Both analyses correctly clustered the chemically most similar compounds, increasing the confidence that this biological approach may provide a solution for the grouping of UVCBs.

Analysis of metabolome changes in the bile acid pool in feces and plasma of antibiotic-treated rats.

The bile acid-liver-gut microbiota axis plays an important role in the host's health. The gut microbiota has an impact on the bile acid pool, but also the bile acids themselves can influence the gut microbiota composition. In this study, six antibiotics from five different classes (i.e. lincosamides, glycopeptides, macrolides, fluoroquinolones, aminoglycosides) were used to modulate microbial communities of Wistar rats to elucidate changes in the bile acid metabolism and to identify key metabolites in the bile acid pool related to gut microbial changes. 20 primary and secondary bile acids were analyzed in plasma and feces of control and treated animals. Antibiotics treatment induced significant changes in primary and secondary bile acids in both matrices. Taurine-conjugated primary bile acids significantly increased in plasma and feces. Contrary, cholic acid and most of the analyzed secondary bile acids significantly decreased in plasma, and cholic acid accumulated in the feces after treatment with all antibiotics but roxithromycin. Despite the different activity spectra of the antibiotics applied against gut microbes, the overall effect on the bile acid pool tended to be similar in both matrices except for streptomycin. These results show that changes in the gut microbial community affect the bile acid pool in plasma and feces and that changes in the bile acid profile can be indicative of alterations of the gut microbiome. Due to the important role of bile acids for the host, changes in the bile acid pool can have severe consequences for the host.

Metabolomics as read-across tool: An example with 3-aminopropanol and 2-aminoethanol.

Read-across and grouping is one of the most commonly used alternative approaches for data gap filling in registrations submitted under the REACH Regulation as defined by the European Chemicals Agency (ECHA) in their 'Read-Across Assessment Framework' (RAAF, 2017). At the same time, the application of read-across is rejected by ECHA frequently due to various reasons. As a major reason hereof, applicants fail to reduce the level of 'remaining uncertainty' intrinsical to every read-across approach compared to testing a substance experimentally. Recently, the use of metabolomics to support read-across cases with biological information has been reported in a case study with phenoxy herbicides (Ravenzwaay et al., 2016). In the present case-study a 'weight-of-evidence' read-across approach from 2-aminoethanol (MEA = 'source') to 3-aminopropanol (3AP = 'target') with metabolomics as 'supporting evidence' reducing the remaining uncertainties is reported. We demonstrate the high structural similarity of the two analogous substances based on the available data and we report how metabolome data add confidence concerning mechanistic similarity in this read-across approach. Finally, the herein described read-across case supported by metabolomics is used to cover the data gaps in repeated dose and reproductive toxicity endpoint of 3AP via weight of evidence for the REACH-registration.

Microbiome-related metabolite changes in gut tissue, cecum content and feces of rats treated with antibiotics.

The metabolic functionality of the gut microbiota contributes to the metabolism and well-being of its host, although detailed insight in the microbiota's metabolism is lacking. Omics technologies could facilitate unraveling metabolism by the gut microbiota. In this study, we performed metabolite profiling of different matrices of the gut, after antibiotic treatment of rats in order to evaluate metabolite changes observed at different dose levels and in different sexes, and to identify the best tissue matrix for further investigations regarding an assessment of metabolic effects of new compounds with antibiotic activity. Three different antibiotics (vancomycin, streptomycin and roxithromycin) were administered orally to rats for 28 days according to the OECD 407 guideline with a subsequent metabolic profiling in feces, cecum content and gut tissue (jejunum, ileum, cecum, colon and rectum). The data were analyzed in the MetaMap®Tox database. Treatment-related effects could be observed in the metabolite profile of feces and cecum content, but not of the different gut tissues. The metabolite profile showed compound specific effects on the microbiome. In line with the activity spectra of the antibiotics tested, vancomycin showed the largest effects, followed by roxithromycin and then by streptomycin for which changes were modest. In general, for all antibiotics the largest changes were observed for the classes of lipids (increase up to 94-fold), bile acids (increase up to 33-fold), amino acids (increase up to 200-fold) and amino acid related (increase up to 348-fold). The most relevant changes in metabolite values were similar in feces and cecum content and among sexes. The results of this targeted analysis indicate that the metabolic profiles of male and female animals in the gut microbiome are comparable. Concluding, taking other samples than feces does not add any extra information. Thus, as a non-invasive sampling method, feces provide a suitable matrix for studies on metabolism by the gut microbiota.

Metabolomics as read-across tool: A case study with phenoxy herbicides.

New technologies, such as metabolomics, can address chemical grouping and read across from a biological perspective. In a virtual case study, we selected MCPP as target substance and MCPA and 2,4-DP as source substances with the goal to waive a 90-day study with MCPP. In order to develop a convincing case to show how biological data can substantiate read across, we used metabolomics on blood samples from the 28-day studies to show the qualitative and quantitative similarity of the substances. The 28-day metabolome evaluation of source substances and the target substance indicate liver and kidneys as target organs. 2,4-DP was identified as the best source substance. Using the information of the 90-day 2,4-DP study, we predicted MCPP's toxicity profile at 2500 ppm: reduced food consumption and body weight gain, liver and kidney weight increases with clinical-pathology changes and a moderate red blood cell parameter reduction. NOEL prediction for MCPP was below that of 2,4-DP (<500 ppm), and similar to that of MCPA (≥150 ppm). Qualitatively, these predictions are comparable to the results of the real MCPP 90-day study in rats (reduced food consumption and body weight gain, weight increases and clinical-pathology changes in liver and kidneys, reduced red blood cells values). Quantitatively, the predicted NOAEL (150 ppm) is similar to the actual study (NOEL = 75 ppm, NOAEL ≤ 500 ppm). Thus, the 90-day rat toxicity study of MCPP could have been waived and substituted by the 90-day results of 2,4-DP by using metabolome data of 28 day studies.

Occupational physicians' perceptions and impact of 2009 GMC consent guidelines.

BACKGROUND: In 2009, the General Medical Council (GMC) updated its guidance on consent, introducing a new statement that employees should be offered the opportunity to view reports, before the reports are sent to the employer. AIMS: To investigate the effects of this change on the perceptions and practice of occupational physicians. METHODS: A cross-sectional survey of UK occupational physicians via the Society of Occupational Medicine e-newsletter, seeking their opinions of the anticipated and actual effects of the guidance on employers, employees, occupational physicians and occupational health services. RESULTS: Two hundred and ninety-five completed questionnaires were returned (estimated response rate 30%). Respondents included 25% of accredited UK specialists. Some reported improved standards and greater transparency, however the change was generally perceived as unfavourable, with employee and employer losses: 50% reported delay providing timely advice to employers and 35% reported delays in employees returning to work. Significant variation in practice and increased costs were reported, with variable effects on different services. Difficulties in areas such as pensions and health surveillance were reported. Some occupational physicians had moved to instant reporting; others had moved away from this to allow more care with wording of reports. CONCLUSIONS: We found significant variations in practice between occupational physicians and concerns of employee and employer losses as a result of changes to the GMC consent guidance. Clearer guidance on practical implementation was desired. The background ethical reasoning should be stated so that the parameters of the guidance are delineated and its reach should be clarified.

Added value of plasma metabolomics to describe maternal effects in rat maternal and prenatal toxicity studies.

For regulatory purposes prenatal developmental toxicity (OECD No. 414) studies are routinely performed in our laboratories. The suitability of metabolomics as technology to identify maternal toxicity in such studies was investigated. Plasma was sampled from pregnant, non-fasted rats on gestation day 20 before cesarean section. Metabolite profiling was performed by gas- and liquid-chromatography-tandem mass spectrometry techniques. The sensitivity of routinely examined maternal toxicity parameters (OECD No. 414) was compared to those of metabolome analysis. Evaluating 44 studies, the metabolome-derived NOEL was more sensitive in 45% of the cases in detecting maternal toxicity than the maternal NOAEL. Metabolome patterns indicative for liver effects and 4-hydroxyphenylpyruvate dioxygenase (HPPD) enzyme-inhibition were established in pregnant rats based on regulated metabolites using reference compounds. The HPPD inhibition and liver toxicity patterns in pregnant rats were reasonably comparable to the ones established in non-pregnant, fasted rats. Metabolomics is a useful tool for an improved and mechanism-based identification of maternal toxicity in maternal and prenatal toxicity studies. The data suggest that the current classical maternal toxicity parameters may underestimate the extent of effects of compounds on the dams.

Role of DHEA and growth factors in chromaffin cell proliferation.

Dehydroepiandrostreone (DHEA) is a neuroactive steroid produced by the inner layer of the adrenal cortex close to the adrenomedullary cells. Chromaffin cell growth and proliferation are under the control of insulin-like growth factor II (IGF-II) and basic fibroblast growth factor (bFGF). The aim of the present study was to examine the role of DHEA on chromaffin cell proliferation induced by IGF-II and bFGF. In our model, DHEA significantly decreased IGF-II-induced proliferation by 48.7%, whereas it did not affect the proliferation induced by bFGF. These data suggest that DHEA exerts a paracrine function in the control of chromaffin cell growth.

Fluorescence based cell counting in collagen monolayer cultures of primary hepatocytes.

Accurate determination of cell number is essential for the quantitative description of biological processes. The changes should be related to a measurable reference e.g. in the case of cell culture, the viable cell number is a very valuable reference parameter. Indirect methods of cell number/viability measurements may have up to 10 % standard deviation. This can lead to undesirable large deviations in the analysis of "-omics" data as well as time course studies. Such data should be preferably normalized to the exact viable cell number at a given time to allow meaningful interpretation and understanding of the biological processes. Manual counting of cell number is very laborious and not possible in certain experimental setups. We therefore, developed a simple and reliable fluorescence based method with an accuracy of 95-98 % for the determination of the viable cell number in situ. We optimized the seeding cell densities for primary rat hepatocytes for optimal cell adhesion. This will help in efficient use of primary cells which are usually limited in availability. The method will be very useful in the application of "-omics" techniques, especially metabolome analysis where the specific rates of uptake/production of metabolites can be reliably calculated.

Low-dose oral recombinant interferon-alpha A in patients with HIV-1 infection: a blinded pilot study.

OBJECTIVE: To evaluate the efficacy of low-dose oral recombinant interferon-alpha (IFN-alpha A) on clinical parameters, body weight, CD4+ lymphocyte counts and natural killer cell cytolytic activity in HIV-infected patients. DESIGN: Blinded crossover trial with controls for the protein and diluent components of the drug preparation. SETTING: Medical school outpatient referral center. PATIENTS, PARTICIPANTS: Eight patients with HIV-1 infection and a CD4+ lymphocyte count between 150 and 600 x 10(6)/l. Concurrent use of zidovudine was permitted. INTERVENTIONS: Patients received (daily, by mouth) 10 ml of a study solution of 2.5% albumin for 6 weeks, 150 IU IFN-alpha A for 6 weeks, and normal saline for 6 weeks. MAIN OUTCOME MEASURES: After two baseline visits, clinical assessments, vital signs, body weight, and laboratory tests, including enumeration of number and percentage of CD4+ and CD8+ lymphocytes and natural killer cell cytolytic activity, were performed every 3 weeks. Complete physical examinations were conducted every 6 weeks. RESULTS: No significant clinical or laboratory changes were observed during treatment with IFN-alpha A. Peak CD4+ lymphocyte counts were achieved at baseline in one patient, during albumin treatment in two patients, during IFN-alpha A treatment in one patient, and during saline treatment in four patients. All patients remained HIV-seropositive. Treatments were well-tolerated. CONCLUSION: This blinded pilot study of orally administered IFN-alpha A (150 IU daily for 6 weeks) did not demonstrate clinical benefit in HIV-infected patients.

Protective effect of rhinovirus receptor blocking antibody in human fibroblast cells.

Infection of HeLa cells by human rhinoviruses (RV) of the major receptor group is inhibited by a HeLa-derived rhinovirus receptor murine monoclonal antibody (RRMA). In yield reduction studies in human embryonic lung fibroblast cells, pretreatment with 1.0 or 10 micrograms/ml of RRMA partially protected (greater than 90% titer reduction) against infection by RV 39 or coxsackie A21 (members of the major receptor family), but not by RV 1A (member of the minor receptor family). The protection afforded by RRMA persisted at least 72 h after a 2-h exposure. These results suggest that RV receptors can be effectively blocked for prolonged periods in cultured fibroblast cells.

Anti-rhinoviral activity of recombinant and hybrid species of interferon alpha.

To define further differences in antiviral activity as well as to identify candidate interferons for study in the prevention of rhinovirus colds, the antiviral activities of nine species of recombinant interferon alpha (IFN-alpha A, IFN-alpha B, IFN-alpha C, IFN-alpha D, IFN-alpha J, [Ser-116]IFN-alpha J1, IFN-alpha K, IFN-alpha J/C(Fnu4HI), and IFN-alpha A/D(BglII)) were evaluated against rhinovirus types 39 (RV 39) and 1A (RV 1A). WI-38 cells were exposed to various concentrations of each interferon and were then infected with RV 39, RV 1A, or VSV. Efficacy was determined by protection from cytopathic effect using a tetrazolium dye assay. The 50% inhibitory concentrations ranged from 4 +/- 3 pg/ml for IFN-alpha C to > 3000 pg/ml for IFN-alpha D against RV 39, and from 6 +/- 4 pg/ml for IFN-alpha J/C(Fnu4HI) to > 3000 pg/ml for IFN-alpha D against RV 1A. IFN-alpha J/C(Fnu4HI), [Ser-116]IFN-alpha J1, and IFN-alpha C were the most active of the interferons, and were all more active than IFN-alpha A, against RV 39, RV 1A, and VSV. These interferons warrant further study against rhinoviruses and other viruses.

Serologic response and reactogenicity to booster immunization of healthy seropositive adults with live or inactivated varicella vaccine.

The immunogenicity and reactogenicity of live and heat-inactivated varicella vaccine were evaluated in 95 healthy seropositive adults (mean age, 32 yrs). Live-attenuated vaccine containing 28,000 pfu, 2800 pfu, or 280 pfu of Oka/Merck strain virus (8.4, 0.84, and 0.08 antigen units, respectively) or heat-inactivated vaccine with comparable antigen content (7.1, 0.71, and 0.07 antigen units) was administered subcutaneously to 15 to 16 adults per group in a randomized, single-blind study. ELISA antibody responses were dose-dependent but independent of whether the vaccine was live or inactivated. Mean titers reached a peak on day 14 and remained elevated through day 42 in recipients of the highest dosages but declined by followup at eleven months. A minority of vaccinees developed a four-fold or greater increase in antibody titer on day 14 (25 to 31 percent in the high-dose groups [greater than or equal to 7.1 antigen units], less than or equal to 7 percent of other groups). The vaccine was generally well tolerated. Localized erythema and swelling occurred at the injection site in 44 to 56 percent of the high- and middle dose recipients [greater than or equal to 0.71 antigen units], compared with 0 to 6 percent of those receiving the lowest dose. Although statistically significant increases in varicella antibody titer were observed after immunization with high doses of live or inactivated vaccine, the duration and clinical significance of this booster effect remains to be determined.

Facial cellulitis and Pseudomonas luteola bacteremia in an otherwise healthy patient.

Pseudomonas luteola is an aerobic, Gram negative rod, formerly classified as CDC group Ve-1 and Chryseomonas luteola. It is an uncommon clinical isolate. A previously healthy 59-year-old homosexual man with facial cellulitis and Pseudomonas luteola bacteremia is reported. Previously reported cases of P. luteola bacteremia have occurred in association with pancreatic abscess, prosthetic valve endocarditis, cardiac surgery, granulomatous hepatitis, peritonitis, and indwelling vascular catheters. This case suggests that the spectrum of disease caused by this bacteria may continue to expand.

Interventional therapy for the treatment of acute myocardial infarction: thrombolysis with and without angioplasty. A brief review of the literature and the experience at a community hospital.

Thrombolytic therapy is a feasible, practical, and important approach to the treatment of the patient with an acute myocardial infarction. Data have conclusively demonstrated improvement in both postinfarction indices of left ventricular function and overall survival statistics. Definitive treatment for the underlying coronary lesion may be done acutely with percutaneous transluminal coronary angioplasty or the patient may undergo elective bypass surgery or coronary angioplasty at a later time, depending on the clinical circumstances. Direct intracoronary infusion of the thrombolytic agent is the most effective route of administration. However, if it is not possible for the patient to be taken to a cardiac catheterization laboratory, the intravenous route may be used. Further development of newer thrombolytic agents will undoubtedly make this mode of therapy standard and routine for the patient with acute myocardial infarction.

Antiviral chemotherapy and prophylaxis of viral respiratory disease.

Respiratory viruses continue to be major causes of morbidity and mortality. Currently available chemotherapy is limited to oral amantadine for uncomplicated influenza A and aerosolized ribavirin for respiratory syncytial virus (RSV) infections. Amantadine is also efficacious for chemoprophylaxis of influenza A virus infections. Rimantadine has similar clinical efficacy and is better tolerated than amantadine. Aerosolized ribavirin may be useful in the treatment of serious respiratory illness caused by viruses other than RSV. Intranasal application of interferon is effective in interrupting the spread of rhinovirus colds in families, but chronic use is limited by nasal toxicity. Several newer agents and approaches for chemoprophylaxis and therapy are at different stages of clinical investigation. Combinations of antiviral agents may offer the best therapeutic advantage but have not been adequately tested in man. As additional drugs become available and uses expand for the currently available agents, rapid viral diagnosis will assume an increasingly important role in their optimal use.

Otologic effects of interferon beta serine in experimental rhinovirus colds.

To evaluate the efficacy and otologic effects of recombinant interferon beta serine for experimental rhinovirus colds, 38 healthy adults received nasal drops of recombinant interferon beta serine, 12 x 10(6) U, or placebo three times daily for 4.3 days beginning 36 hours after infection. Illness rates and severity did not differ between the groups, but the frequency of virus shedding was reduced on the fourth (37% vs 74%) and sixth (11% vs 42%) postchallenge days in recipients of recombinant interferon beta serine. Abnormal eustachian tube function in at least one ear was identified by the inflation-deflation test during 44% of observations in 13 infected recipients of recombinant interferon beta serine compared with 62% of observations in five placebo recipients. Tympanometry revealed middle-ear pressure abnormalities (less than -50 or greater than 20 mm H2O) in at least one ear during 18% of observations in recipients of recombinant interferon beta serine compared with 38% of observations in placebo recipients. These results suggest that antiviral therapy may alter the course of middle-ear dysfunction associated with experimental colds.

Bee sting anaphylaxis in an urban population of South Australia.

The clinical manifestations and circumstances of bee sting anaphylaxis have been studied retrospectively in 98 subjects. Most reactions occurred in children but the most severe reactions were seen in adult males, of whom 7 lost consciousness and 2 required cardiopulmonary resuscitation. Most stings causing anaphylaxis occurred on the unprotected feet whilst the subject was on lawn in the afternoons in December, January and February when the maximum daily temperature was between 20 and 30 degrees C. This is the temperature range when bees are particularly active in gathering pollen. However, a significantly greater frequency of anaphylactic reactions occurred at higher temperatures when bees are less active, suggesting that high environmental temperature may predispose the individual to greater exposure to bees or possibly to anaphylactic reactions per se. The presence of atopy did not appear to predispose subjects to bee venom hypersensitivity. Considerable anxiety and lifestyle alteration were identified in some subjects. The alleviation of this anxiety is considered an appropriate indication for bee venom immunotherapy.

The genesis of craniofacial biology as a health science discipline.

The craniofacial complex encapsulates the brain and contains the organs for key functions of the body, including sight, hearing and balance, smell, taste, respiration and mastication. All these systems are intimately integrated within the head. The combination of these diverse systems into a new field was dictated by the dental profession's desire for a research branch of basic science devoted and attuned to its specific needs. The traditional subjects of genetics, embryology, anatomy, physiology, biochemistry, dental materials, odontology, molecular biology and palaeoanthropology pertaining to dentistry have been drawn together by many newly emerging technologies. These new technologies include gene sequencing, CAT scanning, MRI imaging, laser scanning, image analysis, ultrasonography, spectroscopy and visualosonics. A vibrant unitary discipline of investigation, craniofacial biology, has emerged that builds on the original concept of 'oral biology' that began in the 1960s. This paper reviews some of the developments that have led to the genesis of craniofacial biology as a fully-fledged health science discipline of significance in the advancement of clinical dental practice. Some of the key figures and milestones in craniofacial biology are identified.