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Target tissue specificity of retinoic acid-induced stress proteins and malformations in mice
J F Anson1, J B Laborde, J L Pipkin
1Division of Genetic Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079.
Abstract:
Retinoic acid (RA) is teratogenic in rodents and also induces the synthesis of stress proteins in fetal mouse limb buds. To determine if the RA induction of stress proteins is target tissue specific, pregnant CD-1 mice were gavaged with 100 mg/kg RA on day 11 of gestation, and nuclei isolated from tissues susceptible to RA-induced malformations (target tissues) as well as nuclei isolated from nontarget tissues were examined for stress protein synthesis and malformations. Forelimb and hindlimb (target tissues), as well as heart and tail (nontarget tissues), were removed from embryos 2.5 hours after RA treatment (1.5 hr after [3H]leucine labeling). Cell nuclei were isolated, stained with a DNA specific fluorochrome, propidium iodide, and sorted from the G0 + G1 and G2 + M phases of the cell cycle. Forelimb and hindlimb target tissues showed the synthesis in these embryonic nuclear proteins of an 84,000 relative molecular mass (Mr) protein and a 90,000 Mr protein following RA treatment. Two 20,000-25,000 Mr stress proteins were also labeled both in forelimb and hindlimb. Forelimb and hindlimb from untreated dams showed no stress protein labeling. Neither heart nor tail, nontarget tissues, showed any stress protein labeling following RA treatment. Classical teratological evaluation of embryos treated on GD 11 and sacrificed on GD 17 showed that 100% of the fetuses had forelimb and/or hindlimb malformations, while no malformations were observed in either the heart or tail. Based on the correlation of teratological anomalies with the identification of stress proteins in target tissue only, we postulate that stress proteins may be involved in the teratogenic process. Further work is necessary to establish whether a causal relationship exists.
Insights
Retinoic acid (RA) causes birth defects by inducing stress proteins in developing limbs. These stress proteins were only found in affected fetal tissues, suggesting a role in teratogenesis.
Area of Science:
- Developmental biology
- Toxicology
- Molecular biology
Background:
- Retinoic acid (RA) is a known teratogen in rodents, causing birth defects.
- RA also induces stress protein synthesis in fetal mouse limb buds.
Purpose of the Study:
- To investigate if RA-induced stress protein synthesis is specific to tissues susceptible to malformations.
- To explore the potential involvement of stress proteins in RA-induced teratogenesis.
Main Methods:
- Pregnant mice were administered retinoic acid (RA) on day 11 of gestation.
- Embryonic tissues (limb buds, heart, tail) were collected 2.5 hours post-treatment for analysis.
- Cell nuclei were isolated, stained, sorted by cell cycle phase, and examined for protein synthesis using [3H]leucine labeling.
Main Results:
- Target tissues (forelimb, hindlimb) showed synthesis of 84,000 Mr and 90,000 Mr proteins, along with two 20,000-25,000 Mr stress proteins after RA treatment.
- Nontarget tissues (heart, tail) did not exhibit stress protein labeling post-RA exposure.
- Embryos treated with RA on day 11 exhibited 100% limb malformations by day 17, while heart and tail remained unaffected.
Conclusions:
- The synthesis of specific stress proteins is localized to RA-affected target tissues.
- A correlation exists between teratological anomalies and the presence of stress proteins in target tissues.
- Stress proteins are postulated to play a role in the teratogenic process induced by retinoic acid.