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.

The regulation of RNA synthesis and processing in the nucleolus during inhibition of protein synthesis.

The effect of protein synthesis inhibition by cycloheximide on nucleolar RNA synthesis and processing has been studied in HeLa cells. Synthesis of 45S RNA precursor falls rapidly after administration of the drug. However, the nucleolar content of 45S RNA remains relatively constant for at least 1 hr because the time required for cleavage of the precursor molecule into its products is lengthened after treatment with cycloheximide. The efficiency of transformation of 45S RNA to 32S RNA remains constant with approximately one molecule of the 32S RNA produced for each cleavage of a molecule of 45S RNA. However, shortly after the cessation of protein synthesis the formation of 18S RNA becomes abortive. The amount of 32S RNA present in the nucleolus remains relatively constant. After long periods of protein synthesis inhibition the 28S RNA continues to be synthesized and exported to the cytoplasm but at a greatly reduced rate. When the protein synthesis inhibitor is removed, a prompt, although partial, recovery in the synthesis rate of 45S RNA occurs. The various aspects of RNA synthesis regulation and processing are discussed.

Patient perceptions of communication with diagnostic radiographers.

INTRODUCTION: The aims of this study were to (1) examine what makes a communication encounter with a diagnostic radiographer effective from the patient's perspective and (2) explore the impact of communication skills on the relationship between a diagnostic radiographer and their patient. METHODS: Semi-structured interviews were conducted with 10 patients immediately after their imaging examination. Completed interviews were analysed using an inductive approach. RESULTS: Five themes were identified in the qualitative data. (1) Introduction and Greeting, (2) Explanation/Instruction, (3) Feeling at ease, (4) Clear communication and (5) Gentle manner. CONCLUSION: These results have identified that effective communication promotes the development of rapport and trust between the radiographer and patient. These findings have implications for practitioners, educators and university programs and should be considered in the development of communication skills training programs for diagnostic radiography students.

Evaluation of the systemic toxicity and mutagenicity of OLIGOPIN®, procyanidolic oligomers (OPC) extracted from French Maritime Pine Bark extract.

The potential systemic toxicity of Oligopin®, a French Maritime Pine Bark extract (FMPBE) rich in procyanidolic oligomers, was evaluated in an acute oral limit test and a 90-day repeated dose oral toxicity study with Sprague Dawley rats. The potential mutagenicity was assessed in a bacterial reverse mutation assay and in vitro mammalian chromosome aberration assay with human lymphocytes. The results indicate that Oligopin® was nongenotoxic in both bacterial and human cell assays, was not acutely toxic via oral administration at up to 2000 mg/kg and was well tolerated following 90 days of oral administration to SD rats, with a no observed adverse effect level of 1000 mg/kg/day. The lack of significant adverse systemic effects in the 90 day study is concordant with findings from several human clinical trials. The acute toxicity and mutagenicity data are consistent with data reported by AFSSA in a summary of FMPBE safety, in which a NOAEL of 100 mg/kg/day was established. In contrast, the NOAEL derived from the 90-day study with Oligopin® was 1000 mg/kg/day, suggesting that it is less systemically toxic than other FMPBE previously evaluated in subchronic studies, and comparable to proanthocyanidins extracted from grape seeds, which are widely used as nutritional supplement ingredients.

Glycosylation and processing of the human immunodeficiency virus type 1 envelope protein.

The human immunodeficiency virus type 1 (HIV-1) envelope protein is synthesized as a gp160 precursor that is cleaved to a 120 kDa exterior glycoprotein (gp120) and a 41 kDa transmembrane glycoprotein (gp41). The HIV-1 envelope protein was stably expressed under the control of the transactivator proteins tat and rev, in wild-type and mutant Chinese hamster ovary (CHO) cells. The mutant, ldlD, is conditionally defective for the addition of galactose and N-acetylgalactosamine to oligosaccharide chains. The effects of glycosylation modification on the HIV-1 envelope's structure and function were examined. The effects of galactosylation on the structure of the envelope proteins suggest that cleavage of the gp160 precursor into gp120 and gp41 occurs intracellularly, apparently concurrent with the addition of galactose to N-linked oligosaccharides of the envelope proteins. No evidence for O-linked glycosylation of the envelope proteins in CHO cells was observed. The envelope protein in the transfected hamster cells mediated the fusion of these cells with CD4-positive lymphocytes, and this fusogenic activity was independent of the addition of either galactose or N-acetylgalactosamine to oligosaccharides in the transfected cells.

A double-blind, randomized, crossover trial of pemoline in fatigue associated with multiple sclerosis.

Fatigue occurs in a majority of patients with MS and is generally independent of measurable neurologic disability. Few options for treatment are available. We conducted a double-blind, placebo-controlled, crossover trial for each of two 4-week treatment periods. Forty-six eligible patients entered and five dropped out due to concurrent exacerbations. Nineteen patients (46.3%) experienced excellent or good relief of fatigue with pemoline, and eight patients (19.5%) with placebo (p = 0.06, Fisher's exact test). One-fourth of patients did not tolerate the drug well, and 7% had to discontinue pemoline during the study due to side effects. The most common side effects were anorexia, irritability, and insomnia. Pemoline may be an effective short-term treatment for fatigue associated with MS, but its adverse effects are not well tolerated by many patients.

An open-trial evaluation of mitoxantrone in the treatment of progressive MS.

We treated 13 patients with progressive MS with mitoxantrone. All patients received a standard IV dose of mitoxantrone (8 mg/m2) every 3 weeks for a total of seven infusions, with dosage adjustments depending on the hematologic profile at the nadir. The treatment was well tolerated, with the most common side effect being mild nausea. Four of seven women developed transient secondary amenorrhea. The postenrollment clinical behavior of these patients was generally more favorable than during the 18 months prior to enrollment (only three of 13 patients developed an increase in the Expanded Disability Status Scale of more than 0.5 points), suggesting a possible treatment effect, but comparison with two historical control groups (both the active and placebo groups from the Canadian Cooperative Trial of Cyclophosphamide and Plasma Exchange) does not suggest that mitoxantrone was efficacious. Eight of 12 patients had evidence of MRI activity on 13 of 29 follow-up visits. This small, open-labeled pilot study did not provide strong support for proceeding with a randomized, controlled trial of this dosage regimen of mitoxantrone in patients with progressive MS.

The mouse spot test. Evaluation of its performance in identifying chemical mutagens and carcinogens.

The published results on 60 chemicals and X-rays investigated in the mouse spot test were compared with data on the same chemicals tested in the bacterial mutation assay (Ames test) and lifetime rodent bioassays. The performance of the spot test as an in vivo complementary assay to the in vitro bacterial mutagenesis test reveals that of 60 agents, 38 were positive in both systems, 6 were positive only in the spot test, 10 were positive only in the bacterial test and 6 were negative in both assays. The spot test was also considered as a predictor of carcinogenesis; 45 chemicals were carcinogenic of which 35 were detected as positive by the spot test and 3 out of 6 non-carcinogens were correctly identified as negative. If the results are regarded in sequence, i.e. that a positive result in a bacterial mutagenicity test reveals potential that may or may not be realized in vivo, then 48 chemicals were mutagenic in the bacterial mutation assay of which 38 were active in the spot test and 31 were confirmed as carcinogens in bioassays. 12 chemicals were non-mutagenic to bacteria of which 6 gave positive responses in the spot test and 5 were confirmed as carcinogens. These results provide strong evidence that the mouse coat spot test is an effective complementary test to the bacterial mutagenesis assay for the detection of genotoxic chemicals and as a confirmatory test for the identification of carcinogens. The main deficiency at present is the paucity of data from the testing of non-carcinogens. With further development and improvement of the test it is probable that the predictive performance of the assay in identifying carcinogens should improve, since many of the false negative responses may be due to inadequate testing.

An assessment of the in vivo rat hepatocyte DNA-repair assay.

The in vivo rat hepatocyte autoradiographic assay for unscheduled DNA synthesis (UDS) described by Mirsalis et al, and its in vitro counterpart described earlier by Williams have been employed by us for 4 years. Our experience is that the in vivo assay performs as described in the literature. We have therefore concentrated in this initial paper on the key practical factors we have found to govern the assay sensitivity and reproducibility. This has been achieved by a discussion of the assay performance with two potent rat hepatocarcinogens [the novel azo compound 6-dimethylaminophenylazobenzthiazole (6BT) and the reference agent 2-acetylaminofluorene (2AAF)] and a non-carcinogen of similar structure to 6BT [5-dimethylaminophenylazoindazole (51)]. Assay responses were compared with the effect of these chemicals in the Salmonella mutation assay. We conclude that the in vivo liver UDS assay has a critical role to play as a complement to rodent bone marrow cytogenic assays when conducting assessment studies on agents defined as genotoxic in vitro. However, the in vivo assay is resource-consuming and false results could consequently arise due to incomplete evaluations. Methods to counteract this danger are discussed and criteria for assessing weak UDS responses are suggested.

When neurological symptoms are not what they appear: the challenge of caring for patients with conversion disorders.

Conversion Disorders involve the psychogenic loss or disturbance of sensory, motor or other physical functions in a manner suggestive of neurologic or other somatic disease but without any actual finding of the latter. These problems are subconscious, with patients not being able to control their symptoms. Jean-Marie Charcot, in the 19th century, was fascinated with this disorder and his work, followed by that of Sigmund Freud, laid the foundation for our modern concept of Conversion Disorder. Multiple Sclerosis, with its fluctuating and unpredictable symptoms has frequently been erroneously diagnosed in these cases. The often bizarre, flamboyant presentations, while interesting, pose many difficult and complex management problems. This paper will describe three of the more severe and disabled examples of this condition seen in the London Multiple Sclerosis Clinic and an attempt will be made to uncover common patient characteristics. Current thoughts regarding the management of these patients will be outlined, the emphasis being on helping neuroscience nurses develop an approach which is informed, positive and compassionate.

Assessing the prevalence of cognitive impairment in multiple sclerosis: implications for patient management.

Many patients with multiple sclerosis exhibit behavioural changes, including alterations cognitive functions and psychiatric abnormalities. Increasingly sophisticated neuropsychologic assessment techniques plus magnetic resonance imaging have added to our knowledge in this area, showing that these alterations may be more common than previously recognized. Research studies have attempted to address the question of whether these changes are primarily function of the demyelinating process itself or a reaction to the disability produced by the disorder. This is an important issue in terms of possible intervention. This paper will include a review of the literature.

Temporal lobectomy: an increasingly viable option for seizure patients.

Over the past ten years, the treatment options available to people with epilepsy have changed drastically, resulting in new concepts, treatment methods and philosophies. Complex partial seizures (temporal lobe or psychomotor) account for one third of all chronic seizure disorders. Following temporal lobectomy, the majority of these patients no longer requires medication. The neuroscience nurse must understand the selection criteria, diagnostic tests and perioperative course in order to teach and support these individuals and their families.

Messenger and heterogeneous nuclear RNA in HeLa cells: differential inhibition by cordycepin.

Cordycepin (3'-deoxyadenosine) suppresses the labeling of messenger RNA in HeLa cells. The drug has no effect on either the labeling of nuclear heterogeneous RNA or on the transport of messenger RNA into the cytoplasm. The results suggest that messenger RNA and nuclear heterogeneous RNA are synthesized separately, and that the transcription of messenger RNA is inhibited by the drug.

Regulation of ribosomal RNA synthesis and processing during inhibition of protein synthesis by 1,3-bis(2-chloroethyl)-1-nitrosourea.

The antineoplastic agent BCNU (1,3-bis(2-chloroethyl)-1nitrosourea) at a concentration of 25 mug/ml inhibits initiation of protein synthesis in HeLa cells. At this low concentration of the drug, the rate of synthesis of 45S ribosomal precursor RNA (pre-rRNA) is selectively inhibited without a marked inhibition of nucleoplasmic RNA. The inhibitory effects of the drug are readily reversible upon removal of BCNU from the growth medium. Pulse-chase analysis of the labeled nucleolar RNA in sucrose-gradients and acrylamide gels indicated that the 45S pre-rRNA synthesized before the addition of BCNU matures normally in the presence of the inhibitor. However, the processing of precursor RNA molecules synthesized following the addition of the drug is inhibited when incubation is continued on in the presence of 25 mug/ml BCNU. Since the formation of mature ribosomes is blocked by BCNU, the data would suggest that the effectiveness of the drug as a potent cell growth inhibitor may result from its inhibition of ribosome formation induced by inhibition of protein synthesis.

A single point mutation in epsilon-COP results in temperature-sensitive, lethal defects in membrane transport in a Chinese hamster ovary cell mutant.

At the nonpermissive temperature of 39.5 degrees C, the Chinese hamster ovary cell conditionally lethal, temperature-sensitive (ts) mutant ldlF exhibits the following defects: rapid degradation of low density lipoprotein receptors, disruption of ER-through Golgi transport, and disintegration of the Golgi apparatus. All of these are corrected by transfection with an expression vector for wild-type epsilon-COP, a subunit of coatomers (Guo, Q., Vasile, E., and Krieger, M. (1994) J. Cell Biol. 125, 1213-1224). We now report the identification in ldlF cells of a point mutation in the epsilon-COP gene, Glu251 to Lys251, which prevents the corresponding cDNA from correcting the defects in transfected ldlF cells and the immunochemical analysis of the synthesis, structure, and stability of epsilon-COP. At the permissive temperature (34 degrees C), the steady state level of ts-epsilon-COP in ldlF cells was about half that of epsilon-COP in wild-type Chinese hamster ovary cells and the isoelectric point of ts-epsilon-COP was 0.14 pH units higher than that of the wild-type protein. The stability but not the biosynthesis of ts-epsilon-COP was temperature-sensitive (t1/2 > 6 h at 34 degrees C and approximately 1-2 h at 39.5 degrees C), and this accounts for the virtual absence of detectable ts-epsilon-COP protein in ldlF cells after incubation at 39.5 degrees C for > 6h. The steady state levels in ldlF cells of another coatomer subunit, beta-COP, and the peripheral Golgi protein ldlCp were not temperature-sensitive. Thus, a mutation in epsilon-COP that causes instability at 39.5 degrees C is responsible for all of the temperature-sensitive defects in ldlF cells, and the stability of beta-COP is not linked directly to that of epsilon-COP. ldlF cells should be useful for the future analysis of the structure and function of epsilon-COP, the assembly of COPs into coatomers, and the participation of coatomers in intracellular membrane transport.

Identification and characterization of naturally occurring variants of the macrophage scavenger receptor (SR-A).

The scavenger receptor (SR) family comprises a group of cell surface proteins functionally defined by their ability to bind chemically modified lipoproteins. In macrophages, the class A Type I and Type II SRs (SR-AI/II) are thought to play a key role in adherence to and phagocytosis of infectious agents. Immunoprecipitation studies show that the rat anti-SR-AI/II monoclonal antibody 2F8 detects the mature, trimeric form of the receptor expressed in peritoneal macrophages from A/J, but not from C57Bl/6J (B6) mice. Subsequent sequencing of cDNA and genomic clones indicates that SR-AI and AII of A/J and B6 mice differ in sequence at nine positions, two in the cytoplasmic domain and seven in the extracellular spacer and alpha-helical coiled coil domains. These sequence polymorphisms are non-conservative and produce distinct receptor molecules that differ by four charged residues and alter recognition of the receptor by the monoclonal 2F8 antibody. The B6 SR-AI/II haplotype appears unique, since most inbred strains analyzed show the A/J-type haplotype. Interestingly, several of the B6 polymorphic variant residues are conserved in human and bovine receptors, suggesting a recent divergence of the A/J haplotype. Initial studies in CHO-derived cells expressing individual receptor isoforms indicate that the A/J and B6 receptors are stable and can mature into oligomers expressed in the membrane fractions of these cells. In these transfectants, no major functional differences were detected between receptors of the two haplotypes with respect to internalization and degradation of (125)I-labeled acetylated LDL. However, since SR-AI/II recognizes a large number of structurally unrelated anionic molecules, the possibility that different haplotypes may affect either binding and release of other ligands, or receptor recycling, cannot be excluded.

Recognition and plasma clearance of endotoxin by scavenger receptors.

Lipid A is the active moiety of lipopolysaccharide (LPS, also referred to as endotoxin), a surface component of Gram-negative bacteria that stimulates macrophage activation and causes endotoxic shock. Macrophages can bind, internalize and partially degrade LPS, lipid A and its bioactive precursor, lipid IVA. We report here that lipid IVA binding and subsequent metabolism to a less active form by macrophage-like RAW 264.7 cells is mediated by the macrophage scavenger receptor. Scavenger-receptor ligands inhibit lipid IVA binding to, and metabolism by, RAW cells, and lipid IVA binds to type I and type II bovine scavenger receptors on transfected Chinese hamster ovary cells. Although in vitro competition studies with RAW cells indicate that scavenger receptor binding is not involved in LPS or lipid IVA-induced stimulation of macrophages, in vivo studies show that scavenger-receptor ligands greatly inhibit hepatic uptake of lipid IVA in mice. Thus, scavenger receptors expressed on macrophages may have an important role in the clearance and detoxification of endotoxin in animals.