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P XII.14 Evaluation of clastogenicity or chemical agents using in vitro assay with human spermatozoa
S-Xtt: Germ Ime mutations in mammals and risk evaluation Since aneuplo idy in human germ cells may lead to deleterious reproductive outcomes, it is important to assess the incidence of meiotic nondisjunction for different chromosomes. Cytogenetic analysis of human gametes represents the most direct method for studying errors in chromosome segregat ion. Recently, the development of multicolour FISH with chromosome-specific alphoid DNA probes allowed nondisjunction in male meiosis to be more easily and correctly detected in sperm nuclei as compared to the complex and time-consurmng techniques previously employed. Aim of the study was to evaluate the frequency of aneuploidy and diploidy in sperm of five healthy donors . Decondensed sperm nuclei were simultaneously hybridized with biotin - and digoxigenin-labeled DNA probes recogniz ing the centromere of chromosomes 2 (green signal), Y (red signal), and X (orange signal). Two spots of different colour (green for the autosome and red or orange depending on the sex chromosome present) indicate haploid sperm . Only one spot, two signals of the same colour or a doubling of the two different spots identify nullisomic , disomic or diploid sperm, respectively. The results of fluorescence analysis indIcate: J) the mean ratio of X- or Y·bearing sperm nuclei agrees with the expected I: I; 2) nullisomi c sperm are more frequently observed than disom ic sperm ; 3) sex chromosome me iotic nondisjunction occurs at an higher rate than autosomal nondisjunction; 4) aneuploid sperm for the sex chromosome are due mainly to errors in meios is I; 5) diploidy occurs at a very low frequency as compared to aneuploidy.
S89
9, December 1995). To determine whether these in vitro results accurately depicted risk for individuals taking chromium picol inate as a supplement. the compound was evaluated by the Ames and the rat in vivo cytogenetic assays. Chromium picolinate was not mutagenic when tested up to a maximum of 5 rug/plate in the Ames assay. Male and female Sprague-Dawley rats were dosed with vehicle, 33, 2S0 or 2000 mg/kg of chromium picol inate, which corresponded to 4.1, 30.8 and 246 mg/kg of chromium. The lowest dose of chromium (4.1 mg/kg ), is approximately 1000 times the average human dose, based on a mglkg comparison. Animals were dosed once and were sacrificed either 18 or 42 hours later. Control animals consisted of vehicle (water) and cyclophosphamide (30 mglkg). There was no induction of chromosomal damage by chromium picolinate . Animals dosed with chromium picolinate had values similar to the vehicle control. group. The mean values for males were 0.4%. 0.8% and 0.4% for the 18 hour time point and 1.4%, 0.8% and 0.4% at the 42-hour time point. Female animals similarly dosed with 33, 250 and 2000 mglkg had mean values of 06%. 0.2% and 0.6% at the 18-hour time point and 0.2%, 0.2% and 0.0% at the 42-hour time point. The positive control induced significant damage at the I8-hour time point with an average of 30% damaged cells for males and 37% for females. Chrom ium picolinate was not found to be mutagenic by the Ames assay or to induce chromosomal aberrations in the rat cytogenetic assay. Keyword(s): Chromium picolinate; Mutagenicity; Chromosomal aberration
Keyword(s) : Human sperm; Aneuploidy; Multicolour FISH
Ip XII.161 Evaluation ofclastogenlclly of chemIcal agents using In vitro assay with human spermatozoa
Blo-anllclastogenic effects of mustard oil and garlic In the first-generation offsprings of sodium arsenite treated mice
Hiroyuki Tateno l , Sumio Iijima 1, Akio Asaka l , Yujiroh Kamiguchi l . J Dept. of Bioi. Sci., Asahikawa Med. Col.. Asahikawa, Japan; l Dept. of Health Sciences, Yamanasiu Med. Uniu, Tamaho, Japan
T. Das, A. Roychoudhury , A. Sharma. Center of Advanced Studies in Cell and Chromosome Research, Department of Botany. University of Calcutta. 35, Ballygunge Circular Road. Calcutta- 700 0/9. India
Clastogen ic effects of four chemical agents, nitrobenzene (NB), bleomycm (BLM ), urethane (U'I) and N-meth yl-N' -nitro-N-nitrosoguanidine (MNNG) on human spermatozoa were studied using an interspecific m vitro fertilization system with hamster oocytes . Frozen semen samples were thawed, and motile spermatozoa were separated by Percoll density gradient centrifugation . The spermatozoa were exposed to 500 Ilglml NB, SO Ilg/ml BLM, 1000 Ilglml UT and 0.5 ug/ml MNNG for 2 h at 37"C. More than 100 spermatozoa were karyoanalyzed to measure the incidence of spermatozoa with structural chromosome aberrat ions and the number of structural chromosome aberrat ions per spermatozoon in each experimental group and its matched control group. No c1astogenic effect was found following treatments with NB and UT. In BLM treatment, the incidence of spermatozoa with chromosome aberrations was not significantly higher than that of the control, but the number of chromosome aberrations per spermatozoon rose to significant level (P < 0.05) because some spermatozoa had multiple chromosome aberrations. Aberrations induced by BLM were breaks, fragments and exchanges, and all contained both chromosometype and chromat id-type aberrations. Human sperm chromosomes were also vulnerable to MNNG, showing a significant increase (P < O.OS) in both the incidence of spermatozoa with chromosome aberrations and the number of chromosome aberrations per spermatozoon. MNNG-induced chromosome aberrations were mainly chromatid breaks and fragments . The difference in type of structural chromosome aberrations observed between BLM and MNNO is probably due to a difference in primary DNA lesions induced by these chemical agents.
The protection afforded by mustard oil and garlic was evaluated in the bone mar row cells of progeny of mice exposed to sodium arsenite. The mice were primed with the two dietary components, singly and in combination for thiny consecutive days and sodium arsenite was administered subcutaneously on day 7, 14,21 and 30 of the experiment. The exposed male and female mice were allowed to mate and their offsprings were studied. The end points scored were chromosomal aberrations and percentage of damaged cells . When mustard oil and garlic were given together the reduct ion of arsenite induced chromosomal aberrations wen: more pronounced than either of the supplements given singly. The magn itude of chromosomal aberration in the offsprings were lower than their exposed parents . The radical scavenging property of the sulfhydryl compounds of garlic and the bio-anticlastogenic activity of the fatty acids present in mustard oil may poss ibly attribute to this reduction.
Ip XII.14!
Keyword(s): Chemicals; Clastogenicity; Human sperm
Ip XII.lsl
Evaluation of chromium plcollnate In the Amesand the rat In vivo chromosomal aberrallon assays
Henry J. Esber", Victor Moreno l • Kenneth S. Loveday", J GTC Mason Laboratories. Won:ester, MA, USA; ZNutrilion 21, San Diego. CA, USA Previously, chromium picolinate was shown to induce chromosomal damage in vitro in the AAB-CHO cell line (Steams et ai, 1995, FASEB Journal, Vol.
Keyword(s): Clastogenicity; Anti-elastogenicity; Dietary Sodium arsenite ; Progeny; Chromosome aberrations
supplements;
S89
9, December 1995). To determine whether these in vitro results accurately depicted risk for individuals taking chromium picol inate as a supplement. the compound was evaluated by the Ames and the rat in vivo cytogenetic assays. Chromium picolinate was not mutagenic when tested up to a maximum of 5 rug/plate in the Ames assay. Male and female Sprague-Dawley rats were dosed with vehicle, 33, 2S0 or 2000 mg/kg of chromium picol inate, which corresponded to 4.1, 30.8 and 246 mg/kg of chromium. The lowest dose of chromium (4.1 mg/kg ), is approximately 1000 times the average human dose, based on a mglkg comparison. Animals were dosed once and were sacrificed either 18 or 42 hours later. Control animals consisted of vehicle (water) and cyclophosphamide (30 mglkg). There was no induction of chromosomal damage by chromium picolinate . Animals dosed with chromium picolinate had values similar to the vehicle control. group. The mean values for males were 0.4%. 0.8% and 0.4% for the 18 hour time point and 1.4%, 0.8% and 0.4% at the 42-hour time point. Female animals similarly dosed with 33, 250 and 2000 mglkg had mean values of 06%. 0.2% and 0.6% at the 18-hour time point and 0.2%, 0.2% and 0.0% at the 42-hour time point. The positive control induced significant damage at the I8-hour time point with an average of 30% damaged cells for males and 37% for females. Chrom ium picolinate was not found to be mutagenic by the Ames assay or to induce chromosomal aberrations in the rat cytogenetic assay. Keyword(s): Chromium picolinate; Mutagenicity; Chromosomal aberration
Keyword(s) : Human sperm; Aneuploidy; Multicolour FISH
Ip XII.161 Evaluation ofclastogenlclly of chemIcal agents using In vitro assay with human spermatozoa
Blo-anllclastogenic effects of mustard oil and garlic In the first-generation offsprings of sodium arsenite treated mice
Hiroyuki Tateno l , Sumio Iijima 1, Akio Asaka l , Yujiroh Kamiguchi l . J Dept. of Bioi. Sci., Asahikawa Med. Col.. Asahikawa, Japan; l Dept. of Health Sciences, Yamanasiu Med. Uniu, Tamaho, Japan
T. Das, A. Roychoudhury , A. Sharma. Center of Advanced Studies in Cell and Chromosome Research, Department of Botany. University of Calcutta. 35, Ballygunge Circular Road. Calcutta- 700 0/9. India
Clastogen ic effects of four chemical agents, nitrobenzene (NB), bleomycm (BLM ), urethane (U'I) and N-meth yl-N' -nitro-N-nitrosoguanidine (MNNG) on human spermatozoa were studied using an interspecific m vitro fertilization system with hamster oocytes . Frozen semen samples were thawed, and motile spermatozoa were separated by Percoll density gradient centrifugation . The spermatozoa were exposed to 500 Ilglml NB, SO Ilg/ml BLM, 1000 Ilglml UT and 0.5 ug/ml MNNG for 2 h at 37"C. More than 100 spermatozoa were karyoanalyzed to measure the incidence of spermatozoa with structural chromosome aberrat ions and the number of structural chromosome aberrat ions per spermatozoon in each experimental group and its matched control group. No c1astogenic effect was found following treatments with NB and UT. In BLM treatment, the incidence of spermatozoa with chromosome aberrations was not significantly higher than that of the control, but the number of chromosome aberrations per spermatozoon rose to significant level (P < 0.05) because some spermatozoa had multiple chromosome aberrations. Aberrations induced by BLM were breaks, fragments and exchanges, and all contained both chromosometype and chromat id-type aberrations. Human sperm chromosomes were also vulnerable to MNNG, showing a significant increase (P < O.OS) in both the incidence of spermatozoa with chromosome aberrations and the number of chromosome aberrations per spermatozoon. MNNG-induced chromosome aberrations were mainly chromatid breaks and fragments . The difference in type of structural chromosome aberrations observed between BLM and MNNO is probably due to a difference in primary DNA lesions induced by these chemical agents.
The protection afforded by mustard oil and garlic was evaluated in the bone mar row cells of progeny of mice exposed to sodium arsenite. The mice were primed with the two dietary components, singly and in combination for thiny consecutive days and sodium arsenite was administered subcutaneously on day 7, 14,21 and 30 of the experiment. The exposed male and female mice were allowed to mate and their offsprings were studied. The end points scored were chromosomal aberrations and percentage of damaged cells . When mustard oil and garlic were given together the reduct ion of arsenite induced chromosomal aberrations wen: more pronounced than either of the supplements given singly. The magn itude of chromosomal aberration in the offsprings were lower than their exposed parents . The radical scavenging property of the sulfhydryl compounds of garlic and the bio-anticlastogenic activity of the fatty acids present in mustard oil may poss ibly attribute to this reduction.
Ip XII.14!
Keyword(s): Chemicals; Clastogenicity; Human sperm
Ip XII.lsl
Evaluation of chromium plcollnate In the Amesand the rat In vivo chromosomal aberrallon assays
Henry J. Esber", Victor Moreno l • Kenneth S. Loveday", J GTC Mason Laboratories. Won:ester, MA, USA; ZNutrilion 21, San Diego. CA, USA Previously, chromium picolinate was shown to induce chromosomal damage in vitro in the AAB-CHO cell line (Steams et ai, 1995, FASEB Journal, Vol.
Keyword(s): Clastogenicity; Anti-elastogenicity; Dietary Sodium arsenite ; Progeny; Chromosome aberrations
supplements;