title: A molecular and phenotypic integrative approach to identify a no-effect dose level for antiandrogen-induced testicular toxicity.
abstract: The safety assessment of chemicals for humans relies on identifying no-observed adverse effect levels (NOAELs) in animal toxicity studies using standard methods. With the advent of high information content technologies, especially microarrays, it is pertinent to determine the impact of molecular data on the NOAELs. Consequently, we conducted an integrative study to identify a no-transcriptomic effect dose using microarray analyses coupled with quantitative reverse transcriptase PCR (RT-qPCR) and determined how this correlated with the NOAEL. We assessed the testicular effects of the antiandrogen, flutamide (FM), in a rat 28-day toxicity study using doses of 0.2-30 mg/kg/day. Plasma testosterone levels and testicular histopathology indicated a NOAEL of 1 mg/kg/day. A no-effect dose of 0.2 mg/kg/day was established based on molecular data relevant to the phenotypic changes. We observed differential gene expression starting from 1 mg/kg/day and a deregulation of more than 1500 genes at 30 mg/kg/day. Dose-related changes were identified for the major pathways (e.g., fatty acid metabolism) associated with the testicular lesion (Leydig cell hyperplasia) that were confirmed by RT-qPCR. These data, along with protein accumulation profiles and FM metabolite concentrations in testis, supported the no-effect dose of 0.2 mg/kg/day. Furthermore, the microarray data indicated a dose-dependent change in the fatty acid catabolism pathway, a biological process described for the first time to be affected by FM in testicular tissue. In conclusion, the present data indicate the existence of a transcriptomic threshold, which must be exceeded to progress from a normal state to an adaptative state and subsequently to adverse toxicity.
authors.affiliation: Department of Research Toxicology, Université Paris-Sud, INSERM UMR 996, Chatenay-Malabry, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Ludwig
authors.forename: Helen
authors.initials: H
authors.lastname: Tinwell
authors.forename: Frédéric
authors.initials: F
authors.lastname: Schorsch
authors.forename: Christel
authors.initials: C
authors.lastname: Cavaillé
authors.forename: Marc
authors.initials: M
authors.lastname: Pallardy
authors.forename: David
authors.initials: D
authors.lastname: Rouquié
authors.forename: Rémi
authors.initials: R
authors.lastname: Bars
mesh_terms: D000726:Androgen Antagonists / Q000633:toxicity*; D000818:Animals; D004305:Dose-Response Relationship, Drug; D005485:Flutamide / Q000633:toxicity*; D005786:Gene Expression Regulation; D007985:Leydig Cells / Q000187:drug effects* / Q000473:pathology; D050356:Lipid Metabolism / Q000187:drug effects; D008297:Male; D046228:Microarray Analysis / Q000379:methods; D019055:No-Observed-Adverse-Effect Level; D010641:Phenotype; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013733:Testicular Diseases / Q000139:chemically induced / Q000473:pathology*; D013739:Testosterone / Q000097:blood; D018675:Toxicity Tests / Q000379:methods; D059467:Transcriptome
chemical_list: D000726:Androgen Antagonists; D011506:Proteins; D013739:Testosterone; D005485:Flutamide