Systems Toxicology of Male Reproductive Development: Profiling 774 Chemicals for Molecular Targets and Adverse Outcomes

Abstract

Background: Trends in male reproductive health have been reported for increased rates of testicular germ cell tumors, low semen quality, cryptorchidism, and hypospadias, which have been associated with prenatal environmental chemical exposure based on human and animal studies.

Objective: In the present study we aimed to identify significant correlations between environmental chemicals, molecular targets, and adverse outcomes across a broad chemical landscape with emphasis on developmental toxicity of the male reproductive system.

Methods: We used U.S. EPA's animal study database (ToxRefDB) and a comprehensive literature analysis to identify 774 chemicals that have been evaluated for adverse effects on male reproductive parameters, and then used U.S. EPA's in vitro high-throughput screening (HTS) database (ToxCastDB) to profile their bioactivity across approximately 800 molecular and cellular features.

Results: A phenotypic hierarchy of testicular atrophy, sperm effects, tumors, and malformations, a composite resembling the human testicular dysgenesis syndrome (TDS) hypothesis, was observed in 281 chemicals. A subset of 54 chemicals with male developmental consequences had in vitro bioactivity on molecular targets that could be condensed into 156 gene annotations in a bipartite network.

Conclusion: Computational modeling of available in vivo and in vitro data for chemicals that produce adverse effects on male reproductive end points revealed a phenotypic hierarchy across animal studies consistent with the human TDS hypothesis. We confirmed the known role of estrogen and androgen signaling pathways in rodent TDS, and importantly, broadened the list of molecular targets to include retinoic acid signaling, vascular remodeling proteins, G-protein coupled receptors (GPCRs), and cytochrome P450s.

Citation: Leung MC, Phuong J, Baker NC, Sipes NS, Klinefelter GR, Martin MT, McLaurin KW, Setzer RW, Darney SP, Judson RS, Knudsen TB. 2016. Systems toxicology of male reproductive development: profiling 774 chemicals for molecular targets and adverse outcomes. Environ Health Perspect 124:1050-1061; http://dx.doi.org/10.1289/ehp.1510385.

Figure 1

In vivo phenotypic analysis: chemical coverage and workflow. (A) Experimental protocols in general follow EPA Health Effects Test Guidelines OPPTS ^a870.3700, ^b870.3100, 870.4100, 870.4200, 870.4300, ^c870.3800, and ^d870.3550. Test strains include ^eAlderly Park, CD, Fischer 344, Long-Evans, Sprague-Dawley, and Wistar for rats; ^fB6C3F1, CD, and Swiss for mice; and ^gChinchilla, Dutch, Himalayan, and New Zealand White for rabbits. The prenatal developmental study type (OPPTS 870.3700 or equivalent) consisted mostly of rabbit (527) and rat studies (731), whereas both one-generational and multigenerational reproductive study types (OPPTS 870.3800, 870.3550, or equivalent) consisted primarily of rat studies (104 and 455, respectively). The ToxRefDB subchronic and chronic study types (OPPT 870.3100, 870.4100, 870.4200, 870.4300, or equivalent) consisted mostly of mouse (568) and rat studies (984). (B) Workflow for data-mining ToxRefDB.

Figure 2

In vitro network analysis: workflow and evidence of effects. (A) Workflow for data-mining ToxCastDB. (B) A total of 2,099 articles were co-annotated with 88 of the 212 male reproductive toxicants in ToxRefDB (Figure 5), including 15 chemicals co-annotated with cryptorchidism, hypospadias, and urogenital abnormalities. The citations were listed in Excel File S1.

Figure 3

Co-occurrence of male reproductive effects in ToxRefDB. A chemical was found to produce a male reproductive effect if it was associated with any of the listed search terms in Excel File S2 in ToxRefDB, regardless of dosage. Chemicals may be associated with multiple effects. Chemicals were ranked in the order of association with the least common effect (i.e., malformations), followed by the next (i.e., testicular tumors), and so on.

Figure 4

Phenotypic hierarchy of 63 male reproductive developmental toxicants in ToxRefDB. An unsupervised two-directional heat map was constructed based on hierarchical clustering of 63 chemicals by their effect scores for malformations, sperm effects, histological effects, and relative weight change in male reproductive tissues, using Euclidean distance for measure and Ward’s method for linkage analysis.

Figure 5

Unsupervised two-directional heatmap from hierarchical clustering of 212 male reproductive toxicants in ToxCastDB. Data for 212 male reproductive toxicants in ToxCastDB were used to construct a heat map to visualize the complex chemical–effects relationships on 213 molecular targets. The heat map was generated with the “gplot” package in R version 3.1.0, using Euclidean distance for measure and Ward’s method for linkage analysis. The intensity of blue color indicated the value of gene score.

Figure 6

Quantitative bipartite network of 54 male reproductive developmental toxicants and 156 molecular targets in ToxCastDB. A bipartite network consisting of 54 male reproductive developmental toxicants (source nodes) and 156 molecular targets (target nodes; bold) was constructed with ForceAtlas layout using Gephi version 0.8.2 beta, with 5 predicted modes of action in different colors. The thickness of each chemical-to-molecular target arrow was proportional to the corresponding gene score.