Alternative Multiorgan Initiation-Promotion Assay for Chemical Carcinogenesis in the Wistar Rat

Toxicol Pathol. 2016 Dec;44(8):1146-1159. doi: 10.1177/0192623316678931.


The medium-term multiorgan initiation-promotion chemical bioassay (diethylnitrosamine, methyl-nitrosourea, butyl-hydroxybutylnitrosamine, dihydroxypropylnitrosamine, dimethylhydrazine [DMBDD]) with the Fischer 344 rat was proposed as an alternative to the conventional 2-year carcinogenesis bioassay for regulatory purposes. The acronym DMBDD stands for the names of five genotoxic agents used for initiation of multiorgan carcinogenesis. The Brazilian Agency for the Environment officially recognized a variation of this assay (DMBDDb) as a valid method to assess the carcinogenic potential of agrochemicals. Different from the original protocol, this DMBDDb is 30-week long, uses Wistar rats and two positive control groups exposed to carcinogenesis promoters sodium phenobarbital (PB) or 2-acetylaminofluorene (2-AAF). This report presents the experience of an academic laboratory with the DMBDDb assay and contributes to the establishment of this alternative DMBDD bioassay in a different rat strain. Frequent lesions observed in positive groups to evaluate the promoting potential of pesticides and the immunohistochemical expressions of liver cytochrome P450 (CYP) 2B1/2B2 and CYP1A2 enzymes were assessed. Commonly affected organs were liver, kidney, intestines, urinary bladder, and thyroid. PB promoting activity was less evident than that of 2-AAF, especially in males. This study provides a repository of characteristic lesions occurring in positive control animals submitted to a modified alternative 2-stage multiorgan protocol for carcinogenesis in Wistar rat.

Keywords: DMBDD bioassay; Wistar rats; agrochemicals; carcinogenesis; histological lesions; initiation–promotion; multiorgan assay; regulatory toxicology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 2-Acetylaminofluorene / toxicity*
  • Animals
  • Aryl Hydrocarbon Hydroxylases / biosynthesis
  • Biological Assay
  • Carcinogenicity Tests / methods*
  • Carcinogens / toxicity*
  • Cytochrome P-450 CYP1A2 / biosynthesis
  • Cytochrome P-450 CYP2B1 / biosynthesis
  • Female
  • Liver / drug effects
  • Liver / enzymology
  • Liver / pathology
  • Male
  • Neoplasms, Experimental / chemically induced*
  • Neoplasms, Experimental / enzymology
  • Organ Size / drug effects
  • Organ Specificity
  • Phenobarbital / toxicity*
  • Precancerous Conditions / chemically induced*
  • Precancerous Conditions / enzymology
  • Rats, Wistar
  • Steroid Hydroxylases / biosynthesis


  • Carcinogens
  • 2-Acetylaminofluorene
  • Steroid Hydroxylases
  • Aryl Hydrocarbon Hydroxylases
  • Cyp1a2 protein, rat
  • Cytochrome P-450 CYP1A2
  • Cytochrome P-450 CYP2B1
  • steroid 16-beta-hydroxylase
  • Phenobarbital