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Retinoic acid teratogenicity in rat congenic and recombinant inbred strains with malformation syndrome
ELSEVIER
Retinoic Acid Teratogenicity in Rat Congenic and Recombinant Strains with Malformation Syndrome
Inbred
V. Bilti and V. Kien
A
MODEL SYSTEM for teratogenicity testing was developed in our laboratory based on congenic and recombinant inbred (RI) strains of the rat carrying polydactyly-luxate syndrome (PLS).‘,’ The phenotypic manifestation of PLS determining lx allele is strongly influenced by genetic background where it operates: one or both pairs of limbs can be affected and the manifestation can include preaxial polydactyly, oligodactyly, and hemimely of the tibia and radius. Compounds with known teratogenic effect in rodents given in appropriate doses on day 11 or 12 of pregnancy were found to induce preaxial leg malformations (polydactyly up to oligodactyly) in +/Lx fetuses which are normodactylous without teratogenic influence. After the same dose of a teratogen the +/+ fetuses remained unaffected, or were affected with a much lower frequency. In +/Lx fetuses, the frequency and intensity of malformations depend on genetic background, the teratogenic effect being increased in the presence of Brown Norwegian (BN) rat genes.’ These results indicate not only the Lx allele- teratogen interaction-but also the teratogen interaction with modifying genes of genetic background. Using the above-mentioned system we succeeded in proving the teratogenic action of thalidomide which was previously never clearly detected in rodents.’ We are presenting the results of experiments in which the interaction of retinoic acid (RA) with mutant allele Lx and its modifiers has been studied. Teratogenic action of RA has been firmly established and numerous molecules mediating its effect are known. Retinoids interact with receptors to initiate a cascade of changes in gene expression, namely in the homeobox gene family. In our experiments, the response to teratogenic influence of all-trans RA given on day 11 of pregnancy differs basically depending on fetus genotype (Table 1). Although the LEW/BN +/+ fetuses remained without specific limb malformations, in the LEW/BN +/Lx fetuses carrying Lx allele in heterozygous condition preaxial polydactyly of hind limbs was induced in nearly 13%. On the contrary, in fetuses homozygous in Lx allele with 100% preaxial polydactyly of hind limbs significant preaxial reduction of number of toes and zeugopodium reduction was caused by RA administration. In LEW/BN Lx/Lx fetuses nearly 95% of hind limbs exhibited reduction of toes from six
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas,
New York, NY 10010
Transplantation Proceedings, 29, 1707-l 708 (1997)
Table Genotype of Fetuses
LEW/BN +I+ LEWIBN +/Lx LEW/BN Lx/Lx SHWBXH2 LX/LX
I. Retinoid Dose (mg/kg)
Acid Influence
LF
Limbs
on Hind Limbs Percent PD
Percent R
NO. Litters
0.0 100.0
68 39
136 78
0.0 0.0
0.0 0.0
6 4
0.0 100.0
88 35
176 70
0.0 12.9*
0.0 0.0
8 4
0.0 100.0
103 28
206 56
100.0 5.4
0.0 94.6’
9 4
0.0 100.0
56 26
112 52
100.0 13.5
0.0 86.5*
6 5
Abbreviations: LF, living fetuses; %PD, % of hind limbs with preaxial polydactyly; %R, % of hind limbs with preaxial reduction of toes. *5 up 6 triphalangeal toes. +5 up 4 triphalangeal toes. t4 up 3 triphalangeal toes.
to five or four. In SHR/BXH2 Lx/Lx group 86.5% of hind limbs were found with number of toes reduced from five to four up to three, the foot morphotype being mostly the same as in BXH2 oligodactylous strain, resembling thus the thalidomide effect. In this group zeugopodium and stylopodium were also severely affected and two cases of sirenomelia occurred. The striking reduction changes after RA administration in the last mentioned group indicate the interaction of RA with modifying gene combination specific for SHR,BXH2 genotype. To elucidate further the teratogenic effect of RA and also of thalidomide it is of interest that the strain BN-Lx and BXH2 differ from the SHR-Lx strain in genetic markers on chromosomes 3, 4, 7, and 10, where Hox and RAR genes were assigned. Moreover, the Lx allele was assigned to chromosome 8 of the rat indicating the
From the Institute of Biology, Faculty of University, Prague, Czech Republic. Supported by Grant #274 from The Grant University and GACR grant #302/96/0604. Address reprint requests to V. BilB, Institute of Medicine, Charles University, 1 Faculty of 4, Prague 2 128 00 Czech Republic.
Medicine, Agency
Charles
of Charles
of Biology, Faculty Medicine, Albertov
0041-1345/97/$17.00 PII so041 -1345(97)00024-9
1707
1708 homology of Lx with mouse lu mutation on MMU 9. The extensive homology of these two chromosomes [RN08MMU9] is indicated.4 The RI strain system with unique set of modifying genes fixed in each strain could provide us with a tool for uncovering of genes affected by RA action and involved in limb morphogenesis.
Biti
AND KhEN
REFERENCES
1. Bila V, Kien V: Sbornik ICk. 90:90, 1988 2. l&n 1996
V, Bila V, KaSparek R, et al: Fol Biol [Praha] 42:159,
3. Bila V, Kien V: Fol Biol [Praha] 40:161, 1994 4. Szpirer C, Szpirer J, Klinga-Levan K, et al: Fol Biol [Praha] 42:175, 1996
Retinoic Acid Teratogenicity in Rat Congenic and Recombinant Strains with Malformation Syndrome
Inbred
V. Bilti and V. Kien
A
MODEL SYSTEM for teratogenicity testing was developed in our laboratory based on congenic and recombinant inbred (RI) strains of the rat carrying polydactyly-luxate syndrome (PLS).‘,’ The phenotypic manifestation of PLS determining lx allele is strongly influenced by genetic background where it operates: one or both pairs of limbs can be affected and the manifestation can include preaxial polydactyly, oligodactyly, and hemimely of the tibia and radius. Compounds with known teratogenic effect in rodents given in appropriate doses on day 11 or 12 of pregnancy were found to induce preaxial leg malformations (polydactyly up to oligodactyly) in +/Lx fetuses which are normodactylous without teratogenic influence. After the same dose of a teratogen the +/+ fetuses remained unaffected, or were affected with a much lower frequency. In +/Lx fetuses, the frequency and intensity of malformations depend on genetic background, the teratogenic effect being increased in the presence of Brown Norwegian (BN) rat genes.’ These results indicate not only the Lx allele- teratogen interaction-but also the teratogen interaction with modifying genes of genetic background. Using the above-mentioned system we succeeded in proving the teratogenic action of thalidomide which was previously never clearly detected in rodents.’ We are presenting the results of experiments in which the interaction of retinoic acid (RA) with mutant allele Lx and its modifiers has been studied. Teratogenic action of RA has been firmly established and numerous molecules mediating its effect are known. Retinoids interact with receptors to initiate a cascade of changes in gene expression, namely in the homeobox gene family. In our experiments, the response to teratogenic influence of all-trans RA given on day 11 of pregnancy differs basically depending on fetus genotype (Table 1). Although the LEW/BN +/+ fetuses remained without specific limb malformations, in the LEW/BN +/Lx fetuses carrying Lx allele in heterozygous condition preaxial polydactyly of hind limbs was induced in nearly 13%. On the contrary, in fetuses homozygous in Lx allele with 100% preaxial polydactyly of hind limbs significant preaxial reduction of number of toes and zeugopodium reduction was caused by RA administration. In LEW/BN Lx/Lx fetuses nearly 95% of hind limbs exhibited reduction of toes from six
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas,
New York, NY 10010
Transplantation Proceedings, 29, 1707-l 708 (1997)
Table Genotype of Fetuses
LEW/BN +I+ LEWIBN +/Lx LEW/BN Lx/Lx SHWBXH2 LX/LX
I. Retinoid Dose (mg/kg)
Acid Influence
LF
Limbs
on Hind Limbs Percent PD
Percent R
NO. Litters
0.0 100.0
68 39
136 78
0.0 0.0
0.0 0.0
6 4
0.0 100.0
88 35
176 70
0.0 12.9*
0.0 0.0
8 4
0.0 100.0
103 28
206 56
100.0 5.4
0.0 94.6’
9 4
0.0 100.0
56 26
112 52
100.0 13.5
0.0 86.5*
6 5
Abbreviations: LF, living fetuses; %PD, % of hind limbs with preaxial polydactyly; %R, % of hind limbs with preaxial reduction of toes. *5 up 6 triphalangeal toes. +5 up 4 triphalangeal toes. t4 up 3 triphalangeal toes.
to five or four. In SHR/BXH2 Lx/Lx group 86.5% of hind limbs were found with number of toes reduced from five to four up to three, the foot morphotype being mostly the same as in BXH2 oligodactylous strain, resembling thus the thalidomide effect. In this group zeugopodium and stylopodium were also severely affected and two cases of sirenomelia occurred. The striking reduction changes after RA administration in the last mentioned group indicate the interaction of RA with modifying gene combination specific for SHR,BXH2 genotype. To elucidate further the teratogenic effect of RA and also of thalidomide it is of interest that the strain BN-Lx and BXH2 differ from the SHR-Lx strain in genetic markers on chromosomes 3, 4, 7, and 10, where Hox and RAR genes were assigned. Moreover, the Lx allele was assigned to chromosome 8 of the rat indicating the
From the Institute of Biology, Faculty of University, Prague, Czech Republic. Supported by Grant #274 from The Grant University and GACR grant #302/96/0604. Address reprint requests to V. BilB, Institute of Medicine, Charles University, 1 Faculty of 4, Prague 2 128 00 Czech Republic.
Medicine, Agency
Charles
of Charles
of Biology, Faculty Medicine, Albertov
0041-1345/97/$17.00 PII so041 -1345(97)00024-9
1707
1708 homology of Lx with mouse lu mutation on MMU 9. The extensive homology of these two chromosomes [RN08MMU9] is indicated.4 The RI strain system with unique set of modifying genes fixed in each strain could provide us with a tool for uncovering of genes affected by RA action and involved in limb morphogenesis.
Biti
AND KhEN
REFERENCES
1. Bila V, Kien V: Sbornik ICk. 90:90, 1988 2. l&n 1996
V, Bila V, KaSparek R, et al: Fol Biol [Praha] 42:159,
3. Bila V, Kien V: Fol Biol [Praha] 40:161, 1994 4. Szpirer C, Szpirer J, Klinga-Levan K, et al: Fol Biol [Praha] 42:175, 1996