To investigate the effects of zinc concentration on palatal development in fetal mice and its association with the aryl hydrocarbon receptor (AhR) signaling pathway.
Methods
Pregnant C57BL/6J mice were fed diets with varying zinc concentrations and randomly divided into a zinc-rich (ZR) group, a normal-zinc (NZ) group, and a zinc-deficient (ZD) group. Embryonic development was observed, and the expression levels of AhR signaling pathway-related factors were examined.
Results
No cleft palate was observed in the ZR group or NZ group, whereas the zinc-deficient group exhibited a cleft palate incidence of 27.45%. Hematoxylin and Eosin (HE) staining results revealed failed palatal shelf contact and fusion in the ZD group, whereas complete fusion occurred in the ZR group and a normal medial edge epithelial formed in the NZ group. Results from Immunohistochemistry (IHC), qRT-PCR, and Western blot analyses collectively demonstrated that, compared to the NZ group, the ZR group exhibited significant upregulation (P < .05) in both mRNA and protein expression levels of AhR, HSP90, CYP1A1, and SP1, whereas the ZD group displayed significant downregulation (P < .05).
Conclusion
Zinc concentration is intimately correlated with fetal mouse palatal development, where zinc deficiency may contribute to cleft palate formation through suppression of AhR signaling pathway, whereas zinc-rich conditions facilitate activation of this pathway. This study reveals the regulatory role of zinc-AhR signaling axis in palatal development through mouse models, offering novel theoretical insights into zinc deficiency-induced cleft palate pathogenesis and establishing a foundational framework for preventive interventions.
YoungJISliferSHechtJT, Blanton SH.DNA Methylation variation is identified in monozygotic twins discordant for non-syndromic cleft lip and palate[J]. Front Cell Dev Biol. 2021;9:656865. doi:10.3389/fcell.2021.656865
2.
PanamontaVPradubwongSPanamontaM, Chowchuen B.Global birth prevalence of orofacial clefts: A systematic review[J]. J Med Assoc Thai. 2015;98(Suppl 7):S11–S21.
3.
ShangCLuruiMFangW, ChengchengJ, QinggaoS. Effect of zn on mouse embryo palate fusion by culture in vitro[J]. J Oral Sci Res. 2012;28(12):1225–1227+1230. doi:10.13701/j.cnki.kqyxyj.2012.12.011
4.
QinggaoSShangCFangW, BinW.The effect of zinc of mother intaking and environmental factors on the incidence of fetus cleft palate of mice[J]. J Pract Stomatol. 2013;29(06):795–798.
5.
FangWQinggaoSBinW, ShangC.Study of the relationship between microelement zinc of mother and the cleft palate of fetus in mice[J]. J Oral Sci Res. 2012;28(3):210–212. doi:10.13701/j.cnki.kqyxyj.2012.03.013
6.
KreiserTZaguriDSachdevaSZamostianoRMograbiJSegalDBacharachEGazitE. Inhibition of respiratory RNA viruses by a composition of ionophoric polyphenols with metal ions[J]. Pharmaceuticals (Basel). 2022;15(3):377–391. doi:10.3390/ph15030377
7.
AngelosMGRuhPNWebberBR, BlumRH, RyanCD, BendzickL, ShimS, YingstAM, TufaDM, VernerisMR, et al.Aryl hydrocarbon receptor inhibition promotes hematolymphoid development from human pluripotent stem cells[J]. Blood. 2017;129(26):3428–3439. doi:10.1182/blood-2016-07-730440
8.
HuangYHeJLiangH, HuK, JiangS, YangL, MeiS, ZhuX, YuJ, KijlstraA, et al.Aryl hydrocarbon receptor regulates apoptosis and inflammation in a murine model of experimental autoimmune uveitis[J]. Front Immunol. 2018;9:1713–1728. doi:10.3389/fimmu.2018.01713
9.
YuHJiangLWanB, ZhangW, YaoL, CheT, GanC, SuN, HeJ, HuangJ, et al.The role of aryl hydrocarbon receptor in bone remodeling[J]. Prog Biophys Mol Biol. 2018;134:44–49. doi:10.1016/j.pbiomolbio.2017.12.005
10.
MimuraJYamashitaKNakamuraK, Morita M , Takagi TN , Nakao K , Ema M , Sogawa K , Yasuda M , Katsuki M, et al.Loss of teratogenic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the ah (dioxin) receptor[J]. Genes Cells. 1997;2(10):645–654. doi:10.1046/j.1365-2443.1997.1490345.x
11.
SoyocakAErgunDDKocG, Ergun, S , Ozsobaci NP.Investigation of aryl hydrocarbon receptor, zinc, and vitamin B12 levels in chronic gastritis with Helicobacter pylori infection[J]. Biol Trace Elem Res. 2021;199(7):2431–2437. doi:10.1007/s12011-021-02667-5
12.
Percie du SertNHurstVAhluwaliaA, Alam S, Avey MT, Baker M, Browne WJ, Clark A, Cuthill IC, Dirnagl U, et al.The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. Br J Pharmacol. 2020;177(16):3617–3624. doi:10.1111/bph.15193
13.
RichardsonRMitchellKHammondNL, Mollo MR, Kouwenhoven EN, Wyatt ND, Donaldson IJ, Zeef L, Burgis T, Blance R, et al.P63 exerts spatio-temporal control of palatal epithelial cell fate to prevent cleft palate[J]. PLoS Genet. 2017;13(6):e1006828. doi:10.1371/journal.pgen.1006828
14.
LeslieEJLiuHCarlsonJC, Shaffer JR, Feingold E, Wehby G, Laurie CA, Jain D, Laurie CC, Doheny KF, et al.A genome-wide association study of nonsyndromic cleft palate identifies an etiologic missense variant in GRHL3[J]. Am J Hum Genet. 2016;98(4):744–754. doi:10.1016/j.ajhg.2016.02.014
15.
KrapelsIPvan RooijIAOckéMCvan CleefBAKuijpers-JagtmanAMSteegers-TheunissenRP. Maternal dietary B vitamin intake, other than folate, and the association with orofacial cleft in the offspring. Eur J Nutr. 2004;43(1):7–14. doi:10.1007/s00394-004-0433-y
16.
LiXWLiHYWangFHeWSongQG. The influence of zinc on apoptosis and cell proliferation in palatal shelves during the fusion phase in mice and identification of a special protein family based on gene expression in cleft palate[J]. Chin J Stomatol. 2023;58(12):1273–1280. doi:10.3760/cma.j.cn112144-20230730-00050
17.
YangYChanWK. Selective autophagy maintains the aryl hydrocarbon receptor levels in HeLa cells: A mechanism that is dependent on the p23 co-chaperone[J]. Int J Mol Sci. 2020;21(10):3449. doi:10.3390/ijms21103449
18.
TsyrlovIShurIRoumakV. The NS1A binding protein in alveolar epithelial cells as centerpiece of influenza virus A lung injury mechanism mediated via AhR/arnt signaling pathway[J]. Eur Respir J.2017;50(s61):352. doi:10.1183/1393003.congress-2017.PA352
19.
LeeWJLiuSHChiangCK, Lin SY, Liang KW, Chen CH, Tien HR, Chen PH, Wu JP, Tsai YC, et al.Aryl hydrocarbon receptor deficiency attenuates oxidative stress-related mesangial cell activation and macrophage infiltration and extracellular matrix accumulation in diabetic nephropathy[J]. Antioxid Redox Signal. 2016;24(4):217–231. doi:10.1089/ars.2015.6310
20.
LinnenkampBDWRaskinSEspositoSE, HeraiRH. A comprehensive analysis of AhRR gene as a candidate for cleft lip with or without cleft palate[J]. Mutat Res Rev Mutat Res. 2020;785:108319. doi:10.1016/j.mrrev.2020.108319
21.
ShiMChristensenKWeinbergCR, Romitti P, Bathum L, Lozada A, Morris RW, Lovett M, Murray JC.Orofacial cleft risk is increased with maternal smoking and specific detoxification-gene variants[J]. J Hum Genet. 2007;80(1):76–90. doi:10.1086/510518
22.
HuangBButlerRMiaoY, Dai Y, Wu W, Su W, Fujii-Kuriyama Y, Warner M, Gustafsson JÅ.Dysregulation of notch and ERα signaling in AhR-/- male mice[J]. Proc Natl Acad Sci U S A. 2016;113(42):11883–11888. doi:10.1073/pnas.1613269113
23.
SihuaXXinSQinggaoS. Study on the mechanism of cleft palate caused by TCDD through the interaction of AHR and wnt/ß-catenin signaling pathway[J].J. Zunyi Med Univ. 2024;47(08):759–767. doi:10.14169/j.cnki.zunyixuebao.2024.0103
24.
IkutaTKawajiriK. Zinc finger transcription factor slug is a novel target gene of aryl hydrocarbon receptor[J]. Exp Cell Res. 2006;312(18):3585–3594. doi:10.1016/j.yexcr.2006.08.002
25.
LiuPZhouYDongX, Zheng B, Liang B, Liang R, Liu Z, Li L, Gong P.ZNF165 Is involved in the regulation of immune microenvironment and promoting the proliferation and migration of hepatocellular carcinoma by AhR/CYP1A1[J]. J Immunol Res. 2022;2022:4446805. doi:10.1155/2022/4446805
26.
KwonYJShinSChunYJ. Biological roles of cytochrome P450 1A1, 1A2, and 1B1 enzymes. Arch Pharm Res[J]. 2021;44(1):63–83. doi:10.1007/s12272-021-01306-w
JangJYShinSChoiBI, Park D, Jeon JH, Hwang SY, Kim JC, Kim YB, Nahm SS.Antiteratogenic effects of alpha-naphthoflavone on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposed mice in utero[J]. Reprod Toxicol. 2007;24(3-4):303–309. doi:10.1016/j.reprotox.2007.08.002
29.
YuanXHeXZhangX, Liu C, Wang C, Qiu L, Pu W, Fu Y.Comparative study of folic acid and α-naphthoflavone on reducing TCDD-induced cleft palate in fetal mice[J]. Cleft Palate Craniofac J. 2017;54(2):216–222. doi:10.1597/15-211
30.
ZhuYRenCWanX, Zhu Y, Zhu J, Zhou H, Zhang T.Gene expression of Hsp70, Hsp90 and Hsp110 families in normal palate and cleft palate during mouse embryogenesis[J]. Toxicol Ind Health. 2013;29(10):915–930. doi:10.1177/0748233712446720
31.
VizcainoCMansillaSPortugalJ. Sp1 transcription factor: A long-standing target in cancer chemotherapy[J]. Pharmacol Ther. 2015;152:111–124. doi:10.1016/j.pharmthera.2015.05.008
32.
XieXJiangJYeW, Chen R, Deng Y, Wen J.Sp1, instead of AhR, regulates the basal transcription of porcine CYP1A1 at the proximal promoter [J]. Front Pharmacol. 2018;9:927–937. doi:10.3389/fphar.2018.00927
33.
YeWChenRChenX, Huang B, Lin R, Xie X, Chen J, Jiang J, Deng Y, Wen J.Ahr regulates the expression of human cytochrome P450 1A1 (CYP1A1) by recruiting Sp1[J]. FEBS J.2019;286(21):4215–4231. doi:10.1111/febs.14956
