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Co-exposure to an Aryl Hydrocarbon Receptor Endogenous Ligand, 6-Formylindolo[3,2-b]carbazole (FICZ), and Cadmium Induces Cardiovascular Developmental Abnormalities in Mice

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Abstract

6-Formylindolo[3,2-b]carbazole (FICZ) is a signal substance and an endogenous activator of aryl hydrocarbon receptor (AHR). Cadmium (Cd) is an environmental pollutant that can activate both AHR and Wnt/β-catenin signaling pathways. We aimed to determine how dysregulated signaling through AHR-Wnt/β-catenin cross-talk can influence mice heart development. Mice fetuses were exposed to Cd alone or in combination with FICZ in gestation day (GD) 0. In GD18, fetuses were harvested and randomly divided into two parts for stereological and molecular studies. Stereological and tessellation results revealed that when fetuses were co-exposed with FICZ and Cd, abnormalities were synergistically raised. In the presence of FICZ, mRNA expression levels of Wnt/β-catenin target genes significantly enhanced, especially when animals co-treated with FICZ and Cd. Based on these findings, we propose that chemical pollutants can interfere with the normal function of AHR that has a physiological role in regulating Wnt/β-catenin during cardiogenesis.

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Acknowledgments

The authors of this manuscript wish to express their appreciation to Shiraz University of Medical Sciences, Shiraz, Iran.

Funding

This work was supported by the Shiraz University of Medical Sciences grant for the accomplishment of the Ph.D. thesis of Mahmoud Omidi [grant number 94-7557].

Author information

Authors and Affiliations

  1. Department of Pharmacology and Toxicology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

    Mahmoud Omidi, Hossein Niknahad & Afshin Mohammadi-Bardbori

  2. Histomorphometry and Stereology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

    Ali Noorafshan & Elham Nadimi

  3. Departments of Anatomy, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

    Ali Noorafshan

  4. Department of Radiology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

    Reza Fardid

  5. Department of Immunology, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran

    Elham Nadimi

  6. Diagnostic Laboratory Science and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

    Samaneh Naderi

  7. Department of Reproductive Biology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran

    Azizollah Bakhtari

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  1. Mahmoud Omidi
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Supplementary Fig. S1

Voronoi tessellation results. A micrograph of the cardiomyocytes and micro-vessels in fetal heart in the control (A) and (C), Cd (4.5 mg/kg) plus FICZ (100 μg/kg) (B) and (D) groups respectively (n = 5–6). After setting the scale, cardiomyocytes nuclei and capillaries center are marked and the polygons were superimposed on them using the ImageJ software. The threshold color changed to clear out the background with black area boundaries (E-H). The areas of the polygons are calculated using the ImageJ software. The microscopic slides have stained with H&E or Heidenhain’s Azan trichrome for cardiomyocytes and micro-vessels, respectively. Lower diagrams (I) and (J) shows the polygon distribution percentage classified into different categories according to their areas (μm2). Lower left and right diagrams present the cardiomyocytes (I) and micro-vessels (J) distribution, respectively. Upper left (E and F) and right (G and H) diagrams show the cardiomyocytes and capillary centers of the histologic sections in the control (A and C) and Cd (4.5 mg/kg) plus FICZ (100 μg/kg) groups (B and D), respectively. Each spot marked a cardiomyocyte or capillary and the geometric center of a polygon. Since the profile of the polygons was determined by the arrangement of the points, hence, the cardiomyocyte or capillary pattern is different between the groups. The altered pattern of cardiomyocyte or capillary led to change in intermyocytic or intercapillary distances followed by the variability of polygon areas in Cd (4.5 mg/kg) plus FICZ (100 μg/kg) group. (JPG 2099 kb)

Supplementary Fig. S2

Delaunay tessellation results. A micrograph of the cardiomyocytes and micro-vessels in fetal heart in the control (A) and (C), Cd (4.5 mg/kg) plus FICZ (100 μg/kg) (B) and (D) groups respectively (n = 5–6). After setting the scale, cardiomyocytes nuclei and capillaries center are marked and the triangles were superimposed on them using the ImageJ software. Each triangle represents the distance between a point and nearest adjacent points were marked by the software. The distances between intermyocytic or intercapillary points are estimated. The microscopic slides have stained with H&E or Heidenhain’s Azan trichrome for cardiomyocytes and micro-vessels, respectively. Lower left (E) and right (F) diagrams show the mean distances (μm) between cardiomyocytes and micro-vessels centers from each other in the control (A and C) and Cd (4.5 mg/kg) plus FICZ (100 μg/kg) groups (B and D), respectively. Since the profile of the triangles was determined by the arrangement of the points, hence, the cardiomyocyte or capillary distances are different between the groups. The altered pattern of cardiomyocyte or capillary led to change in intermyocytic or intercapillary distances followed by the variability of triangles sides in Cd (4.5 mg/kg) plus FICZ (100 μg/kg) group. Values are expressed as means ±S.E; Asterisks denote significant differences (*P < 0.05 and ***P < 0.001) between control and Cd (4.5 mg/kg) plus FICZ. (JPG 1926 kb)

Supplementary Fig. S3

Counting of cardiomyocytes nuclei profiles at light microscopic level. The optical disector method was used to count the cardiomyocytes nuclei. An unbiased counting frame was superimposed onto each sampled test field. Cardiomyocytes nuclei were visualized by H&E staining and counted if nuclei were in focus (A) and (B) and located inside the counting frame (arrows) or touching the inclusion line (dashed line). Cardiomyocytes nuclei profiles touching the exclusion line or its extensions were not counted. (JPG 111 kb)

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Omidi, M., Niknahad, H., Noorafshan, A. et al. Co-exposure to an Aryl Hydrocarbon Receptor Endogenous Ligand, 6-Formylindolo[3,2-b]carbazole (FICZ), and Cadmium Induces Cardiovascular Developmental Abnormalities in Mice. Biol Trace Elem Res 187, 442–451 (2019). https://doi.org/10.1007/s12011-018-1391-1

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