2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) disrupts vitamin A homeostasis in rodents : quantitative and mechanistic studies to support risk assessment

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure is well-known to disrupt Vitamin A (retinoid) homeostasis in rats, resulting in decreased hepatic storage, as well as increased metabolism and excretion of retinoids. The aims of this thesis were (1) to clarify the species and strain sensitivity of TCDDinduced retinoid disruption and (2) to identify cellular targets of TCDD-induced retinoid disruption.

Altered hepatic vitamin A gain was a sensitive measure of TCDD exposure in guinea pigs, rats, mice and hamsters, whereas increased renal vitamin A levels were observed only in the rat. Hepatic vitamin A gain was decreased 25% compared to control at estimated doses of 0.1, 1.1, 3.6 and 0.9 µg/kg bw respectively for guinea pigs, rats, mice and hamsters. In terms of sensitivity compared to CYP1A induction (measured as EROD activity), ED50 values were 0.2, 6.6, 6.5 and 13.7 µg/kg bw, in guinea pigs, rats, mice and hamsters respectively. In Long-Evans rats, long-term low-dose TCDD exposure disrupted both retinoid storage and metabolism of retinoic acid and retinoic acid metabolites in liver, kidney and plasma from doses as low as 1 ng/kg bw/day. Notably, 9-cis-4-oxo-13,14-dihydro-RA, a recently discovered retinoic acid metabolite, was decreased 60% in the liver at 1 ng/kg bw/day.

Because there are a large number of putative retinoid metabolizing enzymes that could ultimately increase retinoic acid oxidation and/or conjugation, the transcriptional response was investigated in rat liver using global expression methods. Single doses of 0.4 µg/kg bw TCDD resulted in greater than 2fold induction of genes coding for a battery of phase 1 and phase 11 metabolizing enzymes. No changes were observed in the expression of genes encoding putatively specific retinoid metabolizing enzymes that could explain the early and low-dose decreases in hepatic retinoid levels. However, at 40 µg/kg bw TCDD, decreased expression of cytochrome P450 7A1, short heterodimer partner (SHP; gene designation nr0b2), farnesoid X receptor (FXR), Ntcp, and S1c21a5 (oatp2) were observed and confirmed by RT-PCR analyses in independent rat liver samples. Altered expression of these genes implies major deregulation of cholesterol metabolism and bile acid synthesis and transport.

Employing another strategy, the role of various retinoid proteins in TCDD-induced retinoid disruption was investigated in transgenic mice lacking retinoid receptors or retinoid binding proteins. RXRbeta knockout mice did not show decreased hepatic vitamin A levels following TCDD exposure, suggesting that the role of RXRbeta in TCDD-induced altered hepatic vitamin A metabolism should be further investigated. On the other hand, results showed that mice deficient in CRBP I, CRABP-I and CRABP-II had significantly lower hepatic retinyl ester, retinol and retinoic acid levels compared to wildtype, and these mice were more sensitive to hepatic retinoid depletion than wildtype mice.

Therefore, together these studies have demonstrated altered hepatic vitamin A levels to be a sensitive marker of TCDD exposure in rodents. Long-term TCDD exposure altered tissue levels of retinoic acid and retinoic acid metabolites; 9-cis-4-oxo-13,14-dihydro-RA was a particularly sensitive indicator of TCDD exposure, thereby extending the database of low-dose dioxin effects. Studies in transgenic mice suggested that the roles of RXRbeta and CRBP I in TCDD-induced retinoid disruption warrant farther investigation.

List of scientific papers

I. Fletcher N, Hanberg A, Hakansson H (2001). "Hepatic vitamin a depletion is a sensitive marker of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in four rodent species. " Toxicol Sci 62(1): 166-75
https://pubmed.ncbi.nlm.nih.gov/11399804

II. Fletcher N, Giese N, Schmidt C, Stern N, Lind PM, Viluksela M, Tuomisto JT, Tuomisto J, Nau H, Hakansson H (2005). "Altered retinoid metabolism in female Long-Evans and Han/Wistar rats following long-term 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treatment. " Toxicol Sci 86(2): 264-72. Epub 2005 Apr 27
https://pubmed.ncbi.nlm.nih.gov/15858224

III. Sand S, Fletcher N, von Rosen D, Victorin K, Viluksela M, Tuomisto JT, Tuomisto J, Falk Filipsson A, Hakansson H (2005). "Quantitative and statistical analysis of differences in sensitivity between Long-Evans and Han/Wistar rats following long-term exposure to2,3,7,8-tetrachlorodibenzo-p-dioxin." (Manuscript)

IV. Hoegberg P, Schmidt CK, Fletcher N, Nilsson CB, Trossvik C, Gerlienke Schuur A, Brouwer A, Nau H, Ghyselinck NB, Chambon P, Hakansson H (2005). "Retinoid status and responsiveness to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking retinoid binding protein or retinoid receptor forms." Chem Biol Interact 156(1): 25-39
https://pubmed.ncbi.nlm.nih.gov/16109390

V. Fletcher N, Wahlstrom D, Lundberg R, Nilsson CB, Nilsson KC, Stockling K, Hellmold H, Hakansson H (2005). "2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters the mRNA expression of critical genes associated with cholesterol metabolism, bile acid biosynthesis, and bile transport in rat liver: a microarray study. " Toxicol Appl Pharmacol 207(1): 1-24
https://pubmed.ncbi.nlm.nih.gov/16054898