Evaluation of potential health effects associated with occupational and environmental exposure to styrene - an update.

The potential chronic health risks of occupational and environmental exposure to styrene were evaluated to update health hazard and exposure information developed since the Harvard Center for Risk Analysis risk assessment for styrene was performed in 2002. The updated hazard assessment of styrene's health effects indicates human cancers and ototoxicity remain potential concerns. However, mechanistic research on mouse lung tumors demonstrates these tumors are mouse-specific and of low relevance to human cancer risk. The updated toxicity database supports toxicity reference levels of 20 ppm (equates to 400 mg urinary metabolites mandelic acid + phenylglyoxylic acid/g creatinine) for worker inhalation exposure and 3.7 ppm and 2.5 mg/kg bw/day, respectively, for general population inhalation and oral exposure. No cancer risk value estimates are proposed given the established lack of relevance of mouse lung tumors and inconsistent epidemiology evidence. The updated exposure assessment supports inhalation and ingestion routes as important. The updated risk assessment found estimated risks within acceptable ranges for all age groups of the general population and workers with occupational exposures in non-fiber-reinforced polymer composites industries and fiber-reinforced polymer composites (FRP) workers using closed-mold operations or open-mold operations with respiratory protection. Only FRP workers using open-mold operations not using respiratory protection have risk exceedances for styrene and should be considered for risk management measures. In addition, given the reported interaction of styrene exposure with noise, noise reduction to sustain levels below 85 dB(A) needs be in place.

Alternative diagnoses at paediatric appendicitis MRI.

As the utilization of MRI in the assessment for paediatric appendicitis increases in clinical practice, it is important to recognize alternative diagnoses as the cause of abdominal pain. The purpose of this review is to share our institution's experience using MRI in the evaluation of 510 paediatric patients presenting with suspected appendicitis over a 30 month interval (July 2011 to December 2013). An alternative diagnosis was documented in 98/510 (19.2%) patients; adnexal pathology (6.3%, n = 32), enteritis-colitis (6.3%, n = 32), and mesenteric adenitis (2.2%, n = 11) comprised the majority of cases. These common entities and other less frequent illustrative cases obtained during our overall institutional experience with MRI for suspected appendicitis are reviewed.

Sebotropic eruption associated with use of oral kava kava supplement.

Supplement use is prevalent, and its use is increasing among older adults. Dermatologists need to be aware of the adverse cutaneous effects that can result from herbal supplement use. A 55-year-old man presented with an eruption in a sebotropic distribution after consuming kava kava for 3 weeks, which resolved after discontinuation of the supplement. This case highlights the need for clinicians to consider kava kava in the differential of sebotropic eruptions. The biology, mechanism of action, and potential systemic and cutaneous effects of kava kava are reviewed.

Screening method to identify inhibitors of siderophore biosynthesis in the opportunistic fungal pathogen, Aspergillus fumigatus.

AIMS: Aspergillus fumigatus is the most common cause of airborne mould infections in immunocompromised patients worldwide. Our aim was to develop a method to identify agents that inhibit siderophore biosynthesis because this pathway is unique to the fungus and is essential for virulence. METHODS AND RESULTS: A high-throughput two-step screening assay was developed using 96-well plates in which fungal growth and siderophore production is assessed spectrophotometrically. If a compound inhibits growth only in iron-limited medium (screen 1), its effect on siderophore production is then determined (screen 2). The proof of concept was demonstrated using a known antifungal agent, amphotericin B, and a strain of A. fumigatus deficient in siderophore production. CONCLUSIONS: The two-stage screening method clearly identified growth defects in A. fumigatus related specifically to siderophore biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: The increasing incidence of life-threatening fungal infections has produced an urgent need for novel antifungal agents. The method described in this report will facilitate the identification of novel antifungal compounds that inhibit a pathway critical for A. fumigatus virulence and have a reduced probability of affecting host metabolism.

Evaluation of a Bead-Based Salmonella Molecular Serotyping Method for Salmonella Isolated from Food and Environmental Samples.

Salmonella is a leading cause of foodborne illness worldwide, and foods containing Salmonella (except raw meat and poultry products) are considered adulterated. Serotyping of Salmonella is an essential part of surveillance and investigation of outbreaks. This study evaluated a bead-based Salmonella molecular serotyping (SMS) method, which included the O-group 1, H-antigen, alternate target, and O-group 2 assays, compared with traditional serotyping. Salmonella was isolated from food, pet food, and environmental samples or were reference strains. A total of 572 isolates were analyzed by using two formats of the SMS method in comparison with traditional methods: 485 were analyzed by using Radix SMS (a custom user-mixed format), 218 were analyzed by using Luminex SMS (a commercial kit format), and 131 of the total isolates were analyzed by both formats for comparison. The SMS method was evaluated on the basis of the successful identification of antigens by the probes included in the method. The method identified 550 (96.2%) isolates as expected, 6 (1.0%) isolates were not identified as initially expected but were shown to be correctly identified by SMS after reanalysis by traditional serotyping, and 16 (2.8%) isolates not identified as expected possessed an antigen that should have been detected by the method but was not. Among the isolates considered correctly identified, 255 (44.6%) were identified to a single serovar, 44 (7.7%) required additional biochemical testing to differentiate variants or subspecies, and 251 (43.9%) were partially serotyped because probes for some antigens were not in the assay or had allelic variation for known serovars. Whole genome sequencing, SeqSero, and the Salmonella In Silico Typing Resource gave added confirmation for three isolates. Addition of the O-group 2 assay enabled the identification of 55 (9.6%) of 572 isolates. The SMS method could fully or partially serotype most isolates within a day. The SMS method should be a valuable tool when faster screening methods are needed, such as outbreaks and screening large numbers of environmental isolates.

Evaluation of U. S. National Toxicology Program (NTP) mouse lymphoma assay data using International Workshop on Genotoxicity Tests (IWGT) and the Organization for Economic Co-Operation and Development (OECD) criteria.

The forward gene mutation mouse lymphoma assay (MLA) is widely used, as part of a regulatory test battery, to identify the genotoxic potential of chemicals. It identifies mutagens capable of inducing a variety of genetic events. During the 1980s and early 1990s, the U.S. National Toxicology Program (NTP) developed a publicly available database (https://tools.niehs.nih.gov/cebs3/ui/) of MLA results. This database is used to define the mutagenic potential of chemicals, to develop structure-activity relationships (SAR), and to draw correlations to animal carcinogenicity findings. New criteria for MLA conduct and data interpretation were subsequently developed by the International Workshop for Genotoxicity Testing (IWGT) and the Organization of Economic Cooperation and Development (OECD). These recommendations are included in a new OECD Test Guideline (TG490). It is essential that early experimental data be re-examined and classified according to the current criteria to build a curated database to better inform chemical-specific evaluations and SAR models. We re-evaluated more than 1900 experiments representing 342 chemicals against the newly defined acceptance criteria for background mutant frequency (MF), cloning efficiency (CE), positive control values (modified for this evaluation due to lack of colony sizing), appropriate dose selection, and data consistency. Only 17% of the evaluated experiments met all acceptance criteria used in this re-evaluation. Results from 211 chemicals were determined to be uninterpretable, 92 were positive, and 39 equivocal. The authors could not classify any responses as negative because colony sizing was not performed for any of these experiments and it is clear, based on many experiment with unacceptably low background and positive control MFs, that mutant colony recovery was often suboptimal. This re-evaluation provides a curated database for the MLA. A similar curation should be done for other widely used genetic toxicology assays, but will be more difficult for certain assays (e.g., in vitro chromosomal aberrations) because important parameters such as level of cytotoxicity were often not evaluated/reported. Environ. Mol. Mutagen. 59:829-841, 2018. © 2018 Wiley Periodicals, Inc.

Cytotoxic T-lymphocyte response to autologous human squamous cell cancer of the lung: epitope reconstitution with peptides extracted from HLA-Aw68.

Cytotoxic T-lymphocytes (CTLs) specific for autologous human squamous cell cancer of the lung were generated by stimulation of peripheral blood lymphocytes with autologous tumor cells in vitro. The CTL line was >97% CD3+, CD8+, CD16- and produced tumor necrosis factor-alpha, gamma-interferon, and granulocyte-macrophage colony-stimulating factor after stimulation with autologous tumor. The CTLs lysed autologous tumor but failed to recognize autologous or histocompatibility leukocyte antigen-matched lymphoid cells, K562, or allogeneic tumor cells of several histological types. Antibody-blocking studies suggested that the CTLs recognized one or more antigens presented by the class I major histocompatibility complex molecule Aw68. To characterize these antigens further, histocompatibility leukocyte antigen Aw68 molecules were extracted from the squamous cell cancer of the lung tumor line by immunoaffinity chromatography, and the associated peptides were eluted in acid and separated by reversed-phase high-performance liquid chromatography. Reconstitution of the CTL epitope was evaluated by adding these peptides to autologous Epstein-Barr virus-transformed B-cells. Two peaks of reconstituting activity were observed, suggesting that these CTLs recognize at least two Aw68-associated peptides. This study confirms the existence of a CTL response against autologous human squamous cell cancer of the lung and suggests that this CTL response is directed against peptide epitopes presented by the class I major histocompatibility complex molecules. It is anticipated that this approach will permit identification of peptide epitopes for lung cancer-specific CTLs.

Relationships between benzo(a)pyrene-DNA adduct levels and genotoxic effects in mammalian cells.

The effectiveness of benzo(a)pyrene [B(a)P]-DNA binding as an internal dosimeter was evaluated. Data were obtained from concurrent studies, measuring B(a)P induced genotoxic effects and DNA adducts in several short-term bioassay systems: cytotoxicity, gene mutation, and sister chromatid exchange in Chinese hamster V79 cells; cytotoxicity, gene mutation, and chromosome aberrations in mouse lymphoma L5178Y TK+/-; cytotoxicity and enhanced virus transformation in Syrian hamster embryo cells; and cytotoxicity and morphological transformation in C3H10T1/2CL8 mouse embryo fibroblasts. Both total B(a)P-DNA binding and specific B(a)P-DNA adducts were measured. N2-(10 beta-[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]yl)deoxyguanosine [BPDE I-dGuo] was one of the major adducts identified in all bioassay systems. DNA binding and genotoxic responses varied significantly between bioassays. Each genetic end point was induced with a differing efficiency on a per adduct basis. However, the relationships between frequency of genetic effect or morphological transformation and B(a)P-DNA binding or BPDE I-dGuo were linear within a given assay. In order to compare biological end points of diverse frequencies in diverse biological systems, a doubling adduct level, expressed as the number of BPDE I-dGuo adducts per unit of DNA required to double the induced frequency of biological response, was applied to the data.

Associations between the IASLC/ATS/ERS lung adenocarcinoma classification and EGFR and KRAS mutations.

We sought to investigate the frequency of mutations in epidermal growth factor receptor (EGFR) and Kirsten-RAS (KRAS) by each pathological subtype for patients with resected pulmonary adenocarcinoma as defined by the IASLC/ATS/ERS classification. Histological examination determined the predominant subtype according to the IASLC/ATS/ERS classification. EGFR and KRAS mutations were determined by high-resolution melting and Sanger sequencing. Clinical data were collected from medical records and clinicians. The 178 consecutive patients consisted of 48% males, median age 68 years (range 20-87) and smoking history 78%. The tumour stage was I in 62%, II in 18% and III in 20%. The mutation rates were: EGFR 30%; KRAS 28%. The rate of EGFR mutations in the acinar predominant reference group (n=76), was 37%. The solid predominant subtype showed significantly fewer EGFR mutations [3/33 (9%), odds ratio 0.17 (0.05-0.61), p=0.007]. No differences in mutation rate were observed in other subtypes. No association was found between KRAS mutations and predominant histological subtype. Advanced stage and solid predominant subtype were negative prognostic factors. EGFR mutations can be present in adenocarcinoma of any predominant subtype, however rarely in solid predominant tumours. No association was found between KRAS mutation and the predominant histological subtype.

Standardized cell sources and recommendations for good cell culture practices in genotoxicity testing.

Good cell culture practice and characterization of the cell lines used are of critical importance in in vitro genotoxicity testing. The objective of this initiative was to make continuously available stocks of the characterized isolates of the most frequently used mammalian cell lines in genotoxicity testing anywhere in the world ('IVGT' cell lines). This project was organized under the auspices of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing. First, cell isolates were identified that are as close as possible to the isolate described in the initial publications reporting their use in genotoxicity testing. The depositors of these cell lines managed their characterization and their expansion for preparing continuously available stocks of these cells that are stored at the European Collection of Cell Cultures (ECACC, UK) and the Japanese Collection of Research Bioresources (JCRB, Japan). This publication describes how the four 'IVGT' cell lines, i.e. L5178Y TK+/- 3.7.2C, TK6, CHO-WBL and CHL/IU, were prepared for deposit at the ECACC and JCRB cell banks. Recommendations for handling these cell lines and monitoring their characteristics are also described. The growth characteristics of these cell lines (growth rates and cell cycles), their identity (karyotypes and genetic status) and ranges of background frequencies of select endpoints are also reported to help in the routine practice of genotoxicity testing using these cell lines.

Asynchronous regional brain volume losses in presymptomatic to moderate AD.

To determine if rates and locations of brain volume loss associated with AD are phase-specific, occurring prior to clinical onset and at later stages, we performed longitudinal volumetric MRI analysis on 155 subjects enrolled in a prospective study of aging and dementia. Subjects were divided by Clinical Dementia Rating (CDR) scale into stages of Normal (CDR 0 --> 0), Very Mild (CDR 0 --> 0.5 and 0.5 --> 0.5), Mild (CDR 0.5 --> 1.0 and 1.0 --> 1.0) and Moderate (CDR 1.0 --> 2.0 and 2.0 --> 2.0) dementia. Rates of volume change in CSF spaces, lobar and medial temporal lobe regions were analyzed for group differences across stages. Annual rates of ventricular volume change differed between non-demented and very mild group (p<0.01). In later severity stages, ventricular, temporal, basal ganglia-thalamic region and total volumes show change. Rates of volume loss increase as dementia progresses, but not uniformly in all regions. These regional and phase-specific volume changes form targets for monitoring disease-modifying therapies at clinically relevant, defined stages of dementia.

An epidemic of avian and human psittacosis.

Psittacosis may occur in epidemic or sporadic form. We report an outbreak of avian and human psittacosis associated with an aviary in Philadelphia, Pa. The epidemic spread among birds in the aviary and then to men and women who were in contact with the birds. Chlamydia psittaci was cultured from infected birds, and the human patients were evaluated clinically and serologically. After proper treatment of the birds, cleaning of their cages, and institution of appropriate bird-handling techniques, the outbreak among the birds resolved. The human patients were treated, and no subsequent cases have been seen.

Brain volume preserved in healthy elderly through the eleventh decade.

OBJECTIVE: To determine which brain regions lose volume with aging over time in healthy, nondemented elderly. BACKGROUND: Cross-sectional studies suggest widespread loss of brain volume with aging. These studies may be biased by significant numbers of preclinically demented elderly in the oldest comparison groups. Longitudinal studies may allow closer determination of the effect of aging unaffected by dementia. METHODS: Quantitative volumetric MRI was performed annually on 46 healthy subjects older than age 65 who had maintained cognitive health a mean of 5 years. Comparisons (analysis of variance) were made of rates of volume loss (slopes) divided into 11 young-old (mean age, 70 years), 15 middle-old (mean age, 81 years), and 20 oldest-old (mean age, 87 years) subjects. Regions of interest included CSF spaces, lobar regions, and limbic-subcortical regions. RESULTS: There were significant differences between groups in intracranial, total brain, left hemisphere, right hemisphere, temporal lobe, basilar-subcortical region, and hippocampus volumes, with oldest-old subjects showing the smallest volumes, followed by middle-old and young-old subjects. Oldest-old subjects had significantly greater subarachnoid volumes than the younger groups. There were no significant differences in rates of change of regions of interest across age groups. CONCLUSIONS: After age 65 there is minimal brain volume loss observed over time in healthy elderly. Brain volume differences seen cross-sectionally, at any age, likely reflect small, constant rates of volume loss with healthy aging. Healthy oldest-old subjects do not show greater rates of brain loss compared with younger elderly, suggesting that large changes seen in cross-sectional studies reflect the presence of preclinical dementia in older groups.

Volume loss of the hippocampus and temporal lobe in healthy elderly persons destined to develop dementia.

OBJECTIVE: To determine initial locus and rate of degeneration of temporal lobe structures (total lobe, hippocampus and parahippocampus) in preclinical dementia. BACKGROUND: Postmortem studies suggest that the earliest changes in Alzheimer's disease are neurofibrillary tangle formation in hippocampus and adjacent cortex. MRI volume analysis of temporal lobe structures over time in subjects prior to developing dementia may allow the identification of when these processes begin, the rate they develop, and which areas are key to symptom development. METHODS: 30 nondemented (NoD), healthy, elderly individuals enrolled in a prospective study of healthy aging evaluated annually over a mean of 42 months. Twelve subjects with subsequent cognitive decline were assigned to the preclinical dementia group (PreD). All 120 annual MRI studies analyzed by volumetric techniques assessed group differences in temporal lobe volumes and rates of brain loss. RESULTS: NoD as well as PreD subjects had significant, time-dependent decreases in hippocampal and parahippocampal volume. Rates of volume loss between the groups did not significantly differ. PreD cases had significantly smaller hippocampi when asymptomatic. Parahippocampal volume did not differ between PreD and NoD cases. Significant time-dependent temporal lobe atrophy was present only in PreD. CONCLUSIONS: Hippocampal and parahippocampal atrophy occurs at a similar rate regardless of diagnostic group. Those who develop dementia may have smaller hippocampi to begin with, but become symptomatic because of accelerated loss of temporal lobe volume. Temporal lobe volume loss may mark the beginning of the disease process within six years prior to dementia onset.

Radiation dose from radiopharmaceuticals contaminated with molybdenum-99.

Sixteen patients undergoing routine nuclear imaging procedures were injected with 99mTc-labeled radiopharmaceuticals containing 99Mo which exceeded the recommended limit of 1 microCi of 99mMo per mCi of 99mTc. The kinetics of the resulting 99Mo distribution in 14 of these patients were studied over a period of several weeks. The mean biologic half-life [T 1/2b] ranged from about 19.3 days to 11.2 days depending on the model used. Similarly, the mean radiation dose to the liver ranged from approximately 0.02 rad/microCi of 99Mo to 0.05 microCi of 99Mo.

Genotoxicity studies of benz[l]aceanthrylene.

The genotoxicity of the cyclopenta-fused polycyclic aromatic hydrocarbon, benz[l]aceanthrylene (B[l]A), was evaluated in vitro using the L5178Y/TK+/- mouse lymphoma assay and in vivo using the mouse peripheral blood lymphocyte (PBL) culture system. The mutagenicity and sister chromatid exchange (SCE) inducing potential of B[l]A was then compared to that of benzo[a]pyrene (B[a]P). B[l]A appeared to be slightly less mutagenic than B[a]P at the TK locus, and each compound produced both small and large colony mutants indicating that they are clastogenic as well as mutagenic. Gross chromosome aberration analysis of treated L5178Y/TK+/- mouse lymphoma cells confirmed the clastogenicity of B[l]A in vitro. In the mouse PBL system, after administration by gavage, B[l]A was more cytotoxic and produced a sharper elevation in SCE frequency than B[a]P.

Mutagenicity of trichloroethylene and its metabolites: implications for the risk assessment of trichloroethylene.

This article addresses the evidence that trichloroethylene (TCE) or its metabolites might mediate tumor formation via a mutagenic mode of action. We review and draw conclusions from the published mutagenicity and genotoxicity information for TCE and its metabolites, chloral hydrate (CH), dichloroacetic acid (DCA), trichloroacetic acid (TCA), trichloroethanol, S-(1, 2-dichlorovinyl)-l-cysteine (DCVC), and S-(1, 2-dichlorovinyl) glutathione (DCVG). The new U.S. Environmental Protection Agency proposed Cancer Risk Assessment Guidelines provide for an assessment of the key events involved in the development of specific tumors. Consistent with this thinking, we provide a new and general strategy for interpreting genotoxicity data that goes beyond a simple determination that the chemical is or is not genotoxic. For TCE, we conclude that the weight of the evidence argues that chemically induced mutation is unlikely to be a key event in the induction of human tumors that might be caused by TCE itself (as the parent compound) and its metabolites, CH, DCA, and TCA. This conclusion derives primarily from the fact that these chemicals require very high doses to be genotoxic. There is not enough information to draw any conclusions for trichloroethanol and the two trichloroethylene conjugates, DCVC and DCVG. There is some evidence that DCVC is a more potent mutagen than CH, DCA, or TCA. Unfortunately, definitive conclusions as to whether TCE will induce tumors in humans via a mutagenic mode of action cannot be drawn from the available information. More research, including the development and use of new techniques, is required before it is possible to make a definitive assessment as to whether chemically induced mutation is a key event in any human tumors resulting from exposure to TCE.

Quantification and molecular characterization of hprt mutants of human T-lymphocytes.

Somatic mutations have been implicated as critical early events in carcinogenesis. Point mutations, deletions, and translocation events have been shown to activate oncogenes or inactivate suppressor oncogenes. In human population monitoring, quantitative analysis of mutation events that affect gene function is limited to those genes whose cellular phenotypes can be identified by selection procedures and to those tissues (like blood) that are accessible for analysis. In an effort to determine the frequency and types of mutations that can be detected at the hypoxanthine guanine phosphoribosyltransferase (hprt) gene, we have used the T-cell cloning assay and have developed a strategy to propagate mutants and screen for point mutations and breakage events. Early in the clonal expansion of mutants, 1-2 x 10(4) cells are prepared as a crude cell lysate, and a sample is analyzed using the multiplex polymerase chain reaction (PCR). Those mutants that yield altered DNA fragments are then expanded for Southern blot hybridization, PCR, flanking probe isolation, and DNA sequencing. To date we have found presumed point mutations, intragenic deletions, and deletions that extend outside of the hprt gene. By analyzing mutations in selectable, nonessential gene markers, it should be possible to understand mechanisms of both spontaneous and induced genetic damage. An association of these specific genetic events with human diseases and the evaluation of the ability of environmental chemicals to induce these specific types of mutations will lead to a rational basis for evaluating risks from various chemical exposures.

Age-related brain changes associated with motor function in healthy older people.

OBJECTIVE: To identify the MRI imaging findings associated with motor changes in healthy older people. DESIGN: A cross-sectional study. SETTING: A study of neurologic function in very healthy older people, the Oregon Brain Aging Study. PARTICIPANTS: Clinical and MRI data were examined in 50 very healthy older subjects (mean age = 85.1, SD = 7.2 years). MEASUREMENTS: Clinical measures (finger tapping, hand opening and closing, steps and time to walk 30 feet and timed standing on one foot) were dependent variables in multiple regression analyses using age and the following MRI measures as independent variables: total brain volume (TBV)/intracranial volume; ventricular volume/TBV; periventricular high signal/TBV; deep high signal/TBV. RESULTS: The number of steps and the time to walk 30 feet were each associated with periventricular high signal (steps: r = .58, P < .001; time: r = .60, P < .001) and ventricular volume (steps: r = .54, P < .001; time: r = .58, P < .001). These associations remained significant after adjusting for age. None of the other clinical variables was associated with the MRI volumes. CONCLUSIONS: Gait measures were associated significantly with periventricular high signal and ventricular volume. These CNS changes contribute to the cause of these important markers of aging.

Excimer laser effects on human corneal endothelium. Modulation by serum factor(s).

OBJECTIVE: To determine the possibility of endothelial cell damage after excimer laser ablation. METHODS: Endothelial cell densities and morphology of human corneas after photoablations or mechanical keratectomy were compared with those of the untreated mates after 1 week of culture with or without serum. RESULTS: Corneas cultured in serum-free medium after ablation to a depth of 150 microns showed endothelial cell densities reduced to 60% of untreated, mate corneas; ultrastructural analysis showed endothelial cell damage not seen in untreated mates. Corneas ablated to the same depth and cultured in serum-enriched medium showed no endothelial cell density loss, nor did corneas cultured in serum-free medium after an ablation to a depth of 50 microns or mechanical keratectomies averaging 95 microns. CONCLUSIONS: Endothelial cell loss in deep laser resections may be prevented by factor(s) in fetal bovine serum. The apparent lack of cell loss in clinical studies may be related to the protective action of similar factors in aqueous humor.