Novel mutations in the sacsin gene in ataxia patients from Maritime Canada.

We ascertained two families in Eastern Canada segregating a form of ataxia consistent with a recessive mode of inheritance. We performed a whole genome scan using dense SNP genotyping, and despite an absence of shared homozygosity in the families we defined linkage to a small region on chromosome 13. Direct DNA resequencing was employed to screen biologically relevant candidate genes in the interval, and two presumptive pathogenic mutations were found in the gene encoding sacsin. One variant is an obligate truncating mutation, the second is a missense variant in a highly conserved residue. Unexpectedly, one family was homozygous for the missense mutation, the other compound heterozygous for the two mutations. Our results expand the genotype phenotype correlation of mutations in the sacsin gene, and highlight the challenge of diagnosing genetically heterogeneous disorders on primarily clinical grounds. We demonstrate that whole genome genotyping on a modest scale can be productive in research, and potentially in a clinical context.

The NURSE Center: A peer mentor-tutor project for disadvantaged nursing students in Appalachia.

To assist disadvantaged Appalachian nursing students, a grant-supported peer mentor-tutor project was initiated in a regional university. Located in the NURSE Center (Nursing Undergraduate Resource for Successful Education), the goals of the project were to improve participants'academic achievement, increase retention, encourage timely academic progression, and improve NCLEX-RN passing rates. The authors describe the project, the first year of operation, and future directions.

An in vitro and an in vivo unscheduled DNA synthesis assay with a zinc oxide/hexachloroethane (Zn/HCE) smoke.

1. Since Zn/HCE smoke has been shown previously to be weakly positive in the Ames test, and negative in the bone marrow micronucleus assay, other assays including a second in vivo assay examining unscheduled DNA synthesis (UDS) in rat hepatocytes has been carried out, as recommended by the UK Department of Health guidelines. 2. Zn/HCE smoke was assessed for its ability to induce DNA repair in an UDS assay both in vitro in cultured rat hepatocytes and in rat hepatocytes after in vivo treatment by inhalation. 3. For the in vitro investigation, two studies were carried out assessing media exposed to Zn/HCE smoke using at least seven concentrations up to a toxic level. At the highest concentration of Zn/HCE smoke, where some viable cells were seen, an increase in UDS was observed in both experiments. However this was not statistically significant, was only seen at a level where toxicity was observed and was therefore considered not to be biologically significant. 4. In the in vivo investigation, one study was carried out in three separate parts, assessing two doses of Zn/HCE smoke characterised by their zinc content as approximately 20 and 56 micrograms l-1 air. A dose-related increase in UDS was observed which was not statistically significant. The positive control behaved as anticipated, showing a highly statistically significant response. 5. It was concluded that Zn/HCE smoke did not induce unscheduled DNA repair in the in vitro or in vivo UDS assays under the conditions used in the studies. The overall lack of genotoxic effect of this smoke in this and previous studies in this laboratory would not suggest a major health hazard.

The effect of antioxidants on bleomycin treatment in in vitro and in vivo genotoxicity assays.

Antioxidants are thought to be important in protecting against damage from active oxygen species. The effects of the antioxidant nutrients vitamins C and E have been investigated after bleomycin treatment in the Salmonella typhimurium bacterial mutation assay, in the human peripheral lymphocyte chromosome aberration assay, and in the mouse micronucleus assay in peripheral blood and bone marrow cells. There were no protective effects from vitamins C and E in the bacterial mutation assay, but vitamin C and not vitamin E abolished chromosome damaging responses in human peripheral lymphocytes, and both vitamins reduced responses in micronuclei from peripheral blood cells in mice. This would suggest that in human cells in vitro and mouse cells in vivo these vitamins could have a protective role.

Use of precision-cut liver slices for studies of unscheduled DNA synthesis.

Precision-cut liver slices were prepared from untreated and Aroclor 1254 (ARO)-treated male Sprague-Dawley rats with a Krumdieck tissue slicer. Liver slices were cultured for 24 hr in medium containing [3H]thymidine and 0-0.1 mM 2-acetylaminofluorene (2-AAF) using a dynamic organ culture system and processed for autoradiographic evaluation of unscheduled DNA synthesis (UDS). Compared with control (i.e. 0 mM 2-AAF) liver slice cultures, 2-AAF produced a concentration-dependent increase in UDS, the effect being more marked in liver slices from ARO-treated than from untreated rats. With liver slices from untreated rats, 2-AAF produced the greatest increase in UDS in centrilobular hepatocytes. 2-AAF-induced UDS in liver slices from ARO-treated rats was most marked in centrilobular hepatocytes but the effect also extended to other areas of the liver lobule. These results demonstrate that precision-cut liver slices may be a valuable alternative in vitro system to hepatocyte cultures for screening chemicals for potential genotoxicity. Unlike hepatocyte cultures, liver slices permit the study of zonal differences in UDS. Moreover, this technique could be applied to other tissues and the study of species differences in response.

A method for the detection of protein-bound mutagens in food.

To investigate the possible presence of protein-bound mutagens in food an analytical procedure has been devised in which the sample is enzymically hydrolysed, fractionated by HPLC and examined by a modified liquid incubation Ames assay. To validate the method MeIQx was added, as a model compound, to beefburger and a recovery of 82% obtained. The limit of detection for protein-bound mutagens was 1 microgram/kg, expressed as equivalents of MeIQx. No detectable mutagenicity was observed when the procedure was applied to samples of well cooked beefburger, irradiated chicken or mycoprotein.

Mutagenicity testing of imidazole and related compounds.

Ames tests have been performed with imidazole and its principal metabolites, hydantoin and hydantoic acid. N-Acetyl-imidazole, a potential metabolite resulting from the action of intestinal bacteria, and histamine, a structurally related compound which is widely distributed in mammalian tissues, have also been tested. Imidazole and histamine were also tested in the UDS assay in primary rat hepatocytes, while imidazole alone was tested in the M2-C3H mouse fibroblast malignant transformation assay. Imidazole gave consistently negative results in the Ames test, the UDS assay and the transformation assay. The three metabolites of imidazole, namely hydantoin, hydantoic acid and N-acetyl-imidazole, all gave negative results in the Ames test. Histamine gave no evidence of mutagenic activity in the Ames test or of genotoxicity in the UDS assay. These results indicate that imidazole and its metabolites are unlikely to present a mutagenic or carcinogenic hazard.

Studies on the mutagenicity of a zinc oxide-hexachloroethane smoke.

1. A suitable method has been developed for generating atmospheres of zinc oxide/hexachloroethane smoke (ZnHCE). 2. The smoke was investigated using the Ames test and the micronucleus assay. 3. It was weakly mutagenic to the bacteria, but in the bone marrow no increases in micronuclei were detected up to toxic levels of the smoke. 4. The method used here could be applied to other pyrotechnic mixtures which give rise to complex mixtures of products.

Effects of the nitrosopeptide N-(N-acetyl-L-prolyl)-N-nitrosoglycine on Salmonella typhimurium TA100 in the host-mediated assay in mice.

The N-nitrosopeptide N-(N-acetyl-L-prolyl)-N-nitrosoglycine (APNG) was examined for mutagenicity in the mouse host-mediated assay using Salmonella typhimurium strain TA100. Administration of APNG orally or as a single ip injection was shown to produce an increase in revertants. This study provides the first evidence that APNG is absorbed after oral administration in mice and demonstrates that the mutagenic product of APNG is short-lived in vivo.

Genotoxic effects in peripheral blood and urine of workers exposed to low level benzene.

Blood samples were obtained from a population of refinery workers representing different age groups. Sixty six men with low average exposure to benzene and 33 male controls were investigated. An examination of cell cycle kinetics and sister chromatid exchange was carried out on control and exposed individuals. No significant differences were found between groups of individuals varying in their drinking and smoking habits or their exposure to diagnostic x rays. Individuals with the lowest and highest phenol values were examined for urine mutagenicity, with urinary phenol used here as an indicator of benzene exposure. There was no difference in the number of revertant colonies in strains TA98 and 100 between the high and low urinary phenol groups. There were also no differences in any of the biochemical measures or haematological parameters investigated in all the individuals except that higher values for mean corpuscular volume were found in exposed than in control individuals. These values, however, were within the normal clinical range.

The in vivo genotoxic properties of the nitrosopeptide N-(N-acetyl-L-prolyl)-N-nitrosoglycine.

The N-nitrosopeptide N-(N-acetyl-L-prolyl)-N-nitrosoglycine (APNG) was investigated for in vivo genotoxicity using the dominant lethal assay and the micronucleus test in mice, and the bone marrow test in rats. APNG was shown to cause definite genetic effects in the mouse but a much lesser effect in the rat, indicating that APNG is a genotoxic agent in vivo.

Chromosomal aberrations and sister chromatid exchanges in lymphocytes and urine mutagenicity of migraine patients: a comparison of chronic feverfew users and matched non-users.

1. Thirty migraine patients who had taken the leaves, tablets or capsules of feverfew daily for more than 11 consecutive months were compared to 30 feverfew non-user migraine patients who had been individually age- and sex-matched. 2. The frequency of chromosomal aberrations and sister chromatid exchanges (SCE) were determined from lymphocyte cultures established from blood samples taken over a period of several months. Matched pairs were sampled on the same date for two-thirds of the cases, and the greatest difference in sampling time of the remainder was 20 days. Also, the mutagenicity of urine samples from 10 feverfew user migraine patients was compared to that from 10 matched non-user migraine patients using the Ames Salmonella mutagenicity test system. Paired samples were given on the same date. 3. The mean frequency of chromosomal aberrations in the feverfew user group was lower than that in the non-user group both in terms of cells with breaks (2.13% vs 2.76%) and in terms of cells with all aberrations (4.34% vs 5.11%). However, this difference was small and not significant. 4. The mean frequency of SCE in the feverfew exposed group was lower than that in the control group (8.78 vs 8.80 SCE/cell), but, this difference was not significant as determined by factorial analysis of variance (P = 0.897). There was a highly significant variance between the frequencies of SCE in the matched pairs of migraine patients but this was not related to age, sex or feverfew exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenic effect of amniotic fluid from smoking women at term.

Concentrated term amniotic fluid samples from 44 women smokers and 44 controls were investigated with respect to mutagenic effect in the Salmonella/mammalian-microsome mutagenicity test, using tester strains TA98 and TA100. Tests with freeze-dried specimens of term amniotic fluid showed increases in the number of revertant colonies over background values, regardless of smoking status. However, samples from heavy smokers produced a higher number of revertants than did samples from nonsmokers in several experiments with tester strain TA98. The increase was statistically significant, using either total tar content or number of cigarettes smoked to identify heavy smokers. Experimental series with tester strain TA100 also resulted in higher group means for heavy smokers than for nonsmokers, but the difference was not statistically significant with the concentrations used in this assay. We conclude that heavy smokers may expose their unborn children to mutagenic substances.

Study of the amniotic fluid from smokers and non-smokers in the Ames test.

Amniotic fluid from smokers and non-smokers was tested by the Salmonella/mammalian microsome test. Concentrated amniotic fluid from heavy smokers at term showed an increase in the number of revertants with increasing exposure to tar. However, some of the non-smokers had a higher number of revertants than the smokers. No significant differences were found between second-trimester samples from smokers and non-smokers, but the limited volumes available at this stage of pregnancy may be a source of error.

The mutagenic properties of N-nitrosopeptides in the Ames test.

N-(N-Acetyl-L-prolyl)-N-nitrosoglycine (APNG) and N-(N-acetylvalyl)-N-nitrosoglycine (AVNG) are shown to exert mutagenic activity in the Salmonella/mammalian microsome mutagenicity (Ames) test. Positive responses are apparent for base-pair substitution mutation-detecting strains (TA1535, TA100 and TA102) both with and without the addition of S9-mix. It is concluded that both APNG and AVNG are direct-acting mutagens.

The effects of S9 mix from rat oesophagus, salivary gland, and liver on the mutagenicity of the rat oesophageal carcinogen N-nitroso-N-methylaniline in the Salmonella typhimurium assay.

The present study examines the feasibility of using the Salmonella typhimurium plate-incorporation assay of Ames for detecting target-organ specificity with N-nitroso-N-methylaniline (NMA), a compound for which the target site for tumour formation in the rat is the oesophagus. Thus it was anticipated that the oesophagus would bioactivate this compound. The compound has been investigated using S9 from Aroclor- and NMA-induced rat oesophagus, salivary gland and liver in the presence and absence of the co-mutagen, norharman. No response to NMA was seen with oesophageal S9 even though benzo[a]pyrene produced a dose-related increase in revertants in strain TA98 and TA100. No response to NMA was seen with salivary-gland S9. However, a response was produced with Aroclor-induced rat-liver S9 in the presence of norharman and with NMA-induced rat-liver S9 in the absence of norharman.