James A. Straw

Glial DNA synthesis and cell proliferation in the lesioned frontal cortex of the rat

Proliferation of rat neurological cells was quantified following a lesion of the frontal cortex, with the rate of incorporation of intraventricularly administered [3H]thymidine ([3H]TdR) into cortical DNA serving as an index of glial proliferation. Incorporation of [3H]uridine into the corresponding RNA fractions did not serve this purpose. The intraventricular route of administration of thymidine greatly reduced the amount of [3H]TdR needed to label DNA relative to systemic injection. The rate of incorporation of [3H]TdR into DNA was linear for 75 min post-injection. Significantly more [3H]TdR was incorporated into DNA of the lesioned frontal cortex than that of the contralateral control cortex, during the first 4 days post-trauma. The majority of the acid-insoluble radioactivity (from [3H]TdR) was localized in the nuclear subcellular fraction of the cortex. Experiments indicated that the enhanced incorporation of [3H]TdR was not the result of altered metabolism or pool sizes of TdR in the lesioned cortex. Histological analysis indicated that there was a significant increase in the number of glial cells in the lesioned cortex by day 4 post-lesion, which corresponded to the increase in DNA synthetic activity. It was concluded that mechanical trauma to the frontal cortex of the rat results in an increase in the number of glial cells at and near the lesion which is accompanied by an increase in incorporation of [3H]TdR into cortical DNA. This method of measuring posttraumatic DNA synthesis has several advantages over autoradiography.

Enhanced cytotoxicity with methotrexate in conjunction with hypoxanthine in L1210 cells in culture

SummaryBy inhibiting dihydrofolate reductase, methotrexate (MTX) depletes cellular stores of reduced folates, resulting in the inhibition of DNA and RNA synthesis. Inhibition of RNA synthesis arrests cells in the G1 phase of the cell cycle, preventing these cells from entering S phase and rendering them insensitive to MTX. Because MTX cytotoxicity can be enhanced by concurrent administration of hypoxanthine (HX), we examined the hypothesis that this modulation can allow normal rates of RNA synthesis and cell cycle progression from G1 to S phase. For L1210 cells exposed to MTX for 12 h or 24 h, the addition of HX enhanced the cytotoxicity of MTX; however, no enhancement was observed with a 6-h exposure. Inhibition of RNA synthesis by MTX was prevented by concurrent administration of HX. The effect of HX on cell cycle progression was first examined using flow cytometry, which indicated that MTX treatment alone or with concurrent HX caused a buildup of cells with aG1 content of DNA. Because this technique may fail to distinguish between cells in late G1 phase, the G1/S border, or early S, the method of premature chromosome condensation was used to determine cell cycle position based on chromatin morphology. A shift to a higher degree of chromatin decondensation was observed when HX was coadministered with MTX during a 12-h exposure, suggesting progression from G1 towards S. This correlated with the enhancement of MTX cytotoxicity by HX after 12 h exposure. The results of these studies suggest that HX potentiates MTX cytotoxicity by maintaining RNA synthesis, allowing cells that mightordinarily be arrested in G1 to progress into the cytotoxic S phase.

Pharmacokinetics of zidovudine in HIV-infected patients with end-stage renal disease.

We determined the pharmacokinetics of zidovudine (AZT) and its metabolite (GAZT) in HIV-infected patients with end-stage renal disease (ESRD), between dialysis sessions, and compared these to HIV-infected patients with normal renal function. Clearance of AZT in ESRD patients was not significantly different from controls. The mean serum AZT levels in ESRD patients were six times greater than the levels in normal controls at 4 h. Serum levels of GAZT were higher in ESRD patients at 90 min, and by 4 h were more than an order of magnitude greater than normal controls. If the AZT serum level is a good index of toxicity, we conclude that the currently recommended dose of 200 mg AZT three times a day is probably safe for use in HIV-infected patients with ESRD. The enterohepatic metabolism of AZT, the effect of such a dosing schedule and the effects of circulating levels of GAZT on outcomes in HIV-infected patients with ESRD must be further investigated.

Characterization of repair inhibition by methotrexate of ethylmethanesulfonate-and ultraviolet irradiation-induced DNA damage in Chinese hamster cells

To further understand the mechanism by which methotrexate (MTX) inhibits DNA repair, we have studied the dependency of EMS repair inhibition on both pretreatment time and concentration of MTX. The basis for the selectivity with which MTX inhibited ethylmethanesulfonate (EMS) repair more effectively than UV repair has also been examined. We found that MTX in combination with Cytosine arabinofuranoside (Ara-C) was as effective as hydroxyurea + Ara-C (H/A) in causing inhibition of UV repair

Computer Modeling of the Pharmacokinetics of Fluorouracil and Thymine and Their Kinetic Interaction in Normal Dogs

The kinetic behavior of thymine and 5-fluorouracil has been shown to be non-linear and mediated largely by saturable metabolic processes. In vivo estimates of the Michaelis-Menten parameters Vmax and Km were obtained from constant infusion data in normal dogs using a system of balance equations that equate drug input with total output at steady-state. These estimates were then successfully used to simulate both steady-state and post-infusion plasma concentration-time curves for both compounds over a range of saturating and non-saturating conditions. It has been shown previously that estimates of Vmax and Km obtained from dynamic data can be incorrect if an inappropriate compartmental model is used in the analysis. Determining the Michaelis-Menten parameters at steady-state eliminates this difficulty. Moreover, the use of steady-state derived values to simulate post-infusion data confirms the validity of this technique. The kinetic interaction between thymine and 5-fluorouracil was investigated as a case of competitive metabolic inhibition in vivo by calculating Ki values from data obtained during simultaneous constant infusions of the two compounds. These values were then used in conjunction with a series of differential equations incorporating reciprocal metabolic effects to simulate the effect of thymine on FU plasma concentration.