. 2019 Sep 19;9(9):701.

doi: 10.3390/ani9090701.

Denatonium Benzoate-Induces Oxidative Stress in the Heart and Kidney of Chinese Fast Yellow Chickens by Regulating Apoptosis, Autophagy, Antioxidative Activities and Bitter Taste Receptor Gene Expressions

Enayatullah Hamdard¹, Zhicheng Shi², Zengpeng Lv³, Ahmadullah Zahir⁴, Quanwei Wei⁵, Mohammad Malyar Rahmani⁶, Fangxiong Shi⁷

Affiliations

¹ College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. 2017105117@njau.edu.cn.
² College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. 2017105023@njau.edu.cn.
³ College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. lvzengpeng@njau.edu.cn.
⁴ College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. 2017108149@njau.edu.cn.
⁵ College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. weiquanwei@njau.edu.cn.
⁶ College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. 2017107186@njau.edu.cn.
⁷ College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. fxshi@njau.edu.cn.

PMID: 31546822
PMCID: PMC6770773
DOI: 10.3390/ani9090701

Denatonium Benzoate-Induces Oxidative Stress in the Heart and Kidney of Chinese Fast Yellow Chickens by Regulating Apoptosis, Autophagy, Antioxidative Activities and Bitter Taste Receptor Gene Expressions

Enayatullah Hamdard et al. Animals (Basel). 2019.

. 2019 Sep 19;9(9):701.

doi: 10.3390/ani9090701.

Authors

Enayatullah Hamdard¹, Zhicheng Shi², Zengpeng Lv³, Ahmadullah Zahir⁴, Quanwei Wei⁵, Mohammad Malyar Rahmani⁶, Fangxiong Shi⁷

Affiliations

¹ College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. 2017105117@njau.edu.cn.
² College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. 2017105023@njau.edu.cn.
³ College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. lvzengpeng@njau.edu.cn.
⁴ College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. 2017108149@njau.edu.cn.
⁵ College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. weiquanwei@njau.edu.cn.
⁶ College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. 2017107186@njau.edu.cn.
⁷ College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. fxshi@njau.edu.cn.

PMID: 31546822
PMCID: PMC6770773
DOI: 10.3390/ani9090701

Abstract

The sense of taste which tells us which prospective foods are nutritious, poisonous and harmful is essential for the life of the organisms. Denatonium benzoate (DB) is a bitter taste agonist known for its activation of bitter taste receptors in different cells. The aim of the current study was to investigate the mRNA expressions of bitter taste, downstream signaling effectors, apoptosis-, autophagy- and antioxidant-related genes and effector signaling pathways in the heart/kidney of chickens after DB dietary exposure. We randomly assigned 240, 1-day-old Chinese Fast Yellow chicks into four groups with five replicates of 12 chicks and studied them for 28 consecutive days. The dietary treatments consisted of basal diet and feed containing DB (5, 20 and 100 mg/kg). The results revealed that dietary DB impaired (p < 0.05) the growth performance of the chickens. Haemotoxylin and eosin staining and TUNEL assays confirmed that medium and high doses of DB damaged the epithelial cells of heart/kidney and induced apoptosis and autophagy. Remarkably, the results of RT-PCR and qRT-PCR indicated that different doses of DB gradually increased (p < 0.05) mRNA expressions of bitter taste, signaling effectors, apoptosis-, autophagy- and antioxidant- related genes on day 7 in a dose-response manner, while, these expressions were decreased (p < 0.05) subsequently by day-28 but exceptional higher (P < 0.05) expressions were observed in the high-dose DB groups of chickens. In conclusion, DB exerts adverse effects on the heart/kidney of chickens in a dose-response manner via damaging the epithelium of the heart/kidney by inducing apoptosis, autophagy associated with bitter taste and effector gene expressions. Correlation analyses for apoptosis/autophagy showed agonistic relationships. Our data provide a novel perspective for understanding the interaction of bitter taste, apoptosis, autophagy and antioxidative genes with bitter taste strong activators in the heart/kidney of chicken. These insights might help the feed industries and pave the way toward innovative directions in chicken husbandry.

Keywords: apoptosis; autophagy; bitter taste receptors; chicken; denatonium benzoate; heart; kidney.

PubMed Disclaimer

Conflict of interest statement

The author declares no conflict of interest is present in this manuscript

Figures

**Figure 1**
Effects of DB supplementation on the body and organ weight gain (gr) of chickens. (A) Body weight of chickens at 0, 07, 14, 28 days of age (n = 10). (B) Organ weights of chickens at day 7 and day 28 of age (n = 10). Data are presented as mean value ± SEM. Values without the same mark (a, b, c) represent statistically significant differences (p < 0.05). Subfigure A uses line graphs with experimental days on the X-axis for body weights, and subfigure B separates the data into two bar graphs for heart and kidney, respectively.

**Figure 2**
Effects of Denatonium benzoate supplementation on chicken heart histomorphology on days 07 and 28 (A,B). On day-07 necrosis (red arrow), apoptotic cells (white arrow), pyknotic cells (yellow arrow) and distortion of the morphological characteristics of the cells (green arrow) due to the toxic effect of DB were seen. On day-28, karyolysis due to severe necrosis which caused autophagy (black arrow), apoptotic cells (white arrow) shrinkage of fibroblasts which led to condensation (green arrow) indicate the effect of DB.

**Figure 3**
Effects of DB supplementation on the histomorphology of chicken kidney on day-07 and day-28 (A,B). On day-07, the black arrow shows that PCTs became swollen (hydrophiid degeneration). The white arrow indicates inflamed infiltrated cells and the green arrow indicates separation of the basement membrane of DCTs; Karyolysis occurred where no cells were found due to the toxic effects of DB. The effects on day 28 showed no major differences.

**Figure 4**
Tunnel assay of heart and kidneys at 07 days (A) and at 28 days (B) of age by immunofluorescence. The blue color represents the total cells in the heart and kidneys, and the green color represents the apoptosis cells in the heart and kidneys with three different doses of DB (low-dose, medium-dose and high-dose).

**Figure 5**
RT and qRT-PCR showed effects of DB supplementation on heart mRNA expressions of bitter taste receptors and downstream effectors at 07 days (A) and 28 days (B) of age. Data are presented as mean value ± SEM (n = 6). Values without the same mark (a–d) represent statistically significant differences (p < 0.05). gg, Gallus gallus; PLCβ2, phospholipase Cβ2; IP3R3, type 3 inositol-1,4,5-trisphosphate receptor; Denatonium benzoate- Low Dose treated group, Denatonium benzoate- Medium Dose treated group, Denatonium benzoate- High dose treated group.

**Figure 6**
Effects of DB supplementation on kidney mRNA expressions of bitter taste receptors and downstream effectors at 07 days (A) and 28 days (B) of age. Data are presented as mean value ± SEM (n = 6). Values without the same mark (a–d) represent statistically significant differences (p < 0.05). gg, Gallus gallus; PLCβ2, phospholipase Cβ2; IP3R3, type 3 inositol-1,4,5-trisphosphate receptor; Denatonium benzoate- Low Dose treated group, Denatonium benzoate- Medium Dose treated group, Denatonium benzoate- High dose treated group.

**Figure 7**
Effects of DB supplementation on kidney mRNA expressions of apoptosis-related genes at 07 days (A) and 28 days (B) of age. Data are presented as mean value ± SEM (n = 6). Values without the same mark (a–d) represent statistically significant differences (p < 0.05). CALPN1, Calpain 1; CALPN2, Calpain 2; CASP-2, Caspase 2; CASP-3, Caspase 3; CASP-7, Caspase 7; CASP9, Caspase 9; BCL2, B-cell CLL/lymphoma 2; BCL2L1, BCL2 like 1; MCL1, myeloid cell leukemia sequence 1; BID, BH3 interacting domain death agonist; NOXA, similar to ATL-derived PMA-responsive peptide; Denatonium benzoate- Low Dose treated group, Denatonium benzoate- Medium Dose treated group, Denatonium benzoate- High dose treated group.

**Figure 8**
Effects of BD supplementation on heart mRNA expressions of apoptosis-related genes at 07 days (A) and 28 days (B) of age. Data are presented as mean value ± SEM (n = 6). Values without the same mark (a–d) represent statistically significant differences (p < 0.05). CALPN1, Calpain 1; CALPN2, Calpain 2; CASP-2, Caspase 2; CASP-3, Caspase 3; CASP-7, Caspase 7; CASP9, Caspase 9; BCL2, B-cell CLL/lymphoma 2; BCL2L1, BCL2 like 1; MCL1, myeloid cell leukemia sequence 1; BID, BH3 interacting domain death agonist; NOXA, similar to ATL-derived PMA-responsive peptide; Denatonium benzoate- Low Dose treated group, Denatonium benzoate- Medium Dose treated group, Denatonium benzoate- High dose treated group.

**Figure 9**
Effects of DB supplementation on heart mRNA expressions of autophagy- related genes at 07 days (A) and 28 days (B) of age. Data are presented as mean value ± SEM (n = 6). Values without the same mark (a–d) represent statistically significant differences (p < 0.05). ATG5; Beclin 1; Dynein; LC3-I; LC3-II, mTOR; Denatonium benzoate- Low Dose treated group, Denatonium benzoate- Medium Dose treated group, Denatonium benzoate- High dose treated group.

**Figure 10**
Effects of DB supplementation on kidney mRNA expressions of autophagy- related genes at 07 days (A) and 28 days (B) of age. Data are presented as mean value ± SEM (n = 6). Values without the same mark (a–d) represent statistically significant differences (p < 0.05). ATG5; Beclin 1; Dynein; LC3-I; LC3-II, mTOR; Denatonium benzoate- Low Dose treated group, Denatonium benzoate- Medium Dose treated group, Denatonium benzoate- High dose treated group.

**Figure 11**
Effects of DB supplementation on heart mRNA expressions of antioxidant-related genes at 07 days (A) and 28 days (B) of age. Data are presented as mean value ± SEM (n = 6). Values without the same mark (a–d) represent statistically significant differences (p < 0.05). GPX1, glutathione peroxidase 1; SOD, superoxide dismutase 1; CAT, catalase; Denatonium benzoate- Low Dose treated group, Denatonium benzoate- Medium Dose treated group, Denatonium benzoate- High dose treated group.

**Figure 12**
Effects of DB supplementation on kidney mRNA expressions of antioxidant- related genes at 07 days (A) and 28 days (B) of age. Data are presented as mean value ± SEM (n = 6). Values without the same mark (a–d) represent statistically significant differences (p < 0.05). GPX1, glutathione peroxidase 1; SOD, superoxide dismutase 1; CAT, catalase; Denatonium benzoate- Low Dose treated group, Denatonium benzoate- Medium Dose treated group, Denatonium benzoate- High dose treated group.

**Figure 13**
Heat map of expression profiles (bitter taste, downstream signaling effectors, apoptosis, autophagy and antioxidant genes) in supplementation with three different doses of DB in two stages (day-07 and day-28) in the heart and kidney of chicken.

See this image and copyright information in PMC

Cited by

A deadly taste: linking bitter taste receptors and apoptosis.
Miller ZA, Carey RM, Lee RJ. Miller ZA, et al. Apoptosis. 2025 Apr;30(3-4):674-692. doi: 10.1007/s10495-025-02091-3. Epub 2025 Feb 20. Apoptosis. 2025. PMID: 39979526 Free PMC article. Review.
Freeze-Dried Royal Jelly Proteins Enhanced the Testicular Development and Spermatogenesis in Pubescent Male Mice.
Shi Z, Enayatullah H, Lv Z, Dai H, Wei Q, Shen L, Karwand B, Shi F. Shi Z, et al. Animals (Basel). 2019 Nov 15;9(11):977. doi: 10.3390/ani9110977. Animals (Basel). 2019. PMID: 31731648 Free PMC article.
Cellular mechanisms and molecular pathways linking bitter taste receptor signalling to cardiac inflammation, oxidative stress, arrhythmia and contractile dysfunction in heart diseases.
Welcome MO, Dogo D, Nikos E Mastorakis. Welcome MO, et al. Inflammopharmacology. 2023 Feb;31(1):89-117. doi: 10.1007/s10787-022-01086-9. Epub 2022 Dec 6. Inflammopharmacology. 2023. PMID: 36471190 Free PMC article. Review.
The health impacts and genetic architecture of food liking in cardio-metabolic diseases.
Jiang W, Wang H, Geng Y, Guo M, Zuo Y, Han S, Liu Z, Chen S, Fan S, Li S, Qiao C, Li Q, Li B, Zhang Y, Wei W, Han T. Jiang W, et al. Nat Commun. 2025 May 23;16(1):4810. doi: 10.1038/s41467-025-59945-2. Nat Commun. 2025. PMID: 40410146 Free PMC article.
The bitter end: T2R bitter receptor agonists elevate nuclear calcium and induce apoptosis in non-ciliated airway epithelial cells.
McMahon DB, Kuek LE, Johnson ME, Johnson PO, Horn RLJ, Carey RM, Adappa ND, Palmer JN, Lee RJ. McMahon DB, et al. Cell Calcium. 2022 Jan;101:102499. doi: 10.1016/j.ceca.2021.102499. Epub 2021 Nov 8. Cell Calcium. 2022. PMID: 34839223 Free PMC article.

References

1. Wong G., Gannon K.S., Margolskee R.F. Transduction of bitter and sweet taste by gustducin. Nature. 1996;381:796–800. doi: 10.1038/381796a0. - DOI - PubMed
1. Gentle M. Taste preference in the chicken (Gallus domesticus L.) Br. Poult. Sci. 1972;13:141–155. doi: 10.1080/00071667208415928. - DOI - PubMed
1. Simitzis P.E., Deligeorgis S.G., Bizelis J.A., Fegeros K. Feeding preferences in lambs influenced by prenatal flavour exposure. Physiol. Behav. 2008;93:529–536. doi: 10.1016/j.physbeh.2007.10.013. - DOI - PubMed
1. Mennella J.A., Forestell C.A., Morgan L.K., Beauchamp G.K. Early milk feeding influences taste acceptance and liking during infancy. Am. J. Clin. Nutr. 2009;90:780S–788S. doi: 10.3945/ajcn.2009.27462O. - DOI - PMC - PubMed
1. Boland M., Fordyce H., Greenwood R. Enzymes of nitrogen metabolism in legume nodules: A comparative study. Funct. Plant Biol. 1978;5:553–559. doi: 10.1071/PP9780553. - DOI

LinkOut - more resources

Full Text Sources
Miscellaneous
- NCI CPTAC Assay Portal

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Denatonium Benzoate-Induces Oxidative Stress in the Heart and Kidney of Chinese Fast Yellow Chickens by Regulating Apoptosis, Autophagy, Antioxidative Activities and Bitter Taste Receptor Gene Expressions

Affiliations

Denatonium Benzoate-Induces Oxidative Stress in the Heart and Kidney of Chinese Fast Yellow Chickens by Regulating Apoptosis, Autophagy, Antioxidative Activities and Bitter Taste Receptor Gene Expressions

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

LinkOut - more resources

Full Text Sources

Miscellaneous