In vivo covalent binding of [14C]trinitrotoluene to proteins in the rat.
When a single dose of [14C]trinitrotoluene was administered intraperitoneally (i.p.) to rats at 1, 10 or 50 mg/kg of body weight, covalently bound radioactivity was detected in globin, plasma proteins and proteins in the liver and kidney. The extent of covalent binding was dose dependent and was highest in plasma and renal proteins at all times up to 4 h after dosing. Covalent adduct levels in globin, however, decline slower than others. At a dose of 50 mg/kg of body weight, globin covalent adduct levels peaked at 1 h after dosing at 182 pmol/mg protein and subsequently decreased to approximately 50 pmol/mg protein between days 1 and 8. Of the covalent adduct levels in liver and kidney, those in the 10,000 x g and microsomal fractions were found to be higher than that in the cytosolic fraction. Radioactivity covalently bound to globin and the hepatic proteins was susceptible to dilute acid hydrolysis from which 2-amino-4,6-dinitrotoluene (2A) and 4-amino 2,6-dinitrotoluene (4A) were the major products recovered by solvent extraction. Upon acetylation, the hydrolysate gave rise to derivatives identified as the acetates of 2A and 4A on the basis of mass spectrometry and HPLC cochromatography with authentic samples. Four hours after an i.p. dose of [14C]TNT at 50 mg/kg of body weight about 0.4% of the dose was found as bound adducts to hemoglobin, of which approximately 48% was recovered as solvent extractable radioactivity after acid hydrolysis. About 2% of the radioactive dose was in the liver, of which approximately 30% was covalently bound to hepatic proteins, and approximately 49% of that was convertible to solvent extractable radioactivity upon acid hydrolysis. In vitro incubation of [14C]TNT with blood showed that there was a linear increase of covalent adducts in globin during the first 2 h of incubation; the concentration of covalent adducts was slightly higher than that with plasma proteins. The major compounds recovered from the hydrolysate of the globin adducts were also 2A and 4A as obtained from globin in the in vivo studies. On the basis of the in vitro and in vivo study results, we have confirmed the formation of protein adducts following a single i.p. administration of [14C]TNT at 1, 10 or 50 mg/kg of body weight to the rat or by in vitro incubation with blood.(ABSTRACT TRUNCATED AT 400 WORDS)
Liu
Y Y
YY
Department of Animal and Exploratory Drug Metabolism, Merck Sharp & Dohme Research Laboratories, Rahway, NJ 07065.
Lu
A Y
AY
Stearns
R A
RA
Chiu
S H
SH
D000818:Animals; D001711:Biotransformation; D001798:Blood Proteins / Q000378:metabolism; D001835:Body Weight; D002250:Carbon Radioisotopes; D005243:Feces / Q000737:chemistry; D005914:Globins / Q000378:metabolism; D006868:Hydrolysis; D007668:Kidney / Q000737:chemistry / Q000378:metabolism; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D008297:Male; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D014303:Trinitrotoluene / Q000097:blood / Q000493:pharmacokinetics*
D001798:Blood Proteins; D002250:Carbon Radioisotopes; D014303:Trinitrotoluene; D005914:Globins