Effects of Prenatal Bisphenol A Exposure on Adrenal Gland Development and Steroidogenic Function

Developmental exposure to bisphenol A (BPA), a ubiquitous endocrine disrupting chemical, is associated with organ dysfunction and diseases in adulthood. However, little is known about its effects on the adrenal glands. Therefore, this thesis addresses this important question using both in vivo and in vitro approaches. BPA at environmentally relevant doses was administrated via diet to pregnant mice from embryonic day 7.5 to birth, following which mice were switched to a standard chow. At two months postnatally, adrenal glands and blood samples were collected from adult mouse offspring for structural and functional analysis. I found that: (a) BPA increased adrenal gland weight as well as plasma corticosterone levels; (b) BPA did not alter plasma levels of ACTH; and (c) BPA stimulated expression of the two key steroidogenic factors, steroidogenic acute regulatory protein (StAR) and cyp11A1 in female but not male offspring. To determine the molecular mechanisms underlying the BPA-induced StAR expression, I used human fetal adrenal cortical H295A cells as an in vitro model system, and showed that BPA increased StAR protein expression likely through an estrogen receptor (ER)-mediated mechanism independent of StAR gene transcription, translation and protein half-life. I then investigated the molecular mechanisms underlying the BPA-induced increase in adrenal gland weight using the same in vitro model system. I demonstrated that (a) BPA increased cell number and protein levels of the three universal markers of proliferation (proliferating cell nuclear antigen (PCNA), cyclin D1 and D2, as well as sonic hedgehog (shh) and its key transcriptional regulator Gli1; (b) cyclopamine, a shh pathway inhibitor, blocked these stimulatory effects of BPA on cell proliferation; (c) BPA increased the nuclear translocation of ERβ; and (d) the ERb-specific agonist DPN mimicked while the ERb antagonist PHTPP abrogated the stimulatory effects of BPA on cell proliferation, and prevented BPA-induced activation of the shh signaling. Taken together, these findings demonstrate that developmental exposure to BPA adversely affects adrenal gland development and steroidogenic function in adult mouse offspring. Furthermore, they reveal novel molecular signaling mechanisms of BPA actions in regulating adrenal steroidogenic function and adrenal cortical cell proliferation

Impact of pretreatment dihydropyrimidine dehydrogenase genotype-guided fluoropyrimidine dosing on chemotherapy associated adverse events

Consensus guidelines exist for genotype-guided fluoropyrimidine dosing based on variation in the gene dihydropyrimidine dehydrogenase (DPYD). However, these guidelines have not been widely implemented in North America and most studies of pretreatment DPYD screening have been conducted in Europe. Given regional differences in treatment practices and rates of adverse events (AEs), we investigated the impact of pretreatment DPYD genotyping on AEs in a Canadian context. Patients referred for DPYD genotyping prior to fluoropyrimidine treatment were enrolled from December 2013 through November 2019 and followed until completion of fluoropyrimidine treatment. Patients were genotyped for DPYD c.1905+1G\u3eA, c.2846A\u3eT, c.1679T\u3eG, and c.1236G\u3eA. Genotype-guided dosing recommendations were informed by Clinical Pharmacogenetics Implementation Consortium guidelines. The primary outcome was the proportion of patients who experienced a severe fluoropyrimidine-related AE (grade ≥3, Common Terminology Criteria for Adverse Events version 5.0). Secondary outcomes included early severe AEs, severe AEs by toxicity category, discontinuation of fluoropyrimidine treatment due to AEs, and fluoropyrimidine-related death. Among 1394 patients, mean (SD) age was 64 (12) years, 764 (54.8%) were men, and 47 (3.4%) were DPYD variant carriers treated with dose reduction. Eleven variant carriers (23%) and 418 (31.0%) noncarriers experienced a severe fluoropyrimidine-related AE (p = 0.265). Six carriers (15%) and 284 noncarriers (21.1%) experienced early severe fluoropyrimidine-related AEs (p = 0.167). DPYD variant carriers treated with genotype-guided dosing did not experience an increased risk for severe AEs. Our data support a role for DPYD genotyping in the use of fluoropyrimidines in North America

Fexofenadine and rosuvastatin pharmacokinetics in mice with targeted disruption of organic anion transporting polypeptide 2b1

Organic anion transporting polypeptide 2B1 (OATP2B1) is a widely expressed membrane transporter with diverse substrate specificity. In vitro and clinical studies suggest a role for intestinal OATP2B1 in the oral absorption of medications. Moreover, OATP2B1 is highly expressed in hepatocytes where it is thought to promote liver drug clearance. However, until now, a shortcoming of studies implicating OATP2B1 in drug disposition has been a lack of in vivomodels.Here,we report the development of a knockout (KO) mousemodel with targeted, global disruption of the Slco2b1 gene to examine the disposition of two confirmed mOATP2B1 substrates, namely, fexofenadine and rosuvastatin. The plasma pharmacokinetics of intravenously administered fexofenadine was not different between KO and wildtype (WT) mice. However, after oral fexofenadine administration, KO mice had 70% and 41% lower maximal plasma concentration (Cmax) and area under the plasmaconcentration-timecurve (AUC0-last) than WT mice, respectively. In WT mice, coadministration of fexofenadine with grapefruit juice (GFJ) or apple juice (AJ) was associated with reduced Cmax by 80% and 88%, respectively, while the AUC0-last values were lower by 35% and 70%, respectively. In KO mice, AJ coadministration reduced oral fexofenadine Cmax and AUC0-last values by 67% and 59%, respectively, while GFJ had no effects. Intravenous and oral rosuvastatin pharmacokinetics were similar among WT and KO mice. We conclude that intestinal OATP2B1 is a determinant of oral fexofenadine absorption, as well as a target for fruit juice interactions. OATP2B1 does not significantly influence rosuvastatin disposition in mice

<i>DPYD</i> Exon 4 Deletion Associated with Fluoropyrimidine Toxicity and Importance of Copy Number Variation

Fluoropyrimidine chemotherapy is associated with interpatient variability in toxicity. A major contributor to unpredictable and severe toxicity relates to single nucleotide variation (SNV) in dihydropyrimidine dehydrogenase (DPYD), the rate-limiting fluoropyrimidine metabolizing enzyme. In addition to SNVs, a study of Finnish patients suggested that a DPYD exon 4 deletion was observed in their population. To better understand the potential generalizability of such findings, we investigated the presence of this exon 4 deletion in our Canadian patient population, using a TaqMan assay. We selected 125 patients who experienced severe fluoropyrimidine-associated toxicity, and 125 matched controls. One patient in the severe toxicity group harbored a haploid DPYD exon 4 deletion, and required a 35% dose reduction after their first fluoropyrimidine treatment cycle due to toxicity and required an additional 30% dose reduction before tolerating treatment. The predicted allele frequency was 0.2% in our cohort, much lower than the 2.4% previously reported. We also carried out a literature review of copy number variation (CNV) in the DPYD gene, beyond fluoropyrimidine toxicity and show that various types of CNV in DPYD are present in the population. Taken together, our findings suggest that CNV in DPYD may be an underappreciated determinant of DPYD-mediated fluoropyrimidine toxicity