National Toxicology Program

Acetaminophen study

Background

The data reported in this initial publication is from a larger experiment in which groups of male F344 rats were administered toxic and non-toxic doses of acetaminophen at two different times: between 12 (noon)-1PM or between 12 (midnight)-1AM. The data being posted in this release is from animals dosed between 12 (noon)-1PM, and therefore some of the associated tables will contain the word "Light" in the title. It is anticipated that the remaining data for animals dosed between 12 (midnight)-1AM will be released in the near future.

Study Description

The animal housing conditions were standard for toxicology experiments and have been described (Toxicol. Pathol. 33: 102-110, 2005).

Male Fischer 344 rats approximately 13 weeks old were administered a single gavage dose (0 [vehicle], 50, 150, 1500 and 2000 mg/kg body weight) of acetaminophen (APAP, purity 101.1%) in 0.5% aqueous methylcellulose at 5 mL/kg. The animals were dosed between 12(noon) and 1 PM. In order to assure prompt collection of tissues for RNA analysis the study was run in two parts, separated by two days, with 3 rats per dose-time group in each part.

For each dose-time group the first three rats listed in Table C-2 (see below) are from part one and are cage mates, while the second three animals are also cage mates but from part two. Rats from part one have numbers that are lower than animal numbers in part two. For example 50 mg/kg rats at the 6 hour time point from part one are numbered 201, 202, 203, while those from part 2 are numbered 213, 214, 215. Clinical pathology Table C-2 is the easiest way to relate rat numbers to dose, time after dosing, and study part 1 or part 2.

At 6, 18, 24 and 48 hours after dosing rats were anesthetized with a carbon dioxide/oxygen mixture and blood collected by cardiac puncture. Promptly after blood collection, the abdominal cavity was opened and the portal vein severed. The left lobe of the liver was promptly removed, divided by cross section into two halves, one half of which was used for RNA isolation and histology and the other half for glutathione analysis. The portion for RNA isolation was placed in RNAlater®, cut into small pieces, and stored over night, and then frozen until RNA isolation. RNA isolation was done using QIAGEN RNeasy® Midi Kit. For more details see Toxicol. Pathol. 33: 102-110, 2005. The samples were shipped to the NTP Repository, inventoried and sent to Paradigm Array Labs (Icoria, Inc.) for hybridization on Agilent Rat Oligo (60mer) Chip (Agilent Technologies, Inc, Wilmington, DE). For each time point, RNA from time matched control rats from both parts of the study was pooled to form a time-matched control. Thus all 6 hour rats (from part 1 or part 2) were hybridized against the same (6hr) control, all 12 hour rats hybridized against the same (12hr) control, etc. A normal and fluor reversal hybridization was performed for each animal. Link to microarray data.

A study pathologist evaluated the left lobe, entered diagnoses and severity grade (1 = minimal, 2 = mild, 3 = moderate, 4 = marked). A reviewing pathologist reviewed the livers with diagnoses. A pathology panel resolved discrepancies between study and reviewing pathologist before the data was entered into the Toxicology Data Management System (TDMS). This procedure for assuring pathology quality and consistency has been previously described (Toxicol. Pathol. 30, 88-92, 2002). The rat number is common between the different files. See the individual pathology diagnoses or download the data.

The clinical pathology results are in the pdf file Table C-2 or can be downloaded. The blood from the cardiac punctures was placed in a tube with no additives, centrifuged and the serum collected. Clinical pathology results were obtained using a centrifugal analyzer (Hitachi 911, Chula Vista, CA). Control samples were included at the beginning and end of each run.

Hepatic glutathione analysis was based on the determination of thiols by high-performance liquid chromatography and has been previously described (Toxicol. Pathol. 33: 102-110, 2005). The glutathione results are found in the pdf file (Table C-1) or downloaded.

The individual animal weights can be found here . Using clinical Chemistry Table C-2 one can relate animal weights to cage mates and to experiment number. In a preliminary analysis for the controls, no relationship between body weights within cage was found for gene expression levels in the untreated animals (Toxicol. Pathol. 33: 102-110, 2005).

Link to microarray data