Understanding The Mode Of Action Of A Food Contaminant Acetamide

September 20, 2022

The Use Of Transcriptomics To Support Risk Assessment For The Food Contaminant Acetamide*

Purpose

As a byproduct of food processing, acetamide is a simple amide which occurs in milk, egg, beef, and roasted coffee bean products. An ammonia fiber expansion (AFEXTM) process produces highly digestible and inexpensive commercial animal feed from crop residue (e.g., corn stalks). This benefits ruminant livestock health and productivity but produces acetamide detectable in animal milk, for example. Acetamide is classified as a Group 2B (“possible human carcinogen”) based on liver tumors in rats. However, little is known of its cellular mode of action (MoA), nor is there significant human exposure data available.

Method

  • Short term (7 and 28 day) in vivo rat whole transcriptome studies were completed to investigate cellular MoA of acetamide, and to use transcriptomic Benchmark Dose (BMD) analyses to help determine safe exposure levels.
  • Whole transcriptome gene expression data was obtained by next generation sequence (RNA-Seq) analysis of rat livers following exposure to five concentrations of acetamide.
  • Differential Gene Expression (DGE) analyses produced lists of genes altered by exposure to acetamide relative to untreated animals for the following MoA analyses and point of departure (POD) derivations:
    • Conventional ontology enrichment analyses to find cellular pathways over-represented with genes whose transcription levels were altered by exposure to acetamide.
    • Gene set enrichment analyses using gene lists specifically associated with DNA transcription factors and their regulated or co-regulated gene networks.
    • BMD estimations from mathematical dose response curves fit to individual gene’s expression data based on current consensus best practices. Combining these gene-based BMD methods with ontology enrichment analysis allowed us to derive BMD values for cellular pathways as well as individual genes.

Results

  • Comparison of acetamide MoA to other known rodent cancer-inducing chemicals indicated a novel MoA for acetamide in rodents.
  • Transcriptomic derived POD estimates—both those derived from pathways and from gene lists (i.e., pathway agnostic)—proved highly consistent across numerous proposed alternative approaches to deriving a single value from transcriptomic BMDs.
  • We found high concordance in transcriptional derived POD estimates and those from more traditionally used apical endpoints such as Ki67 cell proliferation index derived from the same samples.
  • We determined a lowest POD of 190mg/kg-d for acetamide based on cellular proliferation (Ki67) index as this was the most sensitive estimate overall.

Client Value

  • This POD value may serve as a reference point for a formal human risk assessment.
  • The consistency of PODs derived for both transcriptional and apical markers support the concept that transcriptomic derived PODs can be used to support risk assessment.

* Nault R, Bals B, Teymouri F, Black MB, Andersen ME, McMullen PD, Krishnan S, Kuravadi N, Paul N, Kumar S, Kannan K, Jayachandra KC, Alagappan L, Patel BD, Bogen KT, Gollapudi BB, Klaunig JE, Zacharewski TR, Bringi V. A toxicogenomic approach for the risk assessment of the food contaminant acetamide. Toxicol Appl Pharmacol. 2020 Feb 1;388:114872. doi: 10.1016/j.taap.2019.114872. Epub 2019 Dec 24. PMID: 31881176; PMCID: PMC7014822.