Measurement in vivo of proliferation rates of slow turnover cells by 2 H 2 O labeling of the deoxyribose moiety of DNA

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Abstract
We describe here a method for measuring DNA replication and, thus, cell proliferation in slow turnover cells that is suitable for use in humans. The technique is based on the incorporation of 2 H 2 O into the deoxyribose (dR) moiety of purine deoxyribonucleotides in dividing cells. For initial validation, rodents were administered 4% 2 H 2 O in drinking water. The proliferation rate of mammary epithelial cells in mice was 2.9% per day and increased 5-fold during pregnancy. Administration of estradiol pellets (0–200 μg) to ovariectomized rats increased mammary epithelial cell proliferation, according to a dose–response relationship up to the 100 μg dose. Similarly, proliferation of colon epithelial cells was stimulated in a dose–response manner by dietary cholic acid in rats. Bromodeoxyuridine labeling correlated with the 2 H 2 O results. Proliferation of slow turnover cells was then measured. Vascular smooth muscle cells isolated from mouse aorta divided with a half-life in the range of 270–400 days and die-away values after 2 H 2 O wash-out confirmed these slow turnover rates. The proliferation rate of an adipocyte-enriched fraction from mouse adipose tissue depots was 1–1.5% new cells per day, whereas obese ad libitum-fed ob/ob mice exhibited markedly higher fractional and absolute proliferation rates. In humans, stable long-term 2 H 2 O enrichments in body water were achieved by daily 2 H 2 O intake, without toxicities. Labeled dR from fully turned-over blood cells (monocytes or granulocytes) exhibited a consistent amplification factor relative to body 2 H 2 O enrichment (≈3.5-fold). The fraction of newly divided naive-phenotype T cells after 9 weeks of labeling with 2 H 2 O was 0.056 (CD4 + ) and 0.043 (CD8 + ) (replacement rate 2 H 2 O labeling of dR in DNA allows safe, convenient, reproducible, and inexpensive measurement of cell proliferation in humans and experimental animals and is well suited for slow turnover cells.

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