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The Cytotoxicity of Methotrexate in Mouse Small Intestine in Relation to Inhibition of Folic Acid Reductase and of DNA Synthesis¹

Pharmacology Division, Sloan-Kettering Institute for Cancer Research, New York, New York 10021

Lethal doses of methotrexate in mice cause a prolonged inhibition of proliferation in the epithelium of small intestine. As early as 6 hr after such doses, a few karyorrhectic cells are present in the proliferating zone of crypts, but by 24 hr the crypt epithelium is atrophic. It fails to recover before the animals die at 4 days after injection. Doses of the agent equal to 0.1 x 50% lethal dose (LD₅₀) also severely damage crypts during the first 24 hr, but the region recovers by 72 hr. Few or no pathological effects are seen after doses of 0.01 or 0.001 x LD₅₀. Nevertheless, doses as low as 0.001 x LD₅₀, i.e., 0.5 mg/kg, markedly inhibit folic acid reductase in the intestine within the first 8 hr after injection, and the inhibition is as extensive at 24 hr. Moreover, studies of the uptake of the drug by intestine and liver show that shortly after injection of such low doses methotrexate is present in concentrations that are greatly in excess of the amounts required to bind all of the reductase in these tissues.

The incorporation of tritiated deoxyuridine into the DNA of small intestine is inhibited by more than 90% within 30 min after injection of 0.001 x LD₅₀, and recovery does not begin until 2 to 4 hr later. Higher doses, 0.01 and 0.1 x LD₅₀, induce even greater maximal inhibitions lasting 4 to 6 and 16 hr, respectively. At 30 min, even after LD₅₀ doses, thymidine incorporation into DNA is unaffected, a result consistent with the induction by methotrexate of a selective block in the methylation of deoxyuridylic acid. That DNA synthesis is depressed is shown by a concurrent inhibition of the incorporation of ³²P-labeled orthophosphate into DNA.

In spite of the early inhibition of DNA synthesis, methotrexate induces relatively few degenerating cells in the proliferative crypt epithelium during the first 6 hr. By contrast, selective inhibitors of DNA synthesis such as hydroxyurea or 1-ß-D-arabinosylcytosine destroy all S-phase cells within the first 2 to 4 hr. It is therefore concluded that inhibition of DNA synthesis by methotrexate in mouse intestinal crypts is not selective but is balanced by concurrent inhibition of RNA or of protein or of both.

¹ Supported in part by Grant CA 08748 from the National Cancer Institute and by the Elsa U. Pardee Foundation.

² The work reported herein is a portion of a dissertation submitted to the Faculty of the Graduate School of Medical Sciences, Cornell University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Present address: Department of Pharmacology, Mount Sinai School of Medicine of the City University of New York, New York, N. Y. 10029.

Received 5/24/71. Accepted 7/23/71.

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