The development and regional distribution of thymidylate synthetase (TS) (EC 2.1.1.45) in rabbit brain were determined. After optimization of the assay for brain, TS activity in brain was measured by a nonspecific (3H2O release) and specific method. The specific method involved the conversion of [6-3H]deoxyuridine monophosphate (dUMP) to [3H]thymidine phosphate and the subsequent identification of [3H]thymidine.l The specific activity of the enzyme in whole brain of newborn rabbits declined from 10.35 +/- 1.17 units/mg protein to 0.71 +/- 0.09 units/mg protein at 10-12 weeks of age. Two=year-old rabbits had 0.81 +/- 0.04 units/mg protein. The decline in specific activity with age was not due to an inhibitor of TS activity or a change in the Km for dUMP. The Km for dUMP of the unpurified enzyme in the brains of both 19-day-old and young adult rabbits was 0.8 microM. In young adult rabbits (3 months) the specific activity of TS was similar in the various regions of the brain tested except for the cerebellum, which had 40% higher specific activity than the whole brain. The results show that TS is widely distributed in adult rabbit brain, and, although the activity declines with age, it stabilizes at adult levels at 3 months of age.

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The in vitro effects of straight chain alkanes (nC6-nC10), benzene and toluene on pulmonary alveolar macrophages (PAM) of rats and rabbits was studied. The concentrations used ranged from 0.02 to 1.0 mM. All hydrocarbons used in the study were cytotoxic to isolated cultured PAM cells in a dose-dependent manner. The LC50 for these hydrocarbons towards rat PAM cells was estimated to be 1.0 mM for nC8, 2 mM for nC7, 5 mM for nC9 and 10 mM for nC6, nC10, benzene and toluene. Rabbit PAM cells were more sensitive to the hydrocarbons, resulting in and LC50 half that for rat PAM cells. Hydrocarbons also caused extracellular release of the lysosomal enzymes cathepsin D (EC 3.4.23.5) and cathepsin B (EC 3.4.22.1) in a manner corresponding with cell damage. There was more cathepsin D activity released from cells than cathepsin B. In addition, hydrocarbons also caused the release of cathepsin B and D from isolated lysosomes, and there was 10-15% more enzyme activity released in the culture medium of lysosomes exposed to concentrations of 0.5 and 1.0 mM compared to PAM cell cultures of either rats or rabbits. Hydrocarbons also caused loss of cell respiration and stimulated a dose-dependent and a time-dependent increase in lipid peroxidation. The two alkanes nC7 and nC8 caused the greatest increase in lipid peroxidation and the greatest loss of cell respiration. The results indicate that there is a relationship between chain length of alkanes and their cytotoxicity to PAM cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The metabolism of [3H]deoxyuridine by rabbit brain was investigated in vitro and in vivo. In vitro, brain slices from various regions of brain and from all age groups accumulated [3H]deoxyuridine from artificial CSF. Within the slices, a portion of the accumulated [3H]deoxyuridine was metabolized to [3H]deoxyuridine phosphate, with subsequent conversion to [3H]thymidine phosphate, and ultimately [3H]DNA. The percentage of the [3H]deoxyuridine phosphorylated and subsequently converted into [3H]DNA was highest at birth and declined to adult levels in 3-month-old rabbits. Thymidine, when added to the incubation medium with the [3H]deoxyuridine, was approximately 10 times as potent as unlabeled deoxyuridine in inhibiting the intracellular phosphorylation and conversion of [3H]deoxyuridine to [3H]thymidine phosphate in brain slices. In vivo, 2.5 h after intraventricular injection of [3H]deoxyuridine, over 90% of the [3H]deoxyuridine was cleared from the central nervous system at all ages. However, in both newborn and 3-month-old rabbits, approximately 40 and 12%, respectively, of the 3H remaining in brain was phosphorylated and converted to [3H]thymidine phosphates; and 11 and 4%, respectively, of the 3H remaining in brain was converted to [3H]DNA. These results show that both immature and mature rabbit brain is able to incorporate deoxyuridine into DNA. Thus, all the enzymes involved in this conversion, including thymidylate synthetase (EC 2.1.1.45), are present and active in brain throughout life.

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