Effect of zinc oxide nanoparticles on dams and embryo–fetal development in rats

Figures & data

Table 1 Formulation analysis of the dosing solution for ZnO^SM20(−) NPs on GDs 5, 11, and 19

Dosing date	ZnO^SM20(−)
	Target concentration (mg/mL)	Determined concentration (mg/mL)	Difference from target (%)
GD 5	0	Not detected	Not detected
GD 11	0	Not detected	Not detected
GD 19	0	Not detected	Not detected
GD 5	10	9.32	−6.82
GD 11	10	9.93	−0.67
GD 19	10	10.25 9.83±0.47a	2.50
GD 5	20	17.20	−14.00
GD 11	20	20.35	1.77
GD 19	20	20.50 19.35±1.86a	2.50
GD 5	40	38.38	−4.06
GD 11	40	41.40	3.49
GD 19	40	39.79 39.86±1.51a	−0.52

Note:

a Values are expressed as the mean ± standard deviation.

Abbreviations: ZnO^SM20(−), 20 nm negatively-charged ZnO; NPs, nanoparticles; GD, gestation day.

Table 2 The Zn content in fetuses after in utero exposure to ZnO^SM20(−) NPs

Parameters	Unit	Dose (mg/kg body weight/day)
		Nontreatment control group	400 mg/kg treatment (ZnO^SM20[−] NPs)
Number of fetuses/pregnant females	Fetus	28/2	24/2
Concentration	(μg/mL)	9.04	9.24
	(μg/mL)	6.77	8.64
	(μg/mL)	7.91±1.61a	8.94±0.42a
Dilution		100.00	100.00
Sample weight	(g)	63.75	49.71
	(g)	46.05	44.45
	(g)	54.90±0.37a	47.08±3.72a
Conversion concentration	(μg/g)	14.18	18.59
	(μg/g)	14.70	19.44
	(μg/g)	14.44±0.37a	19.02±0.60a

Note:

a Values are expressed as the mean ± standard deviation.

Abbreviations: ZnO^SM20(−), 20 nm negatively-charged ZnO; NPs, nanoparticles.

Figure 1 The total Zn levels measured with ICP-AES.

Notes: To investigate the placenta transfer of ZnO^SM20(−) NPs in vivo, four extra female rats were used in the nontreatment control group (n=2) and 400 mg/kg/day groups (ZnO^SM20[−] NPs; n=2), respectively. Dosing occurred on GDs 5–19 in the same manner as for the main study animals.

Abbreviations: ZnO^SM20(−), 20 nm negatively-charged ZnO; NPs, nanoparticles; ICP-AES, inductive coupled plasma atomic emission spectrometry; n, number; GD, gestational day.

Table 3 Body weights of the pregnant rats treated with ZnO^SM20(−) NPs

Dose (mg/kg body weight/day)	ZnO^SM20(−) NPs
	Vehicle (citrate)	100	200	400
Number of pregnant females	22	21	24	25
Gestation day 0	268.5±13.16a	271.5±15.10	269.7±17.40	266.2±13.99
Gestation day 1	273.3±14.07	277.4±15.77	274.9±16.04	274.7±13.10
Gestation day 2	280.0±13.52	284.2±15.73	282.8±16.33	283.0±13.11
Gestation day 3	285.6±15.09	288.0±16.18	287.7±15.08	287.8±15.18
Gestation day 4	291.3±16.18	293.3±17.02	292.8±15.03	291.6±13.34
Gestation day 5	295.1±14.53	297.0±16.70	297.4±15.88	297.6±13.96
Gestation day 6	298.6±15.25	300.5±17.57	299.6±15.93	298.8±14.69
Gestation day 7	302.7±15.42	303.7±19.13	304.8±15.85	302.9±15.00
Gestation day 8	306.6±16.22	310.5±19.29	308.4±18.74	306.7±14.56
Gestation day 9	310.7±15.33	311.9±19.41	312.5±18.86	309.0±15.38
Gestation day 10	317.8±17.95	318.1±20.85	317.5±17.52	314.7±17.34
Gestation day 11	322.3±14.33	324.5±23.58	325.1±18.82	322.2±16.76
Gestation day 12	328.3±18.76	327.6±22.71	329.9±19.20	325.9±17.21
Gestation day 13	333.4±18.41	333.3±22.34	334.2±19.61	328.4±17.16
Gestation day 14	340.0±18.81	340.4±21.64	341.2±21.27	334.4±19.80
Gestation day 15	347.3±18.16	345.4±21.35	346.7±20.08	341.6±19.29
Gestation day 16	359.2±19.59	359.1±23.33	358.1±20.70	351.9±19.12
Gestation day 17	373.3±18.56	372.8±24.77	371.3±22.73	364.8±21.29
Gestation day 18	389.8±19.50	386.9±24.29	386.5±24.77	380.7±20.49
Gestation day 19	405.1±19.83	402.4±25.97	401.7±26.26	394.1±21.42
Gestation day 20	422.2±20.86	418.3±26.38	417.1±26.73	406.4±23.97*
Weight gain during pregnancy	153.8±13.93	146.9±18.58	147.4±18.83	140.2±20.11*
Corrected body weight	340.5±19.77	338.3±22.70	332.9±22.28	320.6±21.14**
Gravid uterine weight	81.8±8.62	80.1±8.80	84.2±9.67	85.8±12.99

Notes:

a Values are expressed as the mean ± standard deviation (g). Statistically different from the vehicle control group;

* P<0.05;

** P<0.01.

Abbreviations: ZnO^SM20(−), 20 nm negatively-charged ZnO; NPs, nanoparticles.

Table 4 Food consumption of the pregnant rats treated with ZnO^SM20(−) NPs

Dose (mg/kg body weight/day)	ZnO^SM20(−) NPs
	Vehicle (citrate)	100	200	400
Number of pregnant females	22	21	24	25
Gestation day 0	19.00±4.47a	20.94±3.73	21.26±2.30	21.83±3.14
Gestation day 2	23.90±3.00	25.78±3.19	26.52±3.82	25.23±2.87
Gestation day 4	26.32±3.23	26.32±3.28	26.71±3.33	27.43±2.92
Gestation day 6	27.07±3.94	26.02±3.50	25.42±3.34	23.63±3.66
Gestation day 8	27.94±4.48	27.56±3.55	28.26±4.86	27.56±4.08
Gestation day 10	28.10±3.12	26.30±3.62	26.71±5.6	28.70±6.55
Gestation day 12	29.05±4.34	27.77±4.26	27.95±377	28.65±5.64
Gestation day 14	29.48±3.42	29.13±4.14	28.48±2.67	27.77±3.89
Gestation day 16	31.53±2.93	31.44±3.32	30.77±3.94	30.49±5.97
Gestation day 18	30.18±6.17	31.65±2.76	31.12±2.77	29.32±3.56

Note:

a Values are expressed as the mean ± standard deviation (g).

Abbreviations: ZnO^SM20(−), 20 nm negatively-charged ZnO; NPs, nanoparticles.

Table 5 Absolute and relative organ weights of pregnant rats treated with ZnO^SM20(−) NPs

Dose (mg/kg body weight/day)	Unit	ZnO^SM20(−) NPs
		Vehicle (citrate)	100	200	400
Number of pregnant females		22	21	24	25
Body weight at term (g)		422.2±20.86a	418.3±26.38	417.1±26.73	406.4±23.97
Liver	(g)	16.00±1.27	15.20±1.25	14.80±1.51	14.48±1.27**
	(g%)	3.79±0.19	3.63±0.20	3.54±0.20**	3.56±0.18**
Kidney, left	(g)	1.07±0.10	1.06±0.11	1.07±0.19	1.08±0.08
	(g%)	0.25±0.02	0.25±0.03	0.26±0.04	0.27±0.02
Kidney, right	(g)	1.10±0.10	1.09±0.10	1.07±0.09	1.09±0.10
	(g%)	0.26±0.02	0.26±0.02	0.26±0.02	0.27±0.02
Spleen	(g)	0.71±0.11	0.66±0.10	0.65±0.08	0.66±0.12
	(g%)	0.17±0.03	0.16±0.02	0.16±0.02	0.16±0.03
Adrenal gland, left	(g)	0.042±0.007	0.044±0.009	0.045±0.007	0.048±0.007
	(g%)	0.009±0.004	0.011±0.002	0.011±0.002	0.012±0.002**
Adrenal gland, right	(g)	0.039±0.007	0.041±0.008	0.045±0.006*	0.045±0.007*
	(g%)	0.009±0.002	0.010±0.002	0.011±0.002*	0.011±0.002**
Ovary, left	(g)	0.068±0.015	0.070±0.020	0.071±0.015	0.066±0.021
	(g%)	0.016±0.004	0.017±0.005	0.017±0.004	0.016±0.005
Ovary, right	(g)	0.071±0.011	0.077±0.020	0.068±0.016	0.073±0.014
	(g%)	0.017±0.003	0.018±0.005	0.016±0.004	0.018±0.004
Brain	(g)	1.94±0.21	1.97±0.06	1.99±0.09	1.93±0.10
	(g%)	0.46±0.05	0.47±0.04	0.48±0.03	0.48±0.03
Pituitary gland	(g)	0.015±0.005	0.016±0.003	0.016±0.004	0.018±0.016
	(g%)	0.004±0.001	0.004±0.001	0.004±0.001	0.004±0.004
Lung	(g)	1.33±0.15	1.34±0.11	1.32±0.10	1.31±0.10
	(g%)	0.32±0.03	0.32±0.03	0.32±0.02	0.32±0.02
Heart	(g)	1.16±0.27	1.13±0.10	1.05±0.18	1.09±0.12
	(g%)	0.27±0.07	0.27±0.02	0.25±0.05	0.27±0.02
Uterus (gravid)	(g)	81.75±8.62	80.07±8.80	84.18±9.67	85.75±12.99
	(g%)	19.38±1.96	19.14±1.76	20.17±1.83	21.09±2.83

Notes:

a Values are expressed as the mean ± standard deviation (g). Statistically different from the vehicle control group;

* P<0.05;

** P<0.01.

Abbreviations: ZnO^SM20(−), 20 nm negatively-charged ZnO; NPs, nanoparticles; g%, relative weight of organ-to-body weight (g).

Figure 2 Body weight changes of female rats during the gestation period.

Notes: Pregnant rats were orally treated with ZnO^SM20(−) NPs for 15 days (GD 5–GD 19) with doses of 100 mg/kg/day, 200 mg/kg/day, and 400 mg/kg/day. GD 0 means the day of pregnancy. “GD” is the day after gestation. Statistically different from the vehicle control group; *P<0.05.

Abbreviations: GD, gestational day; ZnO^SM20(−), 20 nm negatively-charged ZnO; NPs, nanoparticles.

Figure 3 External alteration in the fetus from a pregnant rat treated with ZnO^SM20(−) NPs at a dose of 100 mg/kg/day.

Note: Hypoplasia tail (absent; arrow).

Abbreviations: ZnO^SM20(−), 20 nm negatively-charged ZnO; NPs, nanoparticles.

Figure 4 Visceral alteration in the fetus from a rat treated with vehicle.

Notes: Misshapen thymus (arrow). Vehicle = 20 nM 4-(2-hydroxyethyl)-1-piperaz-ineethanesulfonic acid (HEPES) buffer containing 1% sodium citrate.

Figure 5 Visceral alteration in the fetus from a rat treated with vehicle.

Notes: Dilated renal pelvis (arrow). Vehicle = 20 nM 4-(2-hydroxyethyl)-1-pipera-zineethanesulfonic acid (HEPES) buffer containing 1% sodium citrate.

Figure 6 Visceral alteration in the fetus from a rat treated with ZnO^SM20(−) NPs (100 mg/kg).

Note: Ectopic kidney (malpositioned; arrow).

Abbreviations: ZnO^SM20(−), 20 nm negatively-charged ZnO; NPs, nanoparticles.

Table 6 Caesarean section data from pregnant rats treated with ZnO^SM20(−) NPs

Dose (mg/kg body weight/day)	ZnO^SM20(−) NPs
	Vehicle (citrate)	100	200	400
Number of dams	22	21	24	25
Number of corpora lutea	16.36±2.36a	17.48±3.27	17.79±3.36	17.56±2.75
Number of implantation sites	14.73±1.55	14.43±1.43	15.21±1.77	15.04±1.93
Implantation rate (%)	90.85±9.27	84.61±13.62	87.34±13.31	86.70±11.53
Fetal deaths	0.73±1.03	0.71±0.85	0.58±0.78	0.52±0.82
Resorption
Early	0.64±1.05	0.62±0.80	0.42±0.58	0.40±0.65
Late	0.09±0.29	0.10±0.44	0.13±0.45	0
Dead fetuses	0	0	0.04±0.20	0.12±0.33
Litter size	14.0±1.7	13.7±1.6	14.6±1.7	14.5±1.9
Male/Female	154/154	151/137	183/168	194/169
Sex ratio	1.00	1.10	1.09	1.15
Fetal weight (g)
Male	4.08±0.18	4.11±0.29	4.05±0.26	4.10±0.38
Female	3.84±0.23	3.94±0.30	3.82±0.23	3.89±0.26
Placental weight (g)	0.58±0.06	0.58±0.05	0.57±0.07	0.56±0.04

Note:

a Values are expressed as the mean ± standard deviation (g).

Abbreviations: ZnO^SM20(−), 20 nm negatively-charged ZnO; NPs, nanoparticles.

Table 7 External alterations in fetuses from pregnant rats treated with ZnO^SM20(−) NPs

Dose (mg/kg body weight/day)	ZnO^SM20(−) NPs
	Vehicle (citrate)	100	200	400
Litters examined	22	21	24	25
External examination
Fetuses examined	308	288	351	363
Fetuses with malformations (%)a	0	0	0	0
Litters affected (%)b	0	0	0	0
Fetuses with variations (%)a	6 (1.92)	5 (1.74)	4 (1.14)	3 (0.83)
Litters affected (%)b	5 (22.73)	3 (14.29)	4 (16.67)	2 (8.00)
Hematoma	6	4	4	2
Hypoplasia of tail	0	1	0	0
Short tail	0	0	0	1

Notes:

a A single fetus may be represented more than once when listing individual defects.

b Includes litters with one or more affected fetuses.

Abbreviations: ZnO^SM20(−), 20 nm negatively-charged ZnO; NPs, nanoparticles.

Table 8 Visceral alterations in fetuses from rats treated with ZnO^SM20(−) NPs

Dose (mg/kg body weight/day)	ZnO^SM20(−) NPs
	Vehicle (citrate)	100	200	400
Litters examined	22	21	24	25
Visceral examination
Fetuses examined	147	139	171	176
Fetuses with malformations (%)	0	0	0	0
Litters affected (%)	0	0	0	0
Fetuses with variations (%)a	120 (81.63)	115 (82.73)	119 (69.59)	139 (78.98)
Litters affected (%)b	18 (81.82)	18 (85.71)	23 (95.83)	23 (92.00)
Misshapen thymus	8	5	14	18
Ureter abnormality
Grade I	52	48	37	59
Grade II	36	49	51	46
Grade III	23	8	13	14
Dilated renal pelvis	1	1	0	1
Large kidney	0	1	0	0
Ectopic kidney	0	3	4	1

Notes:

a A single fetus may be represented more than once when listing individual defects.

b Includes litters with one or more affected fetuses.

Abbreviations: ZnO^SM20(−), 20 nm negatively-charged ZnO; NPs, nanoparticles.

Table 9 Skeletal variations in fetuses from rats treated with ZnO^SM20(−) NPs

Dose (mg/kg body weight/day)	ZnO^SM20(−) NPs
	Vehicle (citrate)	100	200	400
Litters examined	22	21	24	25
Skeletal examination
Fetuses examined	161	149	180	187
Fetuses with malformations (%)a	0	0	0	0
Litters affected (%)b	0	0	0	0
Fetuses with variations (%)a	11 (6.83)	7 (4.70)	11 (6.11)	17 (9.10)
Litters affected (%)b	8 (36.36)	6 (28.57)	7 (29.17)	9 (36.00)
Skull
Incomplete ossification	1	0	0	0
Thoracic centrum
Dumbbell ossification	1	0	0	2
Bipartite ossification	0	0	0	1
Asymmetric	0	1	1	1
Incomplete ossification	2	1	2	6
Rib
Supernumerary	5	2	7	5
Sternebra
Asymmetric	2	3	1	0
Incomplete ossification	1	0	1	2
Lumbar vertebra
Incomplete ossification	0	0	0	1

Notes:

a A single fetus may be represented more than once when listing individual defects.

b Includes litters with one or more affected fetuses.

Abbreviations: ZnO^SM20(−), 20 nm negatively-charged ZnO; NPs, nanoparticles.