NLRP3 inflammasome, oxidative stress, and apoptosis induced in the intestine and liver of rats treated with titanium dioxide nanoparticles: in vivo and in vitro study

Figures & data

Figure 1 The in vitro studies of TiO₂ NPs.

Notes: Effect of TiO₂ NPs on cell viability (A), enzyme activity, ROS generation, GSH levels, and apoptosis rate in Caco-2 cells (B) and HepG2 cells (C) after 24 hours (n=3). Values were expressed as mean ± SD. *P<0.05, ^#P<0.01, and ^ŦP<0.001 as compared to control. Apoptosis rates at the concentrations <150 µg/mL TiO₂ NPs were not significant compared to control.

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GSH, glutathione; LDH, lactate dehydrogenase; TiO₂ NPs, titanium dioxide nanoparticles.

Table 1 Comparison of biochemical factors in the serum of different treated animals

Groups	Control	Control +10 mg/kg NPs	Control +50 mg/kg NPs	Control +100 mg/kg NPs
ALT (U/L)	43.80±6.34	63.80±4.86*	81.80±6.83Ŧ,£	106.60±8.67Ŧ,§
AST (U/L)	57.40±7.56	85.00±12.66*	100.20±6.94Ŧ	126.40±7.05Ŧ,§
LDH (U/L)	402.80±8.31	422.20±15.56	519.00±22.54Ŧ,£	767.60±33.90Ŧ,§
ALP (U/L)	134.80±17.88	155.20±14.30	178.60±11.14#,£	271.40±12.11Ŧ,§
Total bilirubin (mg/dL)	2.00±0.23	2.84±0.33*	3.02±0.31Ŧ	3.28±0.33Ŧ,§
BUN (mg/dL)	18.0±3.0	21.00±2.50	43.00±4.40#,£	55.0±4.0Ŧ,§
Creatinine (mg/dL)	0.60±0.1	0.9±0.05	1.8±0.02#,£	2.10±0.10Ŧ,§

Notes: Data were expressed as mean ± SD.

* P<0.05,

# P<0.01, and

Ŧ P<0.001 compared with the control group.

£ P<0.05 as compared to the “Control +10 mg/kg NPs” group and

§ P<0.05 as compared to the “Control +50 mg/kg NPs”.

Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase; BUN, blood urea nitrogen; LDH, lactate dehydrogenase; NPs, nanoparticles.

Figure 2 Effect of TiO₂ NPs on antioxidant gene expressions and apoptosis pathway.

Notes: Treatment of rats with TiO₂ NPs significantly reduced antioxidant gene expressions and induced apoptosis pathway in the intestine (A) and liver (B) of different treated groups. Values were expressed as mean ± SD. *P<0.05, ^#P<0.01, and ^ŦP<0.001 as compared to the control group. ^£P<0.05 as compared to the “Control +10 mg/kg NPs” group and ^§P<0.05 as compared to the “Control +50 mg/kg NPs” group.

Abbreviations: CAT, catalase; GPx, glutathione peroxidase; SOD, superoxide dismutase; TiO₂ NPs, titanium dioxide nanoparticles.

Table 2 Effects of TiO₂ NPs on antioxidant enzyme activities, antioxidant capacity, lipid peroxidation, and caspase activity in the liver and intestine of different treated animals

Variables	Control group		Control +10 mg/kg NPs group		Control +50 mg/kg NPs group		Control +100 mg/kg NPs group
	Liver	Intestine	Liver	Intestine	Liver	Intestine	Liver	Intestine
SOD (unit/mg pr)	99.2±7.1	87.0±8.3	81.0±9.1#	72.8±8.0	59.80±6.1Ŧ,£	59.0±13.5#,£	35.0±3.5Ŧ,§	32.4±1.6Ŧ,§
CAT (unit/mg pr)	57.0±5.3	51.6±5.9	45.20±7.6*	42.6±6.0	33.40±4.5Ŧ,£	32.8±6.4Ŧ,£	22.4±1.8Ŧ,§	21.0±2.7Ŧ,§
GPx (unit/mg pr)	60.2±7.1	54.8±4.6	38.2±11.9#	39.2±5.2Ŧ	36.20±3.8Ŧ	35.2±5.26Ŧ	19.8±2.1Ŧ,§	16.6±1.8Ŧ,§
GSH (µg/mg pr)	130.2±7.4	148.4±14.7	105.60±4.7Ŧ	116.8±5.0#	72.60±8.9Ŧ,£	111.8±3.0Ŧ	52.4±4.9Ŧ,§	44.8±7.0Ŧ,§
MDA (nmol/mg pr)	12.4±1.3	9.0±1.4	22.80±5.7#	18.9±1.0Ŧ	28.1±2.9Ŧ	24.5±3.6Ŧ,£	38.7±2.9Ŧ,§	34.8±3.0Ŧ,§
TAC (nmol/mg pr)	83.6±3.5	72.4±10.8	56.80±9.1#	49.20±6.2#	31.2±4.6Ŧ,£	33.0±6.2Ŧ,£	22.4±2.7Ŧ,§	17.8±2.38Ŧ,§
TOS (nmol/mg pr)	7.4±0.8	7.9±0.8	11.04±1.2#	14.6±0.9Ŧ	15.3±1.6Ŧ	18.3±2.8Ŧ	22.7±1.8Ŧ,§	23.1±1.6Ŧ,§
Caspase-3 activity (arbitrary unit)	10±0.0	10±0.0	13.6±0.6*	13.8±0.5*	15.5±0.2#,£	14.8±0.3#,£	16.9±0.5#,§	17.0±0.5#,§

Notes: TiO₂ NPs significantly increased SOD, CAT, and GPX activity as well as GSH and TAC levels in rats, whereas TiO₂ NPs reduced TOS and MDA levels in the intestine and liver. Values were expressed as mean ± SD.

* P<0.05,

# P<0.01, and

Ŧ P<0.001 as compared to the control group.

£ P<0.05 as compared to the “Control +10 mg/kg NPs” group and

§ P<0.05 as compared to the “Control +50 mg/kg NPs”.

Abbreviations: CAT, catalase; GSH, glutathione; GPx, glutathione peroxidase; MDA, malondialdehyde; pr, protein; SOD, superoxide dismutase; TAC, total antioxidant capacity; TiO₂ NPs, titanium dioxide nanoparticles; TOS, total oxidant status.

Table 3 Intestinal and liver histopathological changes in different treated animals

Tissues	Pathological assessment	Control	Control + 10 mg/kg NPs	Control + 50 mg/kg NPs	Control + 100 mg/kg NPs
Intestine	Cell infiltration	0	1	1	2
	Mucosal disruption and injury	0	1	2	2
	Shortening and shedding of intestinal villi	0	1	2	3
	Mucosal erosion	0	1	2	2
	Edema	0	1	2	2
	Total grade	0	5*	9Ŧ,£	11Ŧ,§
Liver	Confluent necrosis	0	0	1	2
	Focal necrosis	0	2	2	3
	Portal inflammation	0	1	3	3
	Ballooning degeneration	0	1	2	2
	Fatty changes	0	1	1	2
	Congestion of the sinusoids	0	1	1	2
	Total grade	0	6*	10Ŧ,£	14Ŧ,§

Notes: Data were expressed as mean ± SD.

* P<0.05, and

Ŧ P<0.001 as compared to the control group.

£ P<0.05 as compared to the “Control +10 mg/kg NPs” group and

§ P<0.05 as compared to the “Control +50 mg/kg NPs”. The Ishak system was applied for scoring liver injury.²⁰ The histopathological changes in intestine were evaluated according to the study by Erben et al.³²

Figure 3 Apoptosis determined in the intestine and liver by TUNEL.

Notes: Analysis of apoptosis by TUNEL staining of intestine (A) and liver (B) compared to control rats (magnification, 400×). Counter staining was done using DAPI nuclear staining, and pictures were taken using DAPI (i), fluorescein isothiocyanate (apoptotic cells; ii), and merged (iii) filters. Intestine and liver apoptosis were quantified by counting the number of apoptotic cells (C). Values were expressed as mean ± SD. *P<0.05, and ^ŦP<0.001 as compared to the control group. ^£P<0.05 as compared to the “Control +10 mg/kg NPs” group and ^§P<0.05 as compared to the “Control +50 mg/kg NPs”.

Abbreviation: TiO₂ NPs, titanium dioxide nanoparticles.

Figure 4 Effect of TiO₂ NPs on inflammasome and inflammatory pathways.

Notes: Treatment of rats with TiO₂ NPs significantly increased inflammasome and inflammatory gene expressions in the intestine (A) and liver (B), and the IL-1β protein, TNF-α protein, and nitric oxide levels in the intestine (C) and liver (D). Values were expressed as mean ± SD. *P<0.05, ^#P<0.01, and ^ŦP<0.001 as compared to the control group. ^£P<0.05 as compared to the “Control +10 mg/kg NPs” group and ^§P<0.05 as compared to the “Control +50 mg/kg NPs”.

Abbreviations: iNOS, inducible nitric oxide synthase; NLRP3, NLR family pyrin domain containing 3; TiO₂ NPs, titanium dioxide nanoparticles; TNF-α, tumor necrosis factor-α.

Figure 5 Morphological alteration of intestine.

Notes: Effects of TiO₂ NPs on histological changes of intestine determined by H&E (A), and MT staining (B) (magnification, 400×). Results showed that in TiO₂ NP-treated groups crypt structure is injured and mucosa is eroded. Moreover, edema, necrosis, disruption, shortening, and loosening of intestinal villi are present. Arrow indicates collagen deposition and straight line shows villi altitude.

Abbreviations: MT, Masson’s trichrome; TiO₂ NPs, titanium dioxide nanoparticles; H&E, Hematoxylin and Eosin.

Figure 6 Morphological alteration of liver.

Notes: Effects of TiO₂ NPs on histological changes of liver determined by H&E (A) and MT staining (B) (magnification, 400×). Arrow indicates collagen deposition and straight line shows central vein diameter.

Abbreviations: MT, Masson’s trichrome; TiO₂ NPs, titanium dioxide nanoparticles; H&E, Hematoxylin and Eosin.

Figure S1 Characterization of TiO₂ NPs.

Note: SEM (A), TEM image (B and C), and XRD pattern (D) of TiO₂ NPs.

Abbreviations: SEM, scanning electron microscopy; TEM, transmission electron microscopy; TiO₂ NPs, titanium dioxide nanoparticles; XRD, X-ray diffraction.

IshakKBaptistaABianchiLHistological grading and staging of chronic hepatitisJ Hepatol19952266966997560864

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ErbenULoddenkemperCDoerfelKA guide to histomorphological evaluation of intestinal inflammation in mouse modelsInt J Clin Exp Pathol2014784557457625197329

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