Prepubertal Exposure to Tris(2-chloroethyl) Phosphate Disrupts Blood-Testis Barrier Integrity via Ferritinophagy-Mediated Ferroptosis

Abstract

Tris(2-chloroethyl) phosphate (TCEP) is a representative chlorinated organophosphate flame retardant (OPFR) that demonstrates greater persistence than other non-halogenated alkyl or aryl OPFRs. Although TCEP has been shown to accumulate significantly in the environment and contribute to testicular toxicity and spermatogenic dysfunction, the precise underlying factors and mechanisms of action remain unclear. Herein, male ICR mice were gavaged with corn oil, 50 mg/kg body weight (bw) TCEP, or 100 mg/kg bw TCEP from postnatal day (PND) 22 to PND 35. TCEP exposure resulted in the disruption of blood-testis barrier (BTB) integrity and in abnormal testicular development. Considering that Sertoli cells constitute the primary target of toxicants and that TCEP induces oxidative stress in the testis and other organs, we focused on ferroptosis in Sertoli cells. Our findings revealed a significant increase in ferroptosis in the testes and Sertoli cells following TCEP exposure, and we observed functional restoration of Sertoli cell junctions upon treatment with the ferroptosis inhibitor ferrostatin-1. Furthermore, ferritin heavy chain 1 (FTH1) was markedly reduced in TCEP-exposed testes and Sertoli cells. Since nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy is essential for the degradation of FTH1, we assessed ferritinophagic activity and found significant upregulation of NCOA4, ATG5, ATG7, and LC3B II/I in TCEP-exposed testes and Sertoli cells. These results strongly suggest that TCEP triggers Sertoli cell ferroptosis by activating ferritinophagy that leads to reduced expression of BTB-associated proteins, ultimately causing BTB disruption and testicular developmental toxicity.

Keywords: BTB; TCEP; ferritinophagy; ferroptosis.

Figure 1

TCEP exposure leads to testicular toxicity. (A) The body weight in the three groups. (B) The testis coefficient in the three groups. (C) Testicular histomorphological changes in the three groups after H&E staining. (a,d) The control group. (b,e) The T50 group. (c,f) The T100 group. The red box delineates the magnified region, whereas the green box denotes the isolated germ cells. The scale bars in the top-row images represent 500 μm, and the scale bars in the bottom images represent 50 μm. (D) Percentages of abnormal seminiferous tubules with less than three layers of spermatogenic cells and/or sloughed germ cells in the three groups. (E) Expression levels of PLZF and Stra8 in the testes. * p < 0.05, ** p < 0.01, *** p < 0.001. Each experiment was replicated at least three times with independent samples.

Figure 2

TCEP exposure disrupts BTB integrity. (A) Expression levels of BTB-associated proteins in the testes. (B) Assessment of BTB integrity using TEM. (a) The control group. (b) The T50 group. (c) The T100 group. The yellow arrows denoted the TJ; the red asterisks denoted the basal ES. * p < 0.05, ** p < 0.01, *** p < 0.001. Each experiment was replicated at least three times with independent samples.

Figure 3

TCEP exposure leads to impairment of junctional function and ferroptosis in Sertoli cells. (A) Viability of Sertoli cells after TCEP exposure. (B) Expression levels of BTB-associated proteins in Sertoli cells. (C) Levels of ferrous iron, MDA, and GSH in Sertoli cells. (a) Level of ferrous iron. (b) Level of MDA. (c) Level of GSH. (D) Expression of ferroptosis-associated markers in Sertoli cells. * p < 0.05, ** p < 0.01, *** p < 0.001. Each experiment was replicated at least three times with independent samples.

Figure 4

Treatment with Fer-1 ameliorates the TCEP-induced impairment of junctional functionality in Sertoli cells. (A) Levels of ferrous iron, MDA, and GSH in Sertoli cells after Fer-1 treatment. (a) Level of ferrous iron. (b) Level of MDA. (c) Level of GSH. (B) Expression of ferroptosis-associated markers in Sertoli cells after Fer-1 treatment. (C) Expression levels of BTB-associated proteins in Sertoli cells after Fer-1 treatment. * p < 0.05, ** p < 0.01, *** p < 0.001. Each experiment was replicated at least three times with independent samples.

Figure 5

TCEP induces ferroptosis in Sertoli cells and testis through ferritinophagy. (A) Levels of ferrous iron, MDA, and GSH in the testes. (a) Level of ferrous iron. (b) Level of MDA. (c) Level of GSH. (B) Ferritinophagic activity in the testes. (C) Ferritinophagic activity in Sertoli cells. * p < 0.05, ** p < 0.01, *** p < 0.001. Each experiment was replicated at least three times with independent samples.