[Expression of miR-429 and Its Target Gene HSPA4L in Sperms from Asthenospermia Patients.]

OBJECTIVES: To investigate the expression of miR-429 and its target gene heat shock protein A4L (HSPA4L) in sperms from asthenospermia patients. METHODS: Twenty semen samples from healthy and fertile adults and 20 semen samples from asthenospermia patients were collected,and normal sperm parameters were defined according to World Health Organization criteria.The expression levels of miR-429 and HSPA4L mRNA were determined by qRT-PCR,and the bioinformatics tool (Targetscan) was used to predict the target of miR-429.Luciferase reporter assay and transfection study were performed to confirm target gene of miR-429.The expression levels of HSPA4L mRNA and protein were further determined by qRT-CPR and Western blot,respectively. RESULTS: The motility and viability of sperms from asthenospermia patients were lower than that in control group,and miR-429 was up-regulated in sperms from asthenospermia patients.Bioinformatics analysis revealed that HSPA4L was a target of miR-429.Luciferase reporter assay and transfection study further confirmed that miR-429 suppresses the expressions of HSPA4L mRNA and protein via directly targeting HSPA4L 3'UTR.Results from clinical samples also demonstrated that HSPA4L mRNA and protein were down-regulated in sperms from asthenospermia patients and the expression level of miR-429 was inversely correlated with the expression level of HSPA4L mRNA (r=-0.725, P<0.05). CONCLUSIONS: miR-429 is up-regulated in sperms from asthenospermia patients,and it may modulate the motility and viability of sperms via suppressing the expression of HSPA4L.

Acrylamide-induced molecular mutation spectra at HPRT locus in human promyelocytic leukaemia HL-60 and NB4 cell lines.

Acrylamide (AA) is a compound widely used in many industries around the world. The recent finding that it is formed naturally in foods by heating raises human health concerns. AA is a proven carcinogen in animals and a probable carcinogen in humans, while its mutagenicity detected using in vitro mammalian gene mutation assays is still inconsistent in different cell systems. In the present study, we investigated the mutagenicity of AA in human promyelocytic leukaemia cells, HL-60 and NB4 cells, by examining the mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene locus. In a 6-h treatment without the exogenous activation, AA exerted a weak mutagenic effect at the highest concentration used in the study (700 mg/l) in HL-60 cells (P < 0.01) as well as in NB4 cells (P < 0.05). Molecular analysis of AA-induced mutants revealed a different mutation spectrum, when compared to that of spontaneous mutants. The most frequent spontaneous mutations were point mutations, whereas AA-induced mutations were mainly single exon deletions besides point mutations, and an increase in the proportion of partial deletion was associated with the increase of AA treatment. There was no obvious difference in the mutation spectra observed between the HL-60 and NB4 cell lines. These results showed that AA has a weak mutagenic effect at HPRT gene locus in human promyelocytic leukaemia HL-60 and NB4 cell lines and those molecular mutation spectra (single exon deletions and point mutations) may be related to some specific and precise mechanism.

Differential expression of genes associated with cell proliferation and apoptosis induced by okadaic acid during the transformation process of BALB/c 3T3 cells.

Okadaic acid (OA) is a tumor promoter in two-stage carcinogenesis experiments. Nevertheless, the effects of OA on cell transformation, cell proliferation and apoptosis vary widely, and the molecular events underlying these effects of OA are not well understood. In the present study, we examined the promoting activity and the associated effects on cell growth and apoptosis mediated by OA in BALB/c 3T3 cells, and evaluated alterations of gene transcriptional expression by microarray analysis. The promoting activity of OA was estimated by a two-stage transformation assay, in which cells were treated first with a low dose of the initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and then with OA for 14 days. It showed that OA, at concentrations of 7.8-31.3 ng/ml, enhanced the transformation of MNNG-treated cells. In the promotion phase, cells exposed to OA (7.8 ng/ml) grew slowly for the first 2 days and subsequently died. As determined by Hoechst 33342 fluorescent dye and Annexin-V/PI dual-colored flow cytometry, OA induced morphologically apoptotic cells and increased the percentage of early apoptotic cells. The gene expression profile induced by OA at five time points in the promotion phase was determined by use of a specific mouse toxicological microarray containing 1796 clones, and a total of 177 differentially expressed genes were identified. By gene ontology analysis, 31 of these were determined to be functionally involved with cell growth and/or maintenance. In this group, numerous genes associated with the cell proliferation and cell cycle progression were down-regulated at early and/or middle time points. Among these was a subset of genes associated with apoptosis, in which Bnip3, Cycs, Casp3 and Bag1 genes are involved in the mitochondrial pathway of apoptosis. Ier3, Mdm2 and Bnip3 genes may be p53 targets. Furthermore, real-time PCR confirmed the expression changes of five genes selected at random from the differentially expressed genes. We conclude that OA induces cell growth inhibition and apoptosis in the two-stage, MNNG-initiated transformation of BALB/c 3T3 cells. The results of gene expression profile analysis imply that multiple molecular pathways are involved in OA-induced proliferation inhibition and apoptosis. Mitochondrial and p53-associated apoptotic pathways also may contribute to OA-induced apoptosis.

[Mutational spectra in the tk gene of mouse lymphoma cells induced by acrylamide].

OBJECTIVE: To investigate the mechanism of acrylamide (AA)-induced mutational spectra in the tk gene of mouse lymphoma cells. METHODS: L5178Y3.2.7c-tk(+/-) cells were treated with AA at different concentrations. Mutational spectra of tk locus were analyzed by the mouse lymphoma assay (microtiter procedure), and frequency of loss of heterozygosity (LOH) analysis with allele special PCR. Subsequently determined the DNA sequence of negative LOH's colonies induced by AA. RESULTS: The LOH of mutants derived from AA induction was 78.8%, and showed a good dose-response relationship in large colonies. The occurrence of LOH of large colonies at lower doses (150 microg/ml and 300 microg/ml) were 25.0% and 33.3% respectively which were significantly different from those of control (66.7%), and at higher doses (600 microg/ml and 750 microg/ml) were 77.8% and 85.7%. By Sequence analysis showed that AA-induced point mutations were mainly base substitutions, and most of them were T:G-->G:T transversions. CONCLUSION: Functional tk allele gene loss is major mutational event in both spontaneous and induced tk mutants. And point mutations were base substitution.

Development of a semi-quantitative food frequency questionnaire to determine variation in nutrient intakes between urban and rural areas of Chongqing, China.

Nationwide surveys of food and nutrient intake in China have revealed geographical variation between urban and rural areas. This study developed a semi-quantitative food frequency questionnaire (SQFFQ) for cancer risk assessment suitable for both urban and rural populations by conducting a survey of food intake in Chongqing, China. We recruited 100 urban and 104 rural healthy residents aged from 35 to 55 years in Chongqing, and collected dietary data with 3-day weighed records to assist in the development of the SQFFQ. The intake of 35 nutrients was calculated according to Standard Food Composition Tables for China and Japan. For each nutrient estimated by percentage contribution analysis (CA) and multiple regression analysis (MRA), foods with up to a 90% contribution or a 0.90 cumulative R(2) were selected as items for SQFFQs. The food items of the combined SQFFQ were selected from all items listed in either urban or rural SQFFQs. Mean intake of energy, protein and carbohydrate did not differ between the urban and rural residents. The latter consumed more fat than their urban counterparts. We selected 119 food items for the combined SQFFQ, comprising 22 specific items for the urban SQFFQ, 6 for the rural, and 78 common and 13 additional items. The combined SQFFQ covered 33 nutrients with up to a 90% contribution in each area. We were able to develop a data-based SQFFQ that can estimate nutrient intake of both urban and rural populations, with suitable coverage rates. Further reliability and reproducibility tests are now needed to assess its applicability.

Analysis of spontaneous, gamma ray- and ethylnitrosourea-induced hprt mutants in HL-60 cells with multiplex PCR.

AIM: To explore the molecular spectra and mechanism of human hypoxanthine guanine phosphoribosyl transferase (hprt) gene mutation induced by ethyluitrosourea (ENU) and (60)Co gamma-rays. METHODS: Independent human promyelocytic leukemia cells (HL-60) mutants at the hprt locus were isolated from untreated, ethyluitrosourea (ENU) and (60)Co gamma-ray-exposed cells, respectively, and verified by two-way screening. The genetic changes underlying the mutation were determined by multiplex polymerase chain reaction (PCR) amplification and electrophoresis technique. RESULTS: With dosage increased, survival rate of plated cell reduced (in the group with dosage of ENU with 100-200 micro g/ml, P<0.01; in the group with dosage of (60)Co gamma-ray with 2-4 Gy, P<0.05) and mutational frequency increased (in the group of ENU 12.5-200.0 micro g/ml, P<0.05; in the group of (60)Co gamma-ray with 1-4 Gy, P<0.05) significantly. In the 13 spontaneous mutants analyzed, 92.3 % of mutant clones did not show any change in number or size of exon, a single exon was lost in 7.7 %, and no evidence indicated total gene deletion occurred in nine hprt exons. However, deletions were found in 79.7 % of ENU-induced mutations (62.5-89.4 %, P<0.01) and in 61.7 % of gamma-ray-induced mutations (28.6-76.5 %, P<0.01). There were deletion mutations in all 9 exons of hprt gene and the most of induced mutations were chain deletion with multiplex exons (97.9 % in gamma-ray-induced mutants, 88.1 % in ENU-induced mutants). CONCLUSION: The spectra of spontaneous mutations differs completely from that induced by EUN or (60)Co gamma-ray. Although both ENU and gamma-ray can cause destruction of genetic structure, mechanism of mutagenesis between them may be different.

Molecular analysis of Tripterygium hypoglaucum (level) Hutch-induced mutations at the HPRT locus in human promyelocytic leukemia cells by multiplex polymerase chain reaction.

The genotoxicity and cytotoxicity of a Chinese medicinal herb, Tripterygium hypoglaucum (level) Hutch (THH), was investigated in human promyelocytic leukemia (HL-60) cells using the hypoxanthine-guanine phosphoribosyltransferase mutation assay. THH showed clear cytotoxicity and mutagenicity in HL-60 cells at concentrations between 6.7 and 20.0 mg/ml. When the mutants were characterized by techniques based on multiplex PCR, 46.6% of induced mutants were found to have deletions, whereas only 7.7% of spontaneous mutants showed deletions. The rest were not characterized, but were assumed to be mainly point mutations. Mapping of all intragenic deletion breakpoints showed a random distribution of breakpoints in nine exons. Deletion of exon 1 appeared as the only whole gene deletion, while deletions of exon 7/8 and 9 often occurred concomitantly (71.4%). It is concluded that THH is mutagenic in HL-60 cells, predominantly inducing deletions. Since this herb is widely used as a traditional medicine, its genotoxicity should be assessed in vivo in treated humans.

[Effects of total alkaloids of Tripterygium hypoglaucum Hutch on tk gene of mouse lymphoma cells].

OBJECTIVE: To investigate the effects of total alkaloids of Tripterygium hypoglaucum Hutch (THH) on the tk gene of mouse lymphoma cells. METHOD: L5178Y cells were infected with total alkaloids of THH with different concentrations and put into single-cell wells at different time phases. Then the numbers of positive wells were counted and the cell plating efficiency, relative suspension growth and mutation frequency were determined. RESULT: Total alkaloids of THH (0.1-2.0 g x L(-1)) induced tk locus mutation with mutation frequency 2-9 times higher than that of spontaneous mutation frequency of L5178Y cells. There were two different phenotypes of mutation colonies, large colony and small colony, but the main colony was large colony. This phenomenon might be related with the mutagenesis of THH. CONCLUSION: Total alkaloids of THH can exert toxicity and mutagenic effects on tk gene in L5178Y cells, and there may be range limit in gene mutation.