Effects of dietary exposure to the engineered nanomaterials CeO₂, SiO₂, Ag, and TiO₂ on the murine gut microbiome

Figures & data

Table 1. Characteristics of pristine ENM.

ENM*	Name, source	Mean diameter (nm)	Mode diameter (nm)	Sigma (fit)**
CeO2	NM212, JRC	35.4 ± 17.0	28.7	1.38
SiO2	#S5130, Sigma-Aldrich	12.9 ± 4.9	11.0	1.32
Ag	#576832-5G, Sigma-Aldrich	40.2 ± 17.6	32.0	1.40
TiO2	NM105, JRC	26.2 ± 10.7	21.0	1.39

*Engineered nanomaterials (ENM) used in the respective studies were analyzed by scanning electron microscopy as described in Supplementary material 1.

**Standard deviation of logarithmic values

Figure 1. Design of feeding studies with ENM in C57BL/6J mice. In study 1, female mice from the local breeding facility were exposed to CeO₂ or SiO₂ until sacrificing and collection of stools on day 21. In study 2, purchased male and female mice were exposed to Ag-PVP or TiO₂ P25 until sacrificing and collection of stools on day 28. Study 2 was divided into two rounds.

Table 2. Weekly development of the relative bodyweight in study 1, in percent of the bodyweight on the first day of exposure.

Exposure*	Control	CeO2	SiO2
Day 7	103.3 ± 0.7	102.3 ± 0.9	103.9 ± 1.5
Day 14	105.0 ± 0.8	107.2 ± 1.1	106.2 ± 1.1
Day 21	109.6 ± 1.4	109.4 ± 1.0	111.6 ± 1.9

*No significant differences between the exposure groups and the control on any of the weighing days (Means ± SEM; ANOVA with Dunnett’s post-hoc test).

Table 3. Weekly development of the relative bodyweight in study 2, in percent of the bodyweight on the first day of exposure.

Exposure*	Control ♀	Ag ♀	TiO2 ♀	Control ♂	Ag ♂	TiO2 ♂
Day 7	105.7 ± 1.1	106.2 ± 1.0	105.4 ± 1.0	109.0 ± 0.8	109.1 ± 0.7	111.0 ± 0.7
Day 14	109.4 ± 0.9	111.1 ± 0.4	110.2 ± 1.0	116.1 ± 1.1	115.8 ± 1.0	116.9 ± 0.9
Day 21	114.0 ± 1.9	115.8 ± 1.0	116.1 ± 0.8	124.1 ± 1.2	122.4 ± 1.6	125.4 ± 1.4
Day 28	116.9 ± 1.2	119.1 ± 0.8	118.3 ± 1.0	126.1 ± 1.3	126.9 ± 1.9	128.5 ± 1.6

*No significant differences between the exposure groups and the control on any of the weighing days (Means ± SEM; ANOVA with Dunnett’s post-hoc test). Female and male were analyzed separately.

Figure 2. Weighted UniFrac distance analyses of the microbial β-diversity depicted as Principle Coordinates Analyses (PCoA). In study 1, female mice were fed with feed pellets containing either no additive, 1.0% CeO₂ (A) or 1.0% SiO₂ (B) (N = 10 each). All 60 animals of study 2, including two artifacts that were removed before doing further analyses, are depicted together in C. In study 2, female and male mice were fed with feed pellets containing either no additive (N = 19, 10 female and 9 male), 0.2 % Ag (D) (N = 20, 10 per sex) or 1.0% TiO₂ (E) (N = 19, 10 female and 9 male). The comparison of the female control animals of study 1 and study 2 is shown in F. The different shapes of the dots represent the different cages. p Values were calculated using multivariate nested analysis of variance for study 1 and PERMANOVA based on cage average profiles for study 2.

Figure 3. Abundances of phyla in the intestinal microbiome of mice after ENM exposure. In study 1, female mice were fed with feed pellets containing either no additive, 1.0% CeO₂ or 1.0% SiO₂ (N = 10 each). In study 2, female and male mice were fed with feed pellets containing either no additive (N = 19, 10 female and 9 male), 0.2 % Ag (N = 20, 10 per sex) or 1.0% TiO₂ (N = 19, 10 female and 9 male) (A). In the scatter plots (B–D) the abundances of selected phyla in ENM exposed mice are displayed compared to the respective control. The different shapes of the dots represent the different cages. p-Values were calculated based on mixed effect models. In study 2, the interaction between sex and ENM was removed when not significant. Q Values were obtained by FDR correction. p Values ≤0.05 and Q values ≤0.10 were considered significant. E = ENM, S = Sex, f = female, m = male, C = Control, T = Treatment.

Figure 4. Abundances of genera in the intestinal microbiome of mice after ENM exposure. In study 1, female mice were fed with feed pellets containing either no additive, 1.0% CeO₂ or 1.0% SiO₂ (N = 10 each). In study 2, female and male mice were fed with feed pellets containing either no additive (N = 19, 10 female and 9 male), 0.2% Ag (N = 20, 10 per sex) or 1.0% TiO₂ (N = 19, 10 female and 9 male) (A). In the scatter plots (B–D) the abundances of selected genera in ENM exposed mice are displayed compared to the respective control. The different shapes of the dots represent the different cages. p Values were calculated based on mixed effect models. In study 2, the interaction between sex and ENM was removed when not significant. Q Values were obtained by FDR correction. p Values ≤0.05 and Q values ≤0.10 were considered significant. E = ENM, S = Sex, f = female, m = male, C = Control, T = Treatment.

Figure 5. α-diversity of the intestinal microbiome of mice after ENM exposure. In study 1, female mice were fed with feed pellets containing either no additive, 1.0% CeO₂ (A) or 1.0% SiO₂ (B) (N = 10 each). In study 2, female and male mice were fed with feed pellets containing either no additive (N = 19, 10 female and 9 male), 0.2% Ag (C) (N = 20, 10 per sex) or 1.0% TiO₂ (D) (N = 19, 10 female and 9 male). The richness presented as the number of ASVs observed per sample was assessed. p Values were calculated based on mixed effect models. In study 2 the interaction between sex and ENM was removed when not significant. Q values were obtained by FDR correction accounting for the three endpoints richness, Shannon entropy and Simpson's index. Q values ≤0.10 were considered significant.

Data availability statement

The next generation sequencing raw data that were obtained in this investigation, are openly available in figshare at doi: 10.6084/m9.figshare.14499822.