Evaluation of immunologic and intestinal effects in rats administered an E 171-containing diet, a food grade titanium dioxide (TiO2)

Abstract

The toxicity of dietary E 171, a food grade titanium dioxide was evaluated. A recent study reported rats receiving E 171 in water developed inflammation and aberrant crypt foci (ACF) in the gastrointestinal tract. Here, rats received food containing E 171 (7 or 100 days). The 100-day study included feeding E 171 after dimethylhydrazine (DMH) or vehicle only pretreatment. Food consumption was similar between treatment groups with maximum total cumulative E 171 exposure being 2617 mg/kg in 7 days and 29,400 mg/kg in 100 days. No differences were observed due to E 171 in the percentage of dendritic, CD4⁺ T or T_reg cells within Peyer's patches or the periphery, or in cytokine production in plasma, sections of jejunum, and colon in 7- or 100-day E 171 alone fed rats. Differences were observed for IL-17A in colon (400 ppm E 171 + DMH) and IL-12p70 in plasma (40 ppm E 171 + DMH). E 171 had no effect on histopathologic evaluations of small and large intestines, liver, spleen, lungs, or testes, and no effects on ACF, goblet cell numbers, or colonic gland length. Dietary E 171 administration (7- or 100-day), even at high doses, produced no effect on the immune parameters or tissue morphology.

Keywords: Dietary titanium dioxide; E 171; Gastrointestinal inflammation; Intestinal carcinogenesis; T(regulatory) cells.

Figure 1.. Dietary exposure to E 171 did not significantly alter systemic or gastrointestinal tract resident CD103⁺ dendritic cell frequency.

The frequency of gut resident and circulating CD103⁺ DC was determined in rats fed dietary E 171 for 7 or 100 consecutive days. Rats in the 100 day study were pretreated with 180 mg/kg of DMH or Vh control by i.p. injection. Following the indicated feeding period, animals were euthanized and PBMC, spleen, and Peyer’s patches were collected and processed into single cell suspension. Cells were then stained for CD11b/c, CD103, and MHCII and analyzed by flow cytometry to quantify CD103⁺ DC populations. CD103⁺ DC from the 7 day study are shown in panel A and from the 100 day study are shown in panel B. Data are presented as a percentage of the live cells within the lymphocyte gate. Data were analyzed for statistical significance using a one-way ANOVA with a Dunnett’s multiple comparisons test when there were no significant differences in the variance of the standard deviation.

Figure 2.. Dietary E 171 exposure did not change the frequency of CD4⁺ T_helper cells systemically or in intestinal Peyer’s patches.

The frequency of gut resident and circulating total and activated T_helper cells were determined in rats fed dietary E 171 for 7 or 100 days. Rats in the 100 day study were pretreated with 180 mg/kg of DMH or Vh control by i.p. injection. After the feeding period, animals were euthanized and PBMC, spleen, and Peyer’s patches were collected and processed into single cell suspensions. Cells were then stained for CD4 and CD25 and analyzed by flow cytometry. Activated T_helper cells were identified as being CD4/CD25 double positive. Total Thelper from 7 (A) and 100 (C) day animals and activated Thelper from 7 (B) and 100 (D) day studies are shown. Data are presented as a percentage of the live cells in the lymphocyte gate staining positive. Data were analyzed for statistical significance using a one-way ANOVA with a Dunnett’s multiple comparisons test when there were no significant differences in the variance of the standard deviation.

Figure 3.. Acute or chronic exposure to dietary E 171 did not significantly affect the frequency of circulating or GI-resident T_regulatory (T_reg) cells.

The frequency of gut resident and circulating total and activated T_reg cells were determined in rats fed dietary E 171 for a total of 7 or 100 days. Rats in the 100 day study were pretreated with 180 mg/kg of DMH or Vh control by i.p. injection. Following the 7- and 100-day feeding period, animals were euthanized and PBMC, spleen, and Peyer’s patches were collected and processed into single cell suspensions. Cells were then surface stained for CD4 and CD25 and subsequently stained intracellularly for FoxP3. Following antibody stains, cells were analyzed via flow cytometry to identify T_reg cell populations. Data are presented as a percentage of the live cells falling in the lymphocyte gate. Total Treg from 7 (A) and 100 (C) as well as activated Treg from 7 (B) and 100 (D) day studies are shown. Data were analyzed for statistical significance using a one-way ANOVA with a Dunnett’s multiple comparisons test when there were no significant differences in the variance of the standard deviation.

Figure 4.. Chronic dietary E 171 exposure did not alter systemic inflammation as evidenced by inflammatory cytokine accumulation.

Rats were pretreated with 180 mg/kg DMH or Vh control by i.p. injection and fed dietary E 171 for a total of 100 days. Following the 100 day feeding period, animals were euthanized and plasma was collected and assayed for inflammatory cytokines and chemokines using the LEGENDplex rat inflammation panel. Data are shown as cytokine/chemokine concentration (pg/ml) for plasma.. Data were analyzed for statistical significance using a one-way ANOVA with a Dunnett’s multiple comparisons test when there were no significant differences in the variance of the standard deviation. * = p<0.05 indicates significance compared to no E 171 control.

Figure 5.. Chronic dietary E 171 exposure did not modulate small intestine inflammatory cytokine accumulation.

Rats were pretreated with 180 mg/kg DMH or Vh control by i.p. injection and fed dietary E 171 for a total of 100 days. Following the 100 day feeding period, animals were euthanized and plasma was collected and assayed for inflammatory cytokines and chemokines using the LEGENDplex rat inflammation panel. Data are shown as cytokine/chemokine concentration (pg/ml) for small intestine. Cytokine protein levels were normalized to total protein levels for each sample of small intestine. Data were analyzed for statistical significance using a one-way ANOVA with a Dunnett’s multiple comparisons test when there were no significant differences in the variance of the standard deviation.

Figure 6.. Dietary E 171 exposure did not alter colonic inflammation as evidenced by inflammatory cytokine accumulation.

Rats were pretreated with 180 mg/kg DMH or Vh control by i.p. injection and fed dietary E 171 for a total of 100 days. Following the 100 day feeding period, animals were euthanized and plasma was collected and assayed for inflammatory cytokines and chemokines using the LEGENDplex rat inflammation panel. Data are shown as cytokine/chemokine concentration (pg/ml) for colon. Cytokine protein levels were normalized to total protein levels for each sample of colon. Data were analyzed for statistical significance using a one-way ANOVA with a Dunnett’s multiple comparisons test when there were no significant differences in the variance of the standard deviation. *=p<0.05 indicates significance compared to respective no E 171 control.