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P XII.7 Detection of DNA damage and its repair in germ cells of male mice treated with TCDD
S-XII: Oerm line mutatlDns in mammals and risk evaluation The insecticides S-2-ethylsulphinylethyl O,o-dimethyl phosphorothioate (Oxydemeton.methyl - OPI) and O.O·dimethyl S-melhyltarbamaylethyl phosphorothioate (Omethoate - On) induce positive results in the mouse spot test . Consequently, it was our aim to investigate whether germ cells arc also affected. Therefore, young adult male Sprague Dawley ral3 (4 per dose group) were treated by gavage with either of the compounds. Doses ranged from 4 mglkg to 40 mglkg (MTD) for OPI and from 8 mglkg to 30 mg/kg (MTD) for OP2. Primary testes cells were prepared 4 h after treatment and evaluated for the induction of DNA single strandbreaks (SSB) via alkaline elut ion. In addition, in vitro expenments were performed to prove the sens itivity of the method to detect test substance related DNA damage. CHO cells were exposed to different concentrations of up to 5000 g/m1 of OPI or OP2 in the absence or presence of S9 mix. The same concentration range was tested in freshly isolated rat testes cells. Whereas all in vitro experiments revealed a dose dependent induction of SSB, there was no indication of SSB induction after in vivo exposure. Since the dominant lethal test showed also negative results in both cases, OPI and OP2 are considered to be nongenetoxic in male germ cells in vivo.
S81
University, Uchinada; kahoku . Ishikawa 920-02, Japan : J Department of Radiation Biology. FaeultyofMedicine. Osaka University, 2-2. Yamada-Olea. Sui/a. Osaka 565. Japan DNA damage induced in male germ cells by 2,3,7 ,8-tetrachloro dibenzo-pdioxin (TCDD) and its repair were measured in C3WHeN mice . Although about 1125 of TCDD in the liver were found in the testis after the intraperitoneal injection, unscheduled DNA synthesis (UDS) and single-strand breaks were not detected at all stages of male germ cells. Chromosome aberrations in the M I stage of spermatocytes were also not detected. After the direct injection of TCDD into the test is, amounts of TCDD bound to the sperm head were max imum at 7th day after the injection, and a little UDS were detected at stages of spermatogonia, spermatocytes and early spermatids. In con trast to the negligible DNA damage to the male germ cells , degeneration of endoplasmatic reuculum system and condensation of nucleus were sign ificantly found in the Sertoli cells (nursing cells of male germ cells) of TCDD-treated mice. Degeneration of Sertoli cells becomes more serious with the time elapsed ane, the treatment, resulting in the ind irec! killing of male germ cells .
Keyword(s): Organophosphates; Testes cells; Alkaline elution Keyword(s): Dioxin ; DNA damage in mouse germ cells ; Degeneration of Sertoli cells
Ip Xn.6J
Comparison of Inherited and somallc human cancen
p~3
mutations In
Tina Hernandez, Pierre Hainaut, Paul Kleihues, Hiroko Ohg aki. Unit of Mechanisms of Carcinogenesis. International Agency for Research on Cancer, 150 Cours Albert Thomas. 69372 Lyon. France Missense mutat ions in the p53 tumor suppressor gene are common in human cancer. Mutations in a specific tumor type may be regarded as "footprints" of specific carcinogens that are involved as causative agents in the development of cancer. DNA-repair and selection of specific mutations act as additional "filters" to gen erate the final mutation spectrum observed lD any parucular tumor type. Two databases of pS3 mutations in human cancer are maintained at the International Agency of Research on Cancer (IARC). One contains about 6000 somatic mutations and the other contains information on germline mutations in 108 families (551 tumors). Comparison of those two databases reveal an exce ss of transitional CpO nucleotides in gerrnline compared to somat ic mutations (37% to 24% respectively, p ~ 0.003) and a higher frequency of G to T transversions in somatic mutations than in germline (16% to 9% respectively, p = 0.03) . CpO transitions can be considered as the result of an endogenous mutat ional mechanism, whereas G to T transversions are a typical molecular signature of environmental carcinogens, such as benzo[a]pyrene. In patients with gerrnline mutations, the most frequent tumor sites arc breast (25%), sarcoma (24%) and brain (12%) . In brain tumors, the spectrum of germline mutations is remarkably similar to that of somatic mutati ons . In breast and sarcoma cancers, however, the spectrum of germline mutations differs significanlly from that of somatic mutations by the proportion of CpG transitions and 0 to T transversions . Both somat ic and germline mutations are located in conserved regions of exons 5 to 8. with major hotspots at codons 17S, 248 , and 213. However. in contrast to somatic mutations, no germline mutations have been identified m codons 176, 179, and 249. The similarities of the germline and somatic mutation spectra in brain tumors suggest that in these neoplasms, pS3 mutat ions are capable of initiat ing the process of malignant transformation. In add ition. in breast cancer and sarcoma, the differences in the mutation spectra suggest an involvement of exogenous carcinogens in the genesis of sporadic tumors. Keyword(s): pH ; Germline; Mutation database
Ip XII.71
Deteetlon of DNA damage .nd"l repair In germ eeus of male mice treated with TeDD
tnoue' , TakayukJ Kurihara", Tadashi Ueda l , Taisei Nomural _ Research Institute, Kanazawa Medical University. Uchinada; kahoku; Ish ikawa 920-02, Japan; 2Department ofAnatomy, KanlJ2a_ Medical Masao
1 Medical
Ip XII.81
Estimating the risk of dominant lethals and heritable translocatlons using chromosome painting of mouse zygotes and two-cell embryo
Marchetti Francesco' , Jack Bishop1, Xiu Lowe', Andrew J. Wyrobek' . 1 BBRP, Lawrence Livermore National Laboratory. Livermore. CA. USA; 2NIEHS, Research '})-ja ngle Parle. NC. USA The object ive of this research was to investigate whether PAINTIDAPI analys is mouse zygoles I-CI) and 2..,ell embryos (2-CI) provides reliable estimates of dominant lethals (DL ) and heritable transloc ations (HT). These are common rodent methods used for evaluat ing the heritable genetic risk posed by occupat ional or environmental mutagens. Our PAINT/DAPI method detects both stable and unstable chromosomal rearrangements us ing a combinat ion of multicolor chromosome painting (PAINT) and DAPI staining. I-CI and 2-e1 metaphases were collected after postmeiotic treatment of male germ cells with 5 x 50 mglkg acrylamide. Analysis of over 700 metaphases showed that the percentages of zygotes and embryos with unstable chromosomal rearrengements were quantitatively sim ilar and followed a sim ilar post-treatment lime course as the percentages of dead implants in standard DL test. Estimates of HT were 28% and 31% in zygotes and embryos. respectively. These percentages compared favorably with the 39% reported by Shelby et al. (1987) using the standard HT test. Analysis of 2·CI metaphases showed that 65 out 16 (85%) of the abnormal embryos were mosaics (either numerical or structural). These results suggest that PAINTIDAPI analysis of mouse zygotes and 2-(:ell embryos can be used to investigate a spectrum of cytogenetic defeclS Ihat are associated with pregnancy loss, birth defects, as well as mosa icism . Worle performed under the auspices of the U.S. DOE by the Lawrence Livermore National Laboratory under contract W-7405-ENG48 and support from NIEHS YO I-ES-10203-OO. Keyword (s): chromosomal damage; germ cells; embryo development
S81
University, Uchinada; kahoku . Ishikawa 920-02, Japan : J Department of Radiation Biology. FaeultyofMedicine. Osaka University, 2-2. Yamada-Olea. Sui/a. Osaka 565. Japan DNA damage induced in male germ cells by 2,3,7 ,8-tetrachloro dibenzo-pdioxin (TCDD) and its repair were measured in C3WHeN mice . Although about 1125 of TCDD in the liver were found in the testis after the intraperitoneal injection, unscheduled DNA synthesis (UDS) and single-strand breaks were not detected at all stages of male germ cells. Chromosome aberrations in the M I stage of spermatocytes were also not detected. After the direct injection of TCDD into the test is, amounts of TCDD bound to the sperm head were max imum at 7th day after the injection, and a little UDS were detected at stages of spermatogonia, spermatocytes and early spermatids. In con trast to the negligible DNA damage to the male germ cells , degeneration of endoplasmatic reuculum system and condensation of nucleus were sign ificantly found in the Sertoli cells (nursing cells of male germ cells) of TCDD-treated mice. Degeneration of Sertoli cells becomes more serious with the time elapsed ane, the treatment, resulting in the ind irec! killing of male germ cells .
Keyword(s): Organophosphates; Testes cells; Alkaline elution Keyword(s): Dioxin ; DNA damage in mouse germ cells ; Degeneration of Sertoli cells
Ip Xn.6J
Comparison of Inherited and somallc human cancen
p~3
mutations In
Tina Hernandez, Pierre Hainaut, Paul Kleihues, Hiroko Ohg aki. Unit of Mechanisms of Carcinogenesis. International Agency for Research on Cancer, 150 Cours Albert Thomas. 69372 Lyon. France Missense mutat ions in the p53 tumor suppressor gene are common in human cancer. Mutations in a specific tumor type may be regarded as "footprints" of specific carcinogens that are involved as causative agents in the development of cancer. DNA-repair and selection of specific mutations act as additional "filters" to gen erate the final mutation spectrum observed lD any parucular tumor type. Two databases of pS3 mutations in human cancer are maintained at the International Agency of Research on Cancer (IARC). One contains about 6000 somatic mutations and the other contains information on germline mutations in 108 families (551 tumors). Comparison of those two databases reveal an exce ss of transitional CpO nucleotides in gerrnline compared to somat ic mutations (37% to 24% respectively, p ~ 0.003) and a higher frequency of G to T transversions in somatic mutations than in germline (16% to 9% respectively, p = 0.03) . CpO transitions can be considered as the result of an endogenous mutat ional mechanism, whereas G to T transversions are a typical molecular signature of environmental carcinogens, such as benzo[a]pyrene. In patients with gerrnline mutations, the most frequent tumor sites arc breast (25%), sarcoma (24%) and brain (12%) . In brain tumors, the spectrum of germline mutations is remarkably similar to that of somatic mutati ons . In breast and sarcoma cancers, however, the spectrum of germline mutations differs significanlly from that of somatic mutations by the proportion of CpG transitions and 0 to T transversions . Both somat ic and germline mutations are located in conserved regions of exons 5 to 8. with major hotspots at codons 17S, 248 , and 213. However. in contrast to somatic mutations, no germline mutations have been identified m codons 176, 179, and 249. The similarities of the germline and somatic mutation spectra in brain tumors suggest that in these neoplasms, pS3 mutat ions are capable of initiat ing the process of malignant transformation. In add ition. in breast cancer and sarcoma, the differences in the mutation spectra suggest an involvement of exogenous carcinogens in the genesis of sporadic tumors. Keyword(s): pH ; Germline; Mutation database
Ip XII.71
Deteetlon of DNA damage .nd"l repair In germ eeus of male mice treated with TeDD
tnoue' , TakayukJ Kurihara", Tadashi Ueda l , Taisei Nomural _ Research Institute, Kanazawa Medical University. Uchinada; kahoku; Ish ikawa 920-02, Japan; 2Department ofAnatomy, KanlJ2a_ Medical Masao
1 Medical
Ip XII.81
Estimating the risk of dominant lethals and heritable translocatlons using chromosome painting of mouse zygotes and two-cell embryo
Marchetti Francesco' , Jack Bishop1, Xiu Lowe', Andrew J. Wyrobek' . 1 BBRP, Lawrence Livermore National Laboratory. Livermore. CA. USA; 2NIEHS, Research '})-ja ngle Parle. NC. USA The object ive of this research was to investigate whether PAINTIDAPI analys is mouse zygoles I-CI) and 2..,ell embryos (2-CI) provides reliable estimates of dominant lethals (DL ) and heritable transloc ations (HT). These are common rodent methods used for evaluat ing the heritable genetic risk posed by occupat ional or environmental mutagens. Our PAINT/DAPI method detects both stable and unstable chromosomal rearrangements us ing a combinat ion of multicolor chromosome painting (PAINT) and DAPI staining. I-CI and 2-e1 metaphases were collected after postmeiotic treatment of male germ cells with 5 x 50 mglkg acrylamide. Analysis of over 700 metaphases showed that the percentages of zygotes and embryos with unstable chromosomal rearrengements were quantitatively sim ilar and followed a sim ilar post-treatment lime course as the percentages of dead implants in standard DL test. Estimates of HT were 28% and 31% in zygotes and embryos. respectively. These percentages compared favorably with the 39% reported by Shelby et al. (1987) using the standard HT test. Analysis of 2·CI metaphases showed that 65 out 16 (85%) of the abnormal embryos were mosaics (either numerical or structural). These results suggest that PAINTIDAPI analysis of mouse zygotes and 2-(:ell embryos can be used to investigate a spectrum of cytogenetic defeclS Ihat are associated with pregnancy loss, birth defects, as well as mosa icism . Worle performed under the auspices of the U.S. DOE by the Lawrence Livermore National Laboratory under contract W-7405-ENG48 and support from NIEHS YO I-ES-10203-OO. Keyword (s): chromosomal damage; germ cells; embryo development