Animals ; Genes, T-Cell Receptor ; genetics ; Humans ; Hypoxanthine Phosphoribosyltransferase ; genetics ; Mice ; Mutation

OBJECTIVE: To carry out genetic testing and prenatal diagnosis for a Chinese pedigree with Lesch-Nyhan syndrome (LNS) but no specimen from the affected probands. METHODS: All affected individuals in this pedigrees were male and had deceased during childhood, with no biological specimen left. Based on their typical neurological dysfunction and tendency for self-mutilation, the diagnosis of LNS was suspected. Sanger sequencing was carried out to detect potential variant of the HPRT1 gene among female members from the pedigree. Following the identification of the pathogenic variant, prenatal diagnosis was provided for a high-risk fetus. RESULTS: The proband's mother and three other females were found to harbor heterozygous c.500_501delGGinsC (p.Arg167fs*23) variant of the HPRT1 gene, which was unreported previously. Prenatal diagnosis showed that the fetus was a male and had inherited the same pathogenic variant. CONCLUSION The c.500_501delGGinsC variant of the HPRT1 gene probably underlay the LNS in this pedigree. Above finding has provided a basis for prenatal diagnosis and genetic counseling for this pedigree.
Male ; Female ; Humans ; Pregnancy ; Lesch-Nyhan Syndrome/genetics* ; Pedigree ; Hypoxanthine Phosphoribosyltransferase/genetics* ; Prenatal Diagnosis ; China ; Mutation

OBJECTIVETo study genetic damage of workers alone occupationally exposed to methotrexate (MTX) with three end-points.

METHODSThe blood samples from 21 workers exposed to MTX and 21 controls were detected with micronucleus test, comet assay, hprt gene mutation test and TCR gene mutation test.

RESULTSThe mean micronuclei rate (MNR) and mean micronucleated cells rate (MCR) in 21 workers were 10.10 per thousand +/- 0.95 per thousand and 8.05 per thousand +/- 0.75 per thousand, respectively, which were significantly higher than those (5.48 per thousand +/- 0.82 per thousand and 4.38 per thousand +/- 0.58 per thousand) in control (P < 0.01). The mean tail length (MTL) of 21 workers and 21 controls were (1.30 +/- 0.06) microm and (0.07 +/- 0.01) microm, respectively, there was significant difference between workers and controls (P < 0.01). But the difference between workers and controls for mean tail moment (MTM) was not significant (P > 0.05). The average mutation frequency (Mf-hprt) of hprt and (Mf-TCR) of TCR in workers were 1.00 per thousand +/- 0.02 per thousand and (6.87 +/- 0.52) x 10(-4), respectively, which were significantly higher than those [0.86 per thousand +/- 0.01 per thousand and (1.67 +/- 0.14) x 10(-4)] in control (P < 0.01).

CONCLUSIONThe genetic damage to some extent appeared in workers occupationally exposed to methotrexate.

Adult ; Comet Assay ; DNA Damage ; Female ; Humans ; Hypoxanthine Phosphoribosyltransferase ; genetics ; Male ; Methotrexate ; toxicity ; Micronucleus Tests ; Middle Aged ; Mutation ; Occupational Exposure

OBJECTIVETo explore the molecular spectra and mechanism of human hypoxanthine guanine phosphoribosyl transferase (HPRT) gene mutation induced by ethyluitrosourea (ENU).

METHODSSingle cell cloning culture, two-way screening, multiple PCR amplification and electrophoresis technique were used.

RESULTSWith dose of ENU increasing, cell plating efficiency reduced (in the group with 100-200 micro g/ml doses, P < 0.01) and mutation frequency increased (in the group with 12.5-200.0 micro g/ml doses, P < 0.05) significantly. There was no all exons deletion in spontaneous mutations, and only 7.7% of them were detected as single exon deletion. But, deletion was found in 79.7% of ENU-induced mutations (62.5%-89.4%, P < 0.01), and deletion mutations in all nine exons of HPRT gene. Most of ENU-induced mutations were chain deletion with multiple exons (88.1%).

CONCLUSIONSThe spectra in spontaneous mutations differed completely from ENU-induced ones. ENU was liable to cause bigger changes in genetic structure, which suggested a stronger ENU's mutagenesis.

Alkylating Agents ; pharmacology ; Ethylnitrosourea ; pharmacology ; HL-60 Cells ; Humans ; Hypoxanthine Phosphoribosyltransferase ; genetics ; metabolism ; Leukemia, Myeloid ; pathology ; Mutation ; drug effects ; Tumor Stem Cell Assay

OBJECTIVETo evaluate the genotoxic and nongenotoxic effects of short-term exposure to glycidyl mathacrylate (GMA) on human lung fibroblast cells (2BS cells) in vitro.

METHODSDNA strand breakage was determined by single cell gel electrophoresis, and DNA ladder formation assay and flow cytometric analysis were carried out to detect apoptic responses of cells to GMA exposure. The HPRT gene mutation assay was used to evaluate the mutagenicity, and the effect of GMA on gap junctional intercellular communication (GJIC) in the exposed cells was examined with the scrape loading/dye transfer technique. The ability of GMA to transform 2BS cells was also tested by an in vitro cell transformation assay.

RESULTSExposure to GMA resulted in a dose-dependent increase in DNA strand breaks but not apoptic responses. GMA was also shown to significantly induce HPRT gene mutations and morphological transformation in 2BS cells in vitro. In contrast, GMA produced a concentration-dependent inhibition of GJIC.

CONCLUSIONSGMA elicits both genotoxic and nongenotoxic effects on 2BS cells in vitro. The induction of DNA damage and gene mutations and inhibition of GJIC by GMA may casually contribute to GMA-induced cell transformation.

Cell Communication ; Cell Differentiation ; Comet Assay ; DNA Damage ; DNA Mutational Analysis ; Epoxy Compounds ; toxicity ; Fibroblasts ; Gap Junctions ; Humans ; Hypoxanthine Phosphoribosyltransferase ; genetics ; Lung ; cytology ; Methacrylates ; toxicity

OBJECTIVETo label embryonic stem (ES) cells with enhanced green fluorescent protein (EGFP) on the hypoxanthineguanine phosphoribosyl transferase (HPRT) gene locus for the first time to provide a convenient and efficient way for cell tracking and manipulation in the studies of transplantation and stem cell therapy.

METHODSHomologous fragments were obtained by polymerase chain reaction (PCR), from which the gene targeting vector pHPRT-EGFP was constructed. The linearized vector was introduced into ES cells by electroporation. The G418(r)6TG(r) cell clones were obtained after selection with G418 and 6TG media. The integration patterns of these resistant cell clones were identified with Southern blotting.

RESULTSEGFP expressing ES cells on the locus of HPRT were successfully generated. They have normal properties, such as karyotype, viability and differentiation ability. The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages and in differentiated cells. Cultured in suspension, the "green" ES cells aggregated and formed embryoid bodies, retaining the green fluorescence at varying developmental stages. The "green" embryoid bodies could expand and differentiate into various types of cells, exhibiting ubiquitous green fluorescence.

CONCLUSIONSThis generation of "green" targeted ES cells is described in an efficient protocol for obtaining the homologous fragments by PCR. Introducing the marker gene in the genome of ES cells, we should be able to manipulate them in vitro and use them as vehicles in cell-replacement therapy as well as for other biomedical and research purposes.

Animals ; Cells, Cultured ; Chromosome Mapping ; Embryo, Mammalian ; cytology ; Green Fluorescent Proteins ; Hypoxanthine Phosphoribosyltransferase ; genetics ; Luminescent Proteins ; metabolism ; Mice ; Recombination, Genetic ; Stem Cells ; metabolism ; Transgenes

Potential toxicological interactions of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and/or dibuthyl phthalate (DBP) on ozone were investigated after 32- and 52-wk exposures using hprt mutation assay. Male and female B6C3F1 mice exposed to ozone (0.5 ppm), NNK (1.0 mg/kg), DBP (5,000 ppm), and two or three combinations of these toxicants 6 h per day for 32- and 52-wk showed increases in the frequencies of TG rlymphocytes compared to the control groups. Additive interactions were noted from two combination groups compared to the ozone alone in both sexes of 32- and 52-wk studies. The most common specific mutation type in the hprt genes of test materials-treated male and female mice was transversion with very few transition. The results indicate that such dominant transversion may be responsible for toxicity and combined exposure to ozone, NNK, and DBP induces additive genotoxicities compared to ozone alone.
Animals ; Carcinogens/*toxicity ; DNA Mutational Analysis ; Dibutyl Phthalate/*toxicity ; Drug Combinations ; Female ; Hypoxanthine Phosphoribosyltransferase/*genetics ; Male ; Mice ; Mutagenicity Tests ; *Mutation/drug effects ; Nitrosamines/*toxicity ; Ozone/*toxicity ; Reverse Transcriptase Polymerase Chain Reaction ; T-Lymphocytes/drug effects/enzymology

OBJECTIVETo explore the differences of the silica-induced inhibition on cellular proliferation and hprt gene mutagenesis between lung fibroblasts and alveolar type II cells.

METHODSThe proliferation inhibitive cytotoxicity was detected by MTT (3-[4,5-Dimethylthiazolzyl]-2,5-Diphenyl Tetrazolium Bromide) colorimetric method. Mutation in the hprt gene was screened by culture in the presence of the toxic purine analog, 6-thioguanine (6-TG).

RESULTSUnder the same circumstances of silica exposure, alveolar type II cells was more sensitive than lung fibroblasts for proliferation inhibition. The median proliferation inhibition concentration (IC50) of silica on epithelial was 140 micrograms/cm2, whereas IC50 of silica on fibroblasts was 282 micrograms/cm2. At the same doses of silica, the hprt gene mutation frequency in type II cells (84.2 x 10(-6))-156.6 x 10(-6) was statistically higher than that in fibroblasts (67.6 x 10(-6)-114.3 x 10(-6), P < 0.05).

CONCLUSIONThere were significant differences of both silica-induced cell proliferation inhibition and hprt gene mutation between rat lung fibroblasts and type II epithelial cells. In vitro, cultured rat alveolar type II cells were more sensitive in cytotoxicity and hprt gene mutagenesis to silica dust than lung fibroblasts were.

Animals ; Cell Proliferation ; drug effects ; Epithelial Cells ; drug effects ; Fibroblasts ; drug effects ; Hypoxanthine Phosphoribosyltransferase ; genetics ; Lung ; cytology ; drug effects ; metabolism ; Mutation ; Pulmonary Alveoli ; cytology ; drug effects ; Rats ; Silicon Dioxide ; toxicity