La₂O₃ Nanoparticles Induce Reproductive Toxicity Mediated by the Nrf-2/ARE Signaling Pathway in Kunming Mice

Figures & data

Table 1 List of Primers Used for Real-Time RT-PCR

Function	Gene Name	Primer Sequence (5ʹ-3ʹ)	Product (bp)	Genbank Accession
Testosterone synthesis-related genes	StAR	F: CGGGTGGATGGGTCAAGTTC R: GCACTTCGTCCCCGTTCTC	188	NM_011485.5
	CYP11A1	F: AGGTCCTTCAATGAGATCCCTT R: TCCCTGTAAATGGGGCCATAC	137	NM_019779.4
	CYP17A1	F: AGTCAAAGACACCTAATGCCAAG R: ACGTCTGGGGAGAAACGGT	83	NM_007809.3
	LHR	F: CTCGCCCGACTATCTCTCAC R: ACGACCTCATTAAGTCCCCTG	77	NM_013582.3
Nrf-2/ARE pathway-related genes	Keap-1	F: CGGGGACGCAGTGATGTATG R: TGTGTAGCTGAAGGTTCGGTTA	85	NM_016679.4
	Nrf-2	F: TAGATGACCATGAGTCGCTTGC R: GCCAAACTTGCTCCATGTCC	153	NM_010902.4
	NQO1	F: AGGATGGGAGGTACTCGAATC R: TGCTAGAGATGACTCGGAAGG	127	NM_008706.5
	HO-1	F: AGGTACACATCCAAGCCGAGA R: CATCACCAGCTTAAAGCCTTCT	86	NM_010442.2
	GSH-Px	F: GAAGTGCGAAGTGAATGG R: TGTCGATGGTACGAAAGC	224	NM_008160.6
Inflammation	iNOS	F: GTTCTCAGCCCAACAATACAAGA R: GTGGACGGGTCGATGTCAC	127	NM_010927.4
	IL-1β	F: GAAATGCCACCTTTTGACAGTG R: TGGATGCTCTCATCAGGACAG	116	NM_008361.4
	TNF-α	F: CATGGATCTCAAAGACAACCAA R: CTCCTGGTATGAAATGGCAAAT	193	NM_013693.3
	COX-2	F: TACAACAACTCCATCCTCCTTG R: TTCATCTCTCTGCTCTGGTCAA	186	NM_011198.4
Apoptosis	Bcl-2	F: GGTCTTCAGAGACAGCCAGG R: GATCCAGGATAACGGAGGCT	113	NM_177410.3
	BAX	F: GATCAGCTCGGGCACTTTAG R: TTGCTGATGGCAACTTCAAC	120	NM_007527.3
Reference	β-actin	F: GTGCTATGTTGCTCTAGACTTCG R: ATGCCACAGGATTCCATACC	174	NM_007393.5

Figure 1 The characterization of the La₂O₃ NPs and La₂O₃ MPs. (A) The SEM images of La₂O₃ NPs (a) and La₂O₃ MPs (b) at high magnification, and the particles were in irregular sheet structure. Scale bar = 500 nm. (B) The TEM images of La₂O₃ NPs (a) and La₂O₃ MPs (b) at high magnification, showing aciniform aggregates and agglomerates in Figure 1Ba, and the average sizes of La₂O₃ NPs and La₂O₃ MPs in (c). (d) and (e): La₂O₃ NPs and La₂O₃ MPs bioaccumulate in scrotal tissues by TEM analysis. Scale bar = 1 μm. (C) The XRD patterns of the La₂O₃ NPs (a) and La₂O₃ MPs (b).

Table 2 Characteristics of La₂O₃ NPs in the Different pH Values Solutions

Particle	PH	Average Diametre (nm)	PdI	Zeta Potential (mV)
La2O3 NPs	2.98	739.72	0.432	32.72
	4.04	462.38	0.375	33.21
	4.74	361.83	0.276	38.86
	In DI water	284.32	0.462	32.17
	In PBS	395.71	0.489	36.65

Figure 2 Effects of La₂O₃ NPs on mouse growth and testicular histology. (A) The body weights of mice in the 5 groups were compared, and the differences were not statistically significant (Figure 2A) (P > 0.05). (B) Histopathological changes in murine testes caused by intragastric administration of La₂O₃ NPs for 90 days. The testes from control (a), NL (b) and WM (e) groups showed normal morphology and spermatogenesis. In the NM and NH (c and d) groups, the testes exhibited vacuole-like changes in the spermatogenic epithelium, as indicated by arrows (×400). Moreover, moderate LCs edema (asterisk) were observed in NM and NH groups.

Table 3 Effects of Different Doses of La₂O₃ NPs and MPs on Coefficients of Mice

	Coefficients of Tissues
Tissue	Con	NL	NM	NH	WM
Liver (mg/g)	49.47±2.24	49.95±2.61	51.21±1.82	55.49±2.43*	52.42±2.67
Kidney (mg/g)	15.19 ±0.79	14.74±0.56	15.01±0.84	15.94±0.92	15.15±0.74
Lung (mg/g)	5.57±0.69	5.36±0.68	5.75±0.47	5.78±0.31	5.45±0.97
Spleen (mg/g)	2.89±0.36	2.96±0.23	3.52±0.38	3.81±0.42*	3.33±0.27
Heart (mg/g)	5.19±0.33	5.14±0.41	5.07±0.65	5.01±0.78	5.22±0.60
Brain (mg/g)	17.89±0.86	17.32±1.54	16.07±0.87	16.89±0.72	16.35±1.02
Testis (mg/g)	2.87±0.35	3.03±0.77	2.86±0.23	2.84±0.18	2.76±0.43

Notes: Values are expressed as mean ± SD for 10 mice per group *Significantly different from vehicle control at P<0.05.

Table 4 The La Contents in Whole Blood and Different Organs

	Con	La2O3 NPs			La2O3 MPs
		NL	NM	NH	WM
Liver (μg/g)	5.32±0.75	82.21±4.35**	148.38±18.47**	434.98±71.12**	84.75±14.25^**∆
Kidney (μg/g)	3.53±0.58	56.17±7.26**	94.52±15.72**	366.52±48.53**	67.31±7.49^**∆
Spleen (μg/g)	2.85±0.31	48.92±7.81**	82.32±13.31**	343.35±37.57**	54.68±8.42^**∆
Testis (μg/g)	3.37±0.54	43.58±6.26**	75.62±15.17**	276.72±34.45**	45.16±11.27^**∆
Lung (μg/g)	4.15±0.62	35.53±6.23**	67.25±18.54**	214.16±37.31**	27.55±8.58^**∆
Brain (μg/g)	3.28±0.38	31.47±4.32**	51.51 ±7.86**	102.87 ±27.62**	24.68±9.40^**∆
Heart (μg/g)	3.62±0.37	9.81±0.89*	21.56±7.34**	63.45±12.37**	8.31±0.45^∆
Blood (μg/mL)	4.28±0.75	84.65±9.52**	156.30±15.97**	468.36±58.72**	86.68±8.82^**∆

Notes: Values are expressed as mean ± SD for 5 mice per group. ^*,**Significantly different from vehicle control at P < 0.05 and P < 0.01; ^∆Significantly different from NH vs WM group at P<0.05.

Figure 3 La₂O₃ NPs and MPs influence on ultrastructure of testis. (A) Control. (B) La₂O₃ NPs (5 mg/kg). (C) La₂O₃ NPs (10 mg/kg). Arrows indicate mitochondria and vacuoles in mitochondria. (D) La₂O₃ NPs (50 mg/kg). (E) La₂O₃ MPs (50 mg/kg) (×8000). The control and La₂O₃ MPs groups showed no morphological changes, but vacuolar changes in the mitochondria were detected in the NM and NH groups (arrows). In affected tubules, vacuolation of the seminiferous epithelium could be detected (red asterisk).

Figure 4 The impact of La₂O₃ NPs and MPs on sperm parameters, levels of testis testosterone, genes and proteins involved in synthesis of testosterone. Changes in sperm count (Aa) and sperm motility percentages (Ab) and sperm survival percentages (Ac) of the mice; (Ba) testis testosterone; (Bb) LH levels; (Bc) FSH levels; (Bd) GnRH levels in the 90 days intragastric exposures. (N=10, mean S.D.). *P <0.05 vs control group. Effects of La₂O₃ NPs and MPs on mRNA expression of StAR, CYP11A1, CYP17A1 and LHR in testes (Ca-d). Effects of La₂O₃ NPs and MPs on protein expression of StAR, CYP11A1, CYP17A1 and LHR (Da-b). β-Actin was used as the internal control. *P<0.05 vs control.

Figure 5 La₂O₃ NPs and MPs induced Nrf-2/ARE signaling changes in mice testes. (A) Effects of La₂O₃ NPs and MPs on MDA level, SOD and CAT activities. (a) SOD level. (b) MDA activity. (c) CAT activity. (B) La₂O₃ NPs and MPs infleuenced on Nrf-2/ARE signaling relative genes in mouse testes. Effects on mRNA expression levels of Keap-1, Nrf-2, NQO1, HO-1 and GSH-Px in mice testes. (Ca-Cb) The protein levels of Nrf-2/ARE signaling relative genes by western blotting.

Figure 6 Effects of La₂O₃ NPs and MPs on testicular inflammation. (A) Inflammation testicular cells in the control group (a), NL group (b), NM group (c), NH group (d) and WM group (e). NF-κB-positive cells (f) were significantly increased in the NM and NH groups (×400, P<0.05). However, in the NL and WM groups no significant differences were found compared with control group (Figure 6A, P>0.05). The expressions of expression levels of inflammatory enzymes iNOS, IL-1β, TNF-α and COX-2 were elevated in NM and NH groups (P<0.05) (Ba-d). Furthermore, the expressions of TNF-α, IL-1β, iNOS and COX-2 were validated by Western-blot analysis shown in (Ca-b), and the expressions of iNOS, IL-1β, TNF-α and COX-2 in NH group were increased by 1.72-, 1.62-, 1.68- and 1.57-fold, respectively (P<0.05).

Figure 7 Effects of La₂O₃ NPs and MPs on testicular cells apoptosis. TUNEL and immunohistochemical staining of BAX (A-B) was detected at × 400 magnification. Apoptotic testicular cells in the control group (a), NL group (b), NM group (c), NH group (d) and WM group (e). Arrows indicated the positive cells, and histograms showing the percentages of TUNEL and BAX-positive cells (f). Exposure of mice to La₂O₃ NPs and MPs down-regulated of Bcl-2, and up-regulated both in the gene and protein levels of BAX in NH group compared with control group (Figure 7C-D) (P<0.05). The results of Bcl-2 and BAX protein expression in mouse testicular tissue were determined by Western blotting. β-Actin was used as the internal control (Da-b). *P< 0.05 vs control.

Figure 8 Schematic representation of possible mechanisms of La₂O₃ NPs contributing to the apoptosis of testes tissues.