TY - JOUR

T1 - DNA-binding and disposition of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the rat

AU - Dragsted, Lars Ove

AU - Frandsen, Henrik

AU - Reistad, Ragnhild

AU - Alexander, Jan

AU - Larsen, John Christian

N1 - (Ekstern)

PY - 1995

Y1 - 1995

N2 - Untreated and Aroclor 1254-pretreated male Wistar rats were given a single dose of 1.0 mg/kg body weight of randomly tritium-labelled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (3H-PhIP) by oral intubation. Urine and faeces were collected at 24, 48 and 72 hours after dosing, and total radioactivity determined. At 2, 4, 6, 16, 26, 48 and 72 h, animals were killed and several organs, including liver, bladder, lungs, kidneys, stomach, large and small intestines, heart, thigh muscle, spleen and blood were collected for DNA extraction and for determination of total radioactivity. Highest total radioactivity at 2 h was not unexpectedly observed in the stomach, small intestines and bladder, whereas radiolabels corresponding to ∼2.5 nmol PhIP/g of kidney and liver showed the highest levels observed at 24 h. Several tissues, including blood, plasma, liver and muscles had a slightly bimodal time-distribution of radioactivity showing a second peak at 16-24 h. At 72 h after a single dose of PhIP, highest radioactivity was observed in the liver and the large intestine (0.4 nmol PhIP/g tissue), whereas most other organs, irrespective of pretreatment had levels at ∼0.2 nmol/g of tissue. At earlier time points, Aroclor 1254-treated rats had lower amounts of radiolabel in all tissues. Radioactivity bound to DNA was determined by high sensitivity scintillation counting. In contrast to total radioactivity, DNA-associated radioactivity was generally higher in the Aroclor 1254-treated rats, most notably in the heart, but levels had decreased to approximately the same level in controls and in Aroclor 1254-treated rats at 72 h. DNA-binding was highest at 2-6 h after dosing, highest in the heart of Aroclor 1254-treated animals at 6 h (120 adducts/108 bases) followed by thigh muscle at 4-6 h (-50 adducts/108 bases, irrespective of pretreatment). Levels were ∼1.5-3 times lower in other organs at 2-6 h after dosing. At 72 h, radioactivity associated with DNA was again highest in the heart of Aroclor 1254-treated rats (20 adducts/108 bases) and 5-10 times lower in most other organs, approaching the detection limit. Total DNA was extracted from the livers of PhIP dosed rats at 4 and 72 h. DNA was hydrolysed, affinityconcentrated, and analysed by liquid chromatography. A radiolabelled peak had identical retention time and UV-spectral characteristics as peaks isolated by affinity chromatography and HPLC of acid-hydrolysed synthetic PhIP-DNA and PhIP-deoxyguanosine adduct. Thus, our data indicate that this is a product derived from the major DNA adduct in the liver of PhIP-dosed rats, and that the level of PhIP-DNA adducts in the various organs is ∼10 times higher than previously published by others, using the 32P-postlabelling method.

AB - Untreated and Aroclor 1254-pretreated male Wistar rats were given a single dose of 1.0 mg/kg body weight of randomly tritium-labelled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (3H-PhIP) by oral intubation. Urine and faeces were collected at 24, 48 and 72 hours after dosing, and total radioactivity determined. At 2, 4, 6, 16, 26, 48 and 72 h, animals were killed and several organs, including liver, bladder, lungs, kidneys, stomach, large and small intestines, heart, thigh muscle, spleen and blood were collected for DNA extraction and for determination of total radioactivity. Highest total radioactivity at 2 h was not unexpectedly observed in the stomach, small intestines and bladder, whereas radiolabels corresponding to ∼2.5 nmol PhIP/g of kidney and liver showed the highest levels observed at 24 h. Several tissues, including blood, plasma, liver and muscles had a slightly bimodal time-distribution of radioactivity showing a second peak at 16-24 h. At 72 h after a single dose of PhIP, highest radioactivity was observed in the liver and the large intestine (0.4 nmol PhIP/g tissue), whereas most other organs, irrespective of pretreatment had levels at ∼0.2 nmol/g of tissue. At earlier time points, Aroclor 1254-treated rats had lower amounts of radiolabel in all tissues. Radioactivity bound to DNA was determined by high sensitivity scintillation counting. In contrast to total radioactivity, DNA-associated radioactivity was generally higher in the Aroclor 1254-treated rats, most notably in the heart, but levels had decreased to approximately the same level in controls and in Aroclor 1254-treated rats at 72 h. DNA-binding was highest at 2-6 h after dosing, highest in the heart of Aroclor 1254-treated animals at 6 h (120 adducts/108 bases) followed by thigh muscle at 4-6 h (-50 adducts/108 bases, irrespective of pretreatment). Levels were ∼1.5-3 times lower in other organs at 2-6 h after dosing. At 72 h, radioactivity associated with DNA was again highest in the heart of Aroclor 1254-treated rats (20 adducts/108 bases) and 5-10 times lower in most other organs, approaching the detection limit. Total DNA was extracted from the livers of PhIP dosed rats at 4 and 72 h. DNA was hydrolysed, affinityconcentrated, and analysed by liquid chromatography. A radiolabelled peak had identical retention time and UV-spectral characteristics as peaks isolated by affinity chromatography and HPLC of acid-hydrolysed synthetic PhIP-DNA and PhIP-deoxyguanosine adduct. Thus, our data indicate that this is a product derived from the major DNA adduct in the liver of PhIP-dosed rats, and that the level of PhIP-DNA adducts in the various organs is ∼10 times higher than previously published by others, using the 32P-postlabelling method.

UR - http://www.scopus.com/inward/record.url?scp=0028844809&partnerID=8YFLogxK

U2 - 10.1093/carcin/16.11.2785

DO - 10.1093/carcin/16.11.2785

M3 - Journal article

C2 - 7586200

AN - SCOPUS:0028844809

VL - 16

SP - 2785

EP - 2793

JO - Carcinogenesis

JF - Carcinogenesis

SN - 0143-3334

IS - 11

ER -