3Rs‐friendly approach to exogenous metabolic activation that supports high‐throughput genetic toxicology testing

MultiFlow® DNA Damage—p53, γH2AX, Phospho‐Histone H3 is a miniaturized, flow cytometry‐based assay that provides genotoxic mode of action information by distinguishing clastogens, aneugens, and nongenotoxicants. Work to date has focused on the p53‐competent human cell line TK6. While mammalian cell genotoxicity assays typically supply exogenous metabolic activation in the form of concentrated rat liver S9, this is a less‐than‐ideal approach for several reasons, including 3Rs considerations. Here, we describe our experiences with low concentration S9 and saturating co‐factors which were allowed to remain in contact with cells and test chemicals for 24 continuous hours. We exposed TK6 cells in 96‐well plates to each of 15 reference chemicals over a range of concentrations, both in the presence and absence of 0.25% v/v phenobarbital/β‐naphthoflavone‐induced rat liver S9. After 4 and 24 hr of treatment cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk‐away flow cytometric data acquisition. PROAST benchmark dose (BMD) modeling was used to characterize the resulting dose–response curves. For each of the 8 reference pro‐genotoxicants studied, relative nuclei count, γH2AX, and/or p53 biomarker BMD values were order(s) of magnitude lower for 0.25% S9 conditions compared to 0% S9. Conversely, several of the direct‐acting reference chemicals exhibited appreciably lower cytotoxicity and/or genotoxicity BMD values in the presence of S9 (eg, resorcinol). These results prove the efficacy of the low concentration S9 system, and indicate that an efficient and highly scalable multiplexed assay can effectively identify chemicals that require bioactivation to exert their genotoxic effects.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154945/1/em22361_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154945/2/em22361.pd

Assessment of Systemic Genetic Damage in Pediatric Inflammatory Bowel Disease.

The etiology of distal site cancers in inflammatory bowel disease (IBD) is not well understood and requires further study. We investigated whether pediatric IBD patients' blood cells exhibit elevated levels of genomic damage by measuring the frequency of mutant phenotype (CD59-/CD55-) reticulocytes (MUT RET) as a reporter of PIG-A mutation, and the frequency of micronucleated reticulocytes (MN-RET) as an indicator of chromosomal damage. IBD patients (n = 18 new onset disease, 46 established disease) were compared to age-matched controls (constipation or irritable bowel syndrome patients from the same clinic, n = 30) and young healthy adults age 19 - 24 (n = 25). IBD patients showed no indication of elevated MUT RET relative to controls (mean ± std. dev. = 3.1 ± 2.3 x 10-6 versus 3.6 ± 5.6 x 10-6 , respectively). In contrast, of 59 IBD patients where %MN-RET measurements were obtained, 10 exceeded the upper bound 90% tolerance interval derived from control subjects (i.e., 0.42%). Furthermore, each of the 10 IBD patients with elevated MN-RET had established disease (10/42), none were new onset (0/17) (p = 0.049). Interestingly, each of the subjects with increased chromosomal damage was receiving anti-TNF based monotherapy at the time blood was collected (10/10, 100%), whereas this therapy was less common (20/32, 63%) among patients that exhibited ≤ 0.42% MN-RET (p = 0.040). The results clearly indicate the need for further work to understand whether the results presented herein are reproducible, and if so, to elucidate the causative factor(s) responsible for elevated MN-RET frequencies in some IBD patients

Dioxin Causes Ventral Prostate Agenesis by Disrupting Dorsoventral Patterning in Developing Mouse Prostate

Prostate ductal development is initiated by androgen-dependent signals in fetal urogenital sinus (UGS) mesenchyme that stimulate prostatic bud formation in UGS epithelium. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, 5 μg/kg maternal dose) inhibited ventral and dorsolateral but not anterior prostatic budding. We sought to determine which stage of budding, specification or initiation, was inhibited. Ventral prostatic bud formation was maximally inhibited when TCDD exposure spanned E15.5–16.5 and dorsolateral prostatic bud formation when it spanned E14.5–15.5. Because ventral and dorsolateral buds are specified at these times, TCDD impaired bud specification. We hypothesized that TCDD inhibited ventral bud specification by forming a continuous smooth muscle barrier between UGS mesenchyme and epithelium in the ventral prostatic UGS region, blocking mesenchymal-epithelial signaling, but no such barrier was found. We hypothesized that increased aryl hydrocarbon receptor (AHR) signaling in ventral and dorsolateral UGS increased their sensitivity to TCDD, but levels of AHR nuclear translocator (ARNT) protein, Ahr mRNA, and AHR-dependent gene expression were not higher than in anterior UGS where budding was unaffected. However, we identified overlapping expression of Ahr, ARNT, and AHR-induced transcripts in the periprostatic mesenchyme which intimately contacts UGS epithelium where buds are specified. This was considered the putative TCDD site of action in the UGS for inhibition of ventral and dorsolateral prostatic bud specification. Thus, hyperactivation of AHR signaling appears to disrupt dorsoventral patterning of the UGS, reprogramming where prostatic buds are specified, and prostate lobes are formed. Disrupted axial patterning provides a new paradigm for understanding how in utero TCDD exposure causes ventral prostate agenesis and may shed light on how TCDD impairs development of other organs