Aryl hydrocarbon receptor activity in human breast milk and cryptorchidism: A case-control study within the prospective Norwegian HUMIS cohort.

BACKGROUND: The etiology of cryptorchidism remains poorly understood. Endocrine disrupting chemicals can impact estrogen signaling by interacting with aryl hydrocarbon receptor (AhR) activity. OBJECTIVE: To evaluate whether AhR activity in breast milk samples is associated with cryptorchidism. METHOD: We conducted a case-control study based on 199 mother-child pairs (n = 91 cases/108 controls) selected from the Norwegian Human Milk Study (2002-2009). We defined cases for cryptorchidism based on maternal reports at 1-, 6-, 12-, and 24- months after birth. Chemically- and biologically stable AhR activity (pg 2,3,7,8-TCDD equivalent (TEQ)/g lipid) was determined by DR- CALUX® assay in the mothers' milk collected at a median of 33 (10th-90th percentile: 18-57) days after delivery. We used multivariate logistic regression to compare AhR activity levels between cases and controls, and linear regression separately, to establish the relationship with the presence of 27 potential EDCs measured in breast milk and AhR activity. RESULTS: The average estimated daily intake (EDI) of dioxin and (dioxin-like (dl)-compounds via breast milk is 33.7 ± 17.9 pg TEQ/kg bodyweight per day among Norwegian children. There were no significant differences in AhR activation in breast milk samples between cases with cryptorchidism and controls. Among the 27 chemicals measured in breast milk, AhR activity was (borderline) significantly associated with all dl-PCBs, three non-dioxin-like (ndl)-PCBs (PCB-74, PCB-180, PCB-194) and two organochlorine pesticides (OCPs; HCB, ß-HCH). No associations between AhR activity and brominated flame retardants (PBDEs) or poly- and perfluoroalkyl substances (PFASs). CONCLUSION: No association between AhR activity and cryptorchidism was found among Norwegian boys. The average EDI of dioxin and dl-compounds in exclusively breastfed Norwegian infants remains above the safety threshold and, therefore requires further reduction measures. Consistent with a possible role in the observed AhR activity, all dl-PCBs were associated with AhR activity whereas the association was null for either PBDEs or PFASs.

Development of a panel of high-throughput reporter-gene assays to detect genotoxicity and oxidative stress.

The lack of toxicological information on many of the compounds that humans use or are exposed to, intentionally or unintentionally, poses a big problem in risk assessment. To fill this data gap, more emphasis is given to fast in vitro screening tools that can add toxicologically relevant information regarding the mode(s) of action via which compounds can elicit adverse effects, including genotoxic effects. By use of bioassays that can monitor the activation of specific cellular signalling pathways, many compounds can be screened in a high-throughput manner. We have developed two new specific reporter-gene assays that can monitor the effects of compounds on two pathways of interest: the p53 pathway (p53 CALUX) for genotoxicity and the Nrf2 pathway (Nrf2 CALUX) for oxidative stress. To exclude non-specific effects by compounds influencing the luciferase reporter-gene expression non-specifically, a third assay was developed to monitor changes in luciferase expression by compounds in general (Cytotox CALUX). To facilitate interpretation of the data and to avoid artefacts, all three reporter-gene assays used simple and defined reporter genes and a similar cellular basis, the human U2OS cell line. The three cell lines were validated with a range of reference compounds including genotoxic and non-genotoxic agents. The sensitivity (95%) and specificity (85%) of the p53 CALUX was high, showing that the assay is able to identify various types of genotoxic compound, while avoiding the detection of false positives. The Nrf2 CALUX showed specific responses to oxidants only, enabling the identification of compounds that elicit part of their genotoxicity via oxidative stress. All reporter-gene assays can be used in a high-throughput screening format and can be supplemented with other U2OS-based reporter-gene assays that can profile nuclear receptor activity, and several other signalling pathways.