Validation study of the combined repeated-dose toxicity and genotoxicity assay using gpt delta rats

Abstract

Transgenic rodents carrying reporter genes to detect organ-specific in vivo genetic alterations are useful for risk assessment of genotoxicity that causes cancer. Thus, the Organization for Economic Co-operation and Development has established the guideline for genotoxicity tests using transgenic animals, which may be combined with repeated-dose toxicity studies. Here, we provide evidence to support equivalence of gpt delta and wild type (WT) rats in terms of toxicological responses to a genotoxic hepatocarcinogen, N-nitrosodiethylamine (DEN), and a non-genotoxic hepatocarcinogen, di(2-ethylhexyl)phthalate (DEHP). gpt delta rats treated with DEHP showed similar increases in liver and kidney weights, serum albumin, albumin/globulin ratios, and incidence of diffuse hepatocyte hypertrophy compared to WT F344 and Sprague-Dawley (SD) rats. DEN-treated gpt delta rats showed equivalent increases in the number and area of precancerous GST-P-positive foci in the liver compared to WT rats. The livers of DEN-treated gpt delta rats also showed increased frequencies of gpt and Spi(-) mutations; such changes were not observed in DEHP-treated gpt delta rats. These results indicated that gpt delta rats (both F344 and SD backgrounds) showed comparable DEHP-induced toxicity and DEN-induced genotoxicity to those observed in WT rats. With regard to the administration period, the general toxicity of 1.2% DEHP was evident throughout the experimental period, and the genotoxicity of 10 p.p.m. DEN could be detected after 2 weeks of administration and further increased at 4 weeks. These results suggested that combined assays using gpt delta rats could detect both general toxicity and genotoxicity by the canonical 4-week administration protocol. Therefore, this assay using gpt delta rats would be applicable for risk assessment including early detection of genotoxic carcinogens and ultimately serve to reduce cancer risks in humans from environmental chemicals.

Keywords: Genotoxicity; gpt delta; mutation; reduction of animal use; repeated-dose toxicity.

Fig 1

Body weight gain in male rats treated with N-nitrosodiethylamine (DEN) or di(2-ethylhexyl)phthalate (DEHP). F344/wild type (WT) and F344/gpt delta rats (a) or Sprague–Dawley (SD)/WT and SD/gpt delta rats (b) administered 0, 0.1, 1, or 10 p.p.m. DEN. F344/WT and F344/gpt delta rats (c) and SD/WT and SD/gpt delta rats (d) administered 0, 0.012, 0.12, or 1.2% DEHP. Four or five animals from each group (except 10 ppm DEN group at week 8 in F344; n=3) were killed at the time points indicated by arrows. *P < 0.05, **P < 0.01 versus respective control group.

Fig 2

Effects of di(2-ethylhexyl)phthalate (DEHP) on relative organ weights in male F344/wild type (WT), F344/gpt delta, Sprague–Dawley (SD)/WT, and SD/gpt delta rats administered 0, 0.012, 0.12, or 1.2% DEHP in the diet for 2, 4, or 8 weeks. (a) Liver; (b) kidneys. *P < 0.05, **P < 0.01 versus 0% DEHP group (control). ^#P < 0.05, ^##P < 0.01, WT versus gpt delta rats under the same conditions. BW, body weight.

Fig 3

Effects of di(2-ethylhexyl)phthalate (DEHP) on serum biochemistry in male F344/wild type (WT), F344/gpt delta, Sprague–Dawley (SD)/WT, and SD/gpt delta rats administered 0, 0.012, 0.12, or 1.2% DEHP in the diet for 2, 4, or 8 weeks. (a) Serum albumin, (b) albumin/globulin (A/G) ratio, and (c) alkaline phosphatase (ALP). Values are mean ± standard deviations. *P < 0.05, **P < 0.01 versus 0% DEHP group (control). ^##P < 0.01, WT versus gpt delta rats under the same conditions.

Fig 4

Immunohistochemical analysis of GST-P-positive foci. The multiplicity (no./cm²) (a) and area (mm²/cm²) (b) of GST-P-positive foci in the livers of N-nitrosodiethylamine (DEN)-treated rats. *P < 0.05, **P < 0.01 versus 0 p.p.m. DEN group (control). SD, Sprague–Dawley; WT, wild type.

Fig 5

(a) Frequency of mutations in the gpt transgene in the livers of male F344/gpt delta and Sprague–Dawley (SD)/gpt delta rats administered 0, 0.1, 1, or 10 p.p.m. N-nitrosodiethylamine (DEN) in drinking water.(b) Spi⁻ mutant frequencies in the livers of male F344/gpt delta and SD/gpt delta rats administered 0, 0.1, 1, or 10 p.p.m. DEN in the drinking water. (c) Frequency of mutations in the gpt transgene in the livers of male F344/gpt delta and SD/gpt delta rats administered 0, 0.012, 0.12, or 1.2% di(2-ethylhexyl)phthalate (DEHP) in the diet. (d) Spi⁻ mutant frequencies in the livers of male F344/gpt delta and SD/gpt delta rats administered 0, 0.012, 0.12, or 1.2% DEHP in the diet. **P < 0.01 versus 0 p.p.m. DEN group (control).

Fig 6

Mutation spectrum of the gpt transgene in the livers of male F344/gpt delta and Sprague–Dawley (SD)/gpt delta rats administered 0, 0.1, 1, or 10 p.p.m. N-nitrosodiethylamine (DEN) in drinking water. *P < 0.05, **P < 0.01 versus 0 p.p.m. DEN group (control).