In vitro and animal validation of bromine-76-bromodeoxyuridine as a proliferation marker.
The potential of 76Br-bromodeoxyuridine as a PET tracer for characterizing proliferation potential was investigated in multicellular tumor aggregates and in healthy rats and pigs.


METHODS
Bromine-76-bromide was produced by proton irradiation of a 76Se-enriched target using a 17-MeV cyclotron and recovered by thermal diffusion. Bromine-76-BrdU was prepared from the corresponding trimethylstannate by an oxidative bromination. Multicellular aggregates from a carcinoid cell line and two bladder cancer cell lines were co-incubated with 76Br-BrdU and 3H-thymidine and the uptake and DNA incorporation analyzed. About 0.5 MBq 76Br-BrdU were injected in the tail vein of unanaesthetised Sprague-Dawley rats. Two to 36 hr later they were decapitated and the radioactivity concentration and fraction of radioactivity incorporated into DNA determined in five different organs and the blood. Parallel studies were performed in animals pretreated with hydroxyurea. In separate experiments, rats were given an injection of 76Br-bromide and organ uptake was evaluated after 20 hr. PET studies were performed in two pigs and the uptake in different organs was investigated after injection of 76Br-BrdU. In these studies, diuresis was induced by furosemide and mannitol and radioactivity in blood and organs was followed during 10 hr.


RESULTS
In the cell aggregates, 30%-90% of the radioactivity was extracted in the DNA fraction. A good correlation was found between 76Br-BrdU and 3H-thymidine with respect to total uptake and DNA fraction. The DNA fraction increased from 2-10 hr after incubation. With in vivo injection in the rat, relatively high uptake of radioactivity was found in all organs, unrelated to the degree of DNA synthesis. However, inhibition by hydroxyurea occurred only in the spleen and intestines, organs which also showed a high degree of incorporation of 76Br-BrdU into DNA. In the pig, the highest in vivo uptake was observed in the red bone marrow and the intestines. In these organs, 70%-80% of the radioactivity was recovered in the DNA fraction. The concentration of radioactivity in the heart, liver and kidney was 3-10 times lower, and here the DNA fraction accounted for 10%-20% of the radioactivity. The decay-corrected radioactivity in blood and nonproliferating organs decreased with diuresis with a half-life of 13 and 16 hr, respectively.


CONCLUSIONS
It is suggested that the radioactivity uptake as seen after the administration of 76Br-BrdU, is constituted by two parts: one relating to incorporation into DNA and one existing as free 76Br- or metabolites of 76Br-BrdU. If sufficient time has passed, 76Br- dominates other metabolites. A correct assessment of DNA-incorporated radioactivity using PET with 76Br-BrdU is not trivial and can only be made with due correction for 76Br-, using either a complementary investigation after hydroxyurea pretreatment (in animal studies) or a separate 76Br-bromide investigation. Alternatively, the free bromide can be eliminated partially through forced diuresis.
Subfemtomole Biorecognition Project, Japan Sciences Technology Corporation and Uppsala University PET Centre, University Hospital, Sweden.
M
M
Bergström
L
L
Lu
K J
KJ
Fasth
F
F
Wu
E
E
Bergström-Pettermann
V
V
Tolmachev
E
E
Hedberg
A
A
Cheng
B
B
Långström
D000818:Animals; D001967:Bromine Radioisotopes; D001973:Bromodeoxyuridine; D002455:Cell Division; D006801:Humans; D066298:In Vitro Techniques; D019275:Radiopharmaceuticals; D051381:Rats; D017207:Rats, Sprague-Dawley; D013552:Swine; D013556:Swine, Miniature; D013997:Time Factors; D014055:Tomography, Emission-Computed; D014407:Tumor Cells, Cultured
D001967:Bromine Radioisotopes; D019275:Radiopharmaceuticals; D001973:Bromodeoxyuridine