Table 3.

Gene symbols, gene descriptions, and pathways for ToxCast assay endpoints with exposure activity ratios from mixtures of pesticides and pesticide transformation products (EAR_Mixture) exceeding 10^–3 in at least two sites

Gene symbol	Orthologs	ToxCast assay endpoints	Chemicals	Method	Gene effects and functional annotations	Panther pathways	Adverse outcome pathways (AOP #)
ADORA1	H. sapiens D. rerio X. tropicalis	NVS_GPCR_hAdoRA1	Atrazine Deisopropylatrazine	Passive and discrete	Adenosine receptor belonging to the G‐protein coupled receptor family. Adenosine receptors regulate a diverse set of physiologic functions, including playing a role in fertilization	Two G‐protein signaling pathways (G i α/G s α and G q α/G o α mediated)
CYP1A1	H. sapiens D. rerio X. tropicalis	CLD_CYP1A1_24hr CLD_CYP1A1_6hr	Diuron Acetochlor 2,4‐D	Passive and discrete	The superfamily of cytochrome P450 (CYP) genes encode for monooxygenases, which catalyze many reactions involved in drug, steroid, and xenobiotic metabolism and synthesis of cholesterol, steroids, and other lipids. CYP3A4 is involved in metabolism of approximately half the drugs in use
CYP1A2	H. sapiens	CLD_CYP1A2_24hr CLD_CYP1A2_6hr	Diuron Atrazine Acetochlor Simazine 2,4‐D	Passive and discrete
CYP2A1		NVS_ADME_rCYP2A1	Diuron	Passive and discrete
CYP2B6	H. sapiens X. tropicalis	CLD_CYP2B6_24hr CLD_CYP2B6_48hr CLD_CYP2B6_6hr NVS_ADME_hCYP2B6	Diuron Atrazine Metolachlor Acetochlor Simazine Metalaxyl Ametryn Sulfentrazone 2,4‐D	Passive and discrete		Bupropion degradation
CYP3A4	H. sapiens X. tropicalis	CLD_CYP3A4_48hr CLD_CYP3A4_6hr	Metolachlor Metalaxyl Sulfentrazone 2,4‐D	Passive and discrete
CYP2J2	H. sapiens D. rerio X. tropicalis	NVS_ADME_hCYP2J2	Simazined	Discrete only
HMGCS2	H. sapiens	CLD_HMGCS2_24hr	2,4‐D	Passive and discrete	Codes for a mitochondrial enzyme that catalyzes the first reaction of ketogenesis, which provides energy during carbohydrate deprivation (i.e., fasting)		Mitochondrial dysfunction and neurotoxicity (3); calcium‐mediated neuronal reactive oxygen species production and energy imbalance (26); liver X receptor activation leading to liver steatosis (34); ionotropic glutamatergic receptors and cognition (48); nAChR activation contributes to colony death (77‐80, 87, 178); phospholipase A inhibitors lead to hepatotoxicity (130); lysosomal uptake induced liver fibrosis (144); vitamin K epoxide reductase inhibition resulting in coagulopathy (187); estrogen receptor activation leading to breast cancer (200); basal cytotoxicity (205); NADPH oxidase activation leading to reproductive failure (207); reactive oxygen species production leading to population decline (238)
NFE2L2	H. sapiens D. rerio X. tropicalis	ATG_NRF2_ARE_CIS_up	Metolachlor Acetochlor	Passive and discrete	Transcription factor that regulates genes containing antioxidant response elements. Many genes encode proteins involved in response to injury and inflammation and cellular response to oxidative stress		NFE2L2/FXR activation leading to hepatic steatosis (61)
NR1I2	H. sapiens D. rerio X. tropicalis	ATG_PXRE_CIS_up	Acetochlor Atrazine Metolachlor Metolachlor ESA Sulfentrazonep Bentazoned Azoxystrobind Simazined	Passive and discrete	Transcription factor regulating CYP3A4, which is involved in xenobiotic metabolism (see above)	Angiogenesis	Nuclear receptor induced thyroid hormone catabolism and developmental hearing loss (8); pentachlorophenol acute response by percellome (11); pregnane X receptor activation to steatosis (60)
PDE4A	H. sapiens D. rerio X. tropicalis	NVS_ENZ_hPDE4A1	Atrazine Simazine	Passive and discrete	Member of the cyclic nucleotide phosphodiesterase family, which hydrolyze the second messenger cAMP. Mediates a number of cellular responses to extracellular signals and plays a role in many important physiological processes
AR	H. sapiens D. rerio X. tropicalis	ACEA_AR_agonist_80hr	Triclopyrd	Discrete only	Steroid‐hormone activated transcription factor for androgen responsive genes. Androgens (e.g., testosterone) are involved in reproduction, sexual differentiation, and many important physiological processes	Gonadotropin releasing hormone receptor pathway	Androgen receptor agonism leading to reproductive dysfunction in fish (23) and hepatocellular adenomas and carcinomas in mice (117)
GLI3	H. sapiens D. rerio X. tropicalis	TOX21_SSH_3T3_GLI3_Antagonist	Atrazined	Discrete only	DNA‐binding transcription factors that mediates Sonic hedgehog signaling. It is also thought to play a role during embryogenesis	Hedgehog signaling pathway
PTGS2	H. sapiens D. rerio X. tropicalis	NVS_ENZ_oCOX2	Atrazined	Discrete only	Codes for a cyclooxygenase, which is a key enzyme in prostaglandin biosynthesis and acts both as a dioxygenase and as a peroxidase. Involved in inflammation and mitogenesis	Inflammation mediated by chemokine and cytokine signaling pathway; toll receptor, endothelin, and CCKR signaling pathways	Cyclooxygenase inhibition leading to reproductive failure or dysfunction (28, 63, 100‐103); gastric ulcer formation (217), aryl hydrocarbon receptor mediated mortality, via COX‐2 (21)

Chemicals were detected in polar organic chemical integrated samplers (POCIS) or discrete water samples in 15 Great Lakes tributaries in June–July 2016. Listed chemicals contributed at least 10% of the EAR_Mixture value in at least two samples. Suffixes after chemical names represent chemicals that only contributed to an EAR_Mixture threshold exceedance in discrete (“d”) or passive (“p”) samples. Gene effects, functional annotations, and pathways are summarized based on the Database for Annotation, Visualization, and Integrated Discovery (DAVID; https://david.ncifcrf.gov), Panther Classification System (http://www.pantherdb.org), and AOP‐wiki (https://aopwiki.org/).

2,4‐D = 2,4‐dichlorophenoxyacetic acid; H. sapiens = humans; D, rerio = zebrafish; X. tropicalis = Western clawed frog.