The role of the endoplasmic reticulum in the metabolism of "xenobiotica"

Short-chain dehydrogenase/reductase (SDR) enzymes play a key role in the metabolism of steroids, fatty acids, prostaglandins and xenobiotic chemicals. This thesis investigated the role of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in the metabolism of xenobiotics. It further addressed species-specific differences of the inhibition of 11β-HSD1 and some related microsomal SDRs by xenobiotics. 11β-HSD1 catalyzes the conversion of the inactive glucocorticoids cortisone and 11-dehydrocorticosterone to the active cortisol and corticosterone, respectively. Recently, studies using microsomes and the unspecific inhibitor glycyrrhetinic acid (GA) suggested that 11β-HSD1 metabolizes the antidepressant drug bupropion to erythrohydrobupropion (EHB) and threohydrobupropion (THB), and the fungicide triadimefon to triadimenol. In the present work, the role of human 11β-HSD1 in the reduction of triadimefon and bupropion was studied in vitro using the recombinant 11β-HSD1 enzyme, a selective 11β-HSD1 inhibitor and microsomes from liver-specific 11β-HSD1 knock-out mice. Activities were determined using microsomes from human, rat and mouse liver to assess species-specific differences. The results suggest that 11β-HSD1 is the major enzyme responsible for triadimenol formation. Surprisingly, 11β-HSD1 exclusively formed THB but not EHB from bupropion. Due to lower activities of rat and mouse 11β-HSD1 towards these xenobiotics, they are models of limited value to study 11β-HSD1-dependent metabolism of bupropion and triadimefon. A comparison of IC50 values suggests that exposure to these compounds is unlikely to impair the 11β-HSD1-dependent activation of glucocorticoids. In contrast, elevated glucocorticoids during stress or upon pharmacological administration are likely to inhibit 11β-HSD1-dependent metabolism of these xenobiotics.
11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) catalyzes the conversion of the active glucocorticoid cortisol to the inactive cortisone. It has been reported that some organotins and dithiocarbamates are potent inhibitors of human 11β-HSD2. We found that the zebrafish enzyme is not inhibited by these organotins. Furthermore, the dithiocarbamate thiram showed a reduced inhibitory effect on zebrafish 11β-HSD2 compared with the human enzyme. Sequence comparison revealed the presence of an alanine at position 253 on zebrafish 11β-HSD2, corresponding to cysteine-264 in the substrate binding pocket of the human enzyme. Substitution of alanine-253 by cysteine resulted in a more than 10-fold increased sensitivity of zebrafish 11β-HSD2 to thiram. These findings are important, as the zebrafish is a widely used model in ecotoxicology, and 11β-HSD2 is catalyzing the conversion of 11β-hydroxytestosterone to 11-ketotestosterone, the main androgen in fish.
The gene encoding 11β-HSD1 in zebrafish is absent. Therefore, the mechanism how the ratio between active and inactive glucocorticoids is controlled in fish is unclear. It was suggested by a phylogenetic analysis that one of the two ancestors of 11β-HSD1 might reduce cortisone to cortisol. These ancestors are 11β-HSD3a and 11β-HSD3b. We cloned both zebrafish cDNAs and tested them for 11-oxosteroid reductase activity. Furthermore, we examined the metabolism of cortisone in zebrafish microsomes. Our results indicate that the 11-oxosteroid reductase activity is completely absent in zebrafish.
17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) catalyzes the conversion of Δ4-androstenedione to testosterone. We reported earlier that some UV filters inhibit the human enzyme. We tested whether these UV filters also inhibit the zebrafish enzyme. We found interesting species-specific differences of the inhibitory potential of UV filters on human and zebrafish 17β-HSD3. Furthermore, we were able to show additive inhibitory effects of UV filter mixtures and bioaccumulation of UV filters in vitro.
In conclusion, the results presented in this thesis significantly extend the knowledge of the role of 11β-HSD1 in the metabolism of xenobiotics. The thesis further emphasizes the importance of considering species-specific differences when trying to extrapolate effects of xenobiotics observed in animal models to humans.