Dose- and time-dependent changes in tissue levels of tetrabromobisphenol A (TBBPA) and its sulfate and glucuronide conjugates following repeated administration to female Wistar Han Rats

Abstract

Tetrabromobisphenol A (TBBPA), a nongenotoxic flame retardant, causes uterine tumors in female rats. A proposed mode of action (MoA) for these tumors involves an increase in the bioavailability of estradiol as a result of TBBPA inhibiting estrogen sulfotransferases (ES), the enzymes responsible for inactivating and enhancing the elimination of estradiol. The objective of this study was to evaluate the effect of dose and repeated administration of TBBPA on the level of TBBPA, TBBPA-glucuronide (GA) and TBBPA-sulfate (S) conjugates in plasma, liver and uterus of female Wistar Han rats administered TBBPA (50, 100, 250, 500 or 1000 mg/kg) for 28 consecutive days. In accordance with this objective, TBBPA sulfation was used as a surrogate for evaluating the potential for estradiol sulfation to be limited at high dose levels of TBBPA. Blood samples were collected at 4 and 8 h post-dosing on study day 7, 14, and 28, while liver and uterus were collected at the same time points following 28 days of dosing. Tissue samples were analyzed for TBBPA, TBBPA-GA and TBBPA-S by LC-MS/MS. A dose-related increase in the concentration of all three analytes occurred in plasma (day 7, 14, and 28) as well as liver and uterus tissue (day 28) at both 4 and 8 h post dose. The plasma concentration of TBBPA-GA and TBBPA-S was higher in animals dosed for 28 days compared to those dosed for 7 or 14 days showing an increase in systemic circulation of these conjugates with repeated administration. The balance of these conjugates was also different in tissues with TBBPA-S > TBBPA-GA at high doses in the liver and TBBPA-GA > TBBPA-S in both plasma and uterus. In all three tissues the ratio of TBBPA-S/TBBPA-GA showed a decreasing trend with dose, suggesting that at high TBBPA dose levels sulfation of TBBPA becomes limited. This effect was most apparent in the liver and plasma at 28 days of administration. Together these data show that administration of high doses of TBBPA associated with the induction of uterine tumors, results in a disruption in the balance of conjugates reflected by a decrease in the TBBPA-S/TBBPA-GA ratio. A limitation in the sulfation of TBBPA in vivo supports in vitro data defining TBBPA as an inhibitor of ES activity, thus providing further support that the proposed MoA occurs under conditions of high dose, chronic TBBPA administration to Wistar Han rats. Given that the uterine tumors observed in rats (250-1000 mg/kg-day) only occur at very high doses that perturb homeostatic control, it is unlikely such effects would occur in humans given that current TBBPA exposure levels are approximately eight orders of magnitude lower than these doses that are associated with exceeding the capacity of conjugation pathways in animal studies.

Keywords: Estrogen sulfotransferases; Glucuronidation; Liver; TBBPA; Uterus.

Fig. 1

Schematic to illustrate the competition of TBBPA with estradiol for sulfation and potentially glucuronidation in vivo. SULT1E1 and SULT1A1 are estrogen sulfotransferases with 3′-phosphoadenosine 5′-phosphosulfate (PAPS) being the cofactor that donates sulfate. Members of the glucuronyltransferase family, UGT1 have preferential recognition for estrogens and have been identified in human and rat uterine tissue , . UGT1A1 is identified as the isoform involved with the conjugation of estradiol and its metabolites using uridine diphosphate glucuronic acid (UDPGA) to donate glucuronic acid. UGT1A7 has broad specificity and glucuronidates both planar and nonplanar compounds, polycyclic aromatics and compounds with bulky side chain ring substitutions , .

Fig. 2

Representative extracted ion chromatogram (EIC) for TBBPA, TBBPA-S and TBBPA-GA conjugates quantitated in plasma, with similar scans observed in the liver and uterus. The x-axis is time (min) and the y-axis is intensity (CPS). * denotes putative conjugates, TBBPA-GA/S (diconjugate of glucuronide and sulfate) and TBBPA-S/S (sulfate diconjugate) as described in Section 2.5.

Fig. 3

Liver; the concentrations (ng/gram of tissue) of A TBBPA, B TBBPA-GA, and C TBBPA-S with dose at 4- and 8-h following dosing on day 28. Each bar represents the mean ± SD (n = 4–6 per group). (*) indicates a significant dose-related increasing trend (p < 0.01). (^) indicates a significant decrease in the analyte at each dose level at 8 h compared to the 4-h time point (p < 0.05).

Fig. 4

Liver; the concentrations (ng/gram of tissue) of TBBPA-GA and TBBPA-S at A 4 and B 8 h= following dosing on day 28. Each bar represents the mean ± SD (n = 4–6 per group). (^) indicates a significantly higher concentration of TBBPA-S compared to TBBPA-GA at each dose level (t-test, p < 0.05).

Fig. 5

Liver; ratio of TBBPA-GA (GA)/TBBPA (A and D), TBBPA-S (S)/TBBPA (B and E), and S/GA (C and F) with dose at 4- and 8-h following dosing on day 28. Each bar represents the mean ± SD (n = 4–6 per group). (*) indicates a significant dose-related increasing (A and D) or decreasing (C and F) trend (p < 0.05).

Fig. 6

Plasma; The concentration (ng/mL) of TBBPA at 4- and 8-h following A 7, B 14, and C 28-days of dosing. Each bar represents the mean ± SD (n = 4–6 per group). (*) indicates a significant dose-related increasing trend (p < 0.01). (^) indicates a significant decrease in TBBPA concentration at each dose level at 8-h compared to the 4-h time point (p < 0.05).

Fig. 7

Plasma; The concentration of TBBPA-S and TBBPA-GA at 4 h following 7, 14, and 28 days of consecutive dosing. Each bar represents the mean ± SD (n = 4–6 per group). (*) indicates a significant dose-related increasing trend in TBBPA-GA and TBBPA-S at A 7, B 14, and C 28-days of dosing (p < 0.01). (^) indicates a significantly higher concentration of TBBPA-GA compared to TBBPA-S at each dose level (p < 0.05).

Fig. 8

The concentration of A TBBPA-GA and B TBBPA-S in plasma 4 h following dosing on study day 7, 14, and 28. Each bar represents the mean ± SD (n = 4–6 per group). (^) indicates a higher concentration of conjugate at day 28 compared to day 7 or day 14 (p < 0.05).

Fig. 9

Plasma; ratio of TBBPA-GA (GA)/TBBPA (A and D), TBBPA-S (S)/TBBPA (B and E), and S/GA (C and F) with dose at the 4- and 8-h time points following 28 days of consecutive dosing. Each bar represents the mean ± SD (n = 4–6 per group). (*) indicates a significant dose-related increasing (A and D) (p < 0.01) or decreasing (C; p < 0.01) (F; p < 0.05) trend.

Fig. 10

Uterus; the concentrations (ng/g of tissue) of A TBBPA, B TBBPA-GA, and C TBBPA-S with dose at 4- and 8-h following dosing on day 28. Each bar represents the mean ± SD (n = 4–6 per group). (*) indicates a significant dose-related increasing trend (p < 0.01). (^) indicates a significantly higher concentration of analyte at 4 h compared to 8 h at each dose level (p < 0.05).

Fig. 11

Uterus; the concentration of TBBPA-GA and TBBPA-S at A 4 and B 8 h following 28 consecutive days of dosing. Each bar represents the mean ± SD (n = 4–6 per group). (^) indicates a higher concentration of TBBPA-GA compared to TBBPA-S at each respective dose (p ≤ 0.05).

Fig. 12

Uterus; ratio of TBBPA-GA (GA)/TBBPA (A and D), TBBPA-S (S)/TBBPA (B and E), and S/GA(C and F) with dose at 4 and 8 h following 28 days of consecutive dosing. Each bar represents the mean ± SD (n = 4–6 per group). (*) indicates a significant dose-related increasing (D) or decreasing (C) trend (p < 0.05).

Fig. 13

The concentration of TBBPA, TBBPA-GA, and TBBPA-S in A liver, B plasma, and C uterus tissue 4 h post dosing on day 28. Each symbol is the mean ± SD (n = 4–6 per group).

Fig. 14

The concentration of TBBPA, TBBPA-GA, and TBBPA-S in A liver, B plasma, and C uterus tissue 4 h post dosing on day 28. Each symbol is the mean ± SD (n = 4–6 per group). (*) indicates a significant dose-related decreasing trend (p < 0.05).