Rapid in vivo inactivation by acivicin of CTP synthetase, carbamoyl-phosphate synthetase II, and amidophosphoribosyltransferase in hepatoma.

A single injection of the anti-glutamine drug, acivicin (NSC 163501), in tumor-bearing rats in 30 min decreased the activities of amidophosphoribosyltransferase, carbamoyl-phosphate synthetase II and CTP synthetase to 56, 50, and 7% of those of the controls. By 1 hr the activities were down to 32, 13 and 3% and they remained low for 12 hr, after which they slowly returned towards normal range in 72 hr. The decline of the activity of CTP synthetase (a loss of 80% in 10 min) was the most rapid, and the activity only returned to 60% of the controls by 3 days after the acivicin injection. In the hepatoma the concentrations of ATP and UTP changed little, but those of GTP and CTP rapidly decreased, reaching at the lowest point 32 and 2%, respectively, of control values 2 hr after acivicin; concentrations started to rise at 12 hr, reaching normal levels by 48 hr. The drop in enzyme activities preceded the decline in the pools of GTP and CTP. The behavior of enzyme activities and nucleotide concentrations in the host liver had a pattern similar to that in the hepatoma; however, the changes were less extensive than those in the tumor. The differential response between tumor and liver is attributed, in part at least, to the tissue L-glutamine concentration which in the hepatoma (0.5 mM) was 9 times lower than in the liver (4.5mM). The selectivity of acivicin action in inhibiting glutamine-utilizing enzymes is also demonstrated by the lack of effect on aspartate carbamoyltransferase, an enzymic activity which resides in the same complex as that of carbamoyl-phosphate synthetase II. The rapid decline in the activities of glutamine-utilizing enzymes is attributed to an inactivation of the enzymes by acivicin which functions as an active sitedirected affinity analog of L-glutamine. The rapid modulation of the enzymic phenotype and ribonucleotide concentrations by acivicin provides a useful tool for elucidating the role of enzymic and nucleotide imbalance in the commitment of cancer cells to replication and in the targeting of anticancer chemotherapy.

Multi-enzyme-targeted chemotherapy by acivicin and actinomycin.

On the basis of our observation of the increased specific activities of glutamine-utilizing enzymes in purine and pyrimidine metabolism in hepatoma 3924A, and because the concentration of glutamine is ten times lower in the hepatomas than in the liver, the biochemical pharmacology of the anti-glutamine agent, acivicin, was examined. (1) Acivicin competitively inhibited the activities of amidophosphoribosyl-transferase, CTP synthetase and carbamoyl-phosphate synthetase II from extracts of liver and hepatoma 3924A. (2) In addition to the competitive inhibition exerted by acivicin, evidence was obtained that this drug also irreversibly inactivated in vitro the glutamine-utilizing enzymes. It is particularly relevant for the selectivity of acivicin that the activity of aspartate carbamoyltransferase, an enzyme present in the same complex as carbamoyl-phosphate synthetase II, was not affected by the anti-glutamine agent. (3) Acivicin in vivo brought down the activities of glutamine-utilizing enzymes in a period of 10 min to 1 hr after injection. CTP synthetase activity declined to less than 10% of that observed in the uninjected rats. The decreases were not reversible by various in vitro methods, but in vivo the activities returned to normal range in 72 hr. (4) The activity of aspartate carbamoyltransferase, which exists as a multi-enzyme complex with synthetase II, was not altered by acivicin injection. Similar results were observed in transplantable sarcoma in the rat. (5) The acivicin-induced decrease in enzymic activities could not be restored by purification of the enzymes. (6) In vitro studies indicated that addition of acivicin to liver or hepatoma extracts or purified enzymes rapidly decreased enzymic activities; the activities could not be restored. These results are consistent with an interpretation that acivicin acts either as a tight-binding inhibitor or as an inactivator through alkylation of the enzymes of glutamine utilization. (7) Acivicin in combination with actinomycin provided a synergistic kill of hepatoma cells in tissue culture and also inhibited the growth of transplantable solid hepatoma 3924A in the rat. (8) The synergistic biological results of combination chemotherapy with acivicin and actinomycin can be accounted for by the action of acivicin in inhibiting GMP and CTP synthetases, resulting in a decrease in GTP and CTP content, and by the actinomycin-caused inhibition of RNA polymerase in selectively blocking the utilization of GTP and CTP.

Effects of lead on benzene metabolism.

The possibility that Pb could affect benzene metabolism, through inhibition of enzyme synthesis, was examined by studies of the effects of chronic oral Pb treatments on benzene conversion to phenol. Rats were given either distilled deionized water or 0.05, 0.58, 17, or 352 ppm Pb solutions as drinking water. After 6, 9, 12, and 15 wk of treatment, rats from each group were sacrificed and in vitro benzene metabolism by benzene hydroxylase was measured in liver enzyme preparations. After 24 wk, the remaining animals were injected ip with 400 mg/kg benzene and urinary phenol levels measured daily for 4 d. The enzyme activity, studied in vitro, was significantly increased in animals that ingested the 352 ppm solution for 6 wk. Also, Pb levels in all groups of animals were significantly higher in tissues from the animals that ingested 352 ppm Pb. Although urinary phenol levels accounted for 30-40% of the ip benzene dose, Pb ingestion had no significant effect on phenol excretion. These results suggested that oral Pb treatments had a significant effect on the enzyme responsible for benzene hydroxylation to phenol. However, other unknown factors appear to compensate for these changes in vivo.

Response of prepubertal girls and college women to work in the heat.

Five prepubertal females and five college women, matched for aerobic power, walked on a treadmill at approximately 30% VO2 max for two 50-min periods in three environments: 1) 28 degrees C, 45% rh, 2) 35 degrees C, 65% rh, and 3) 48 degrees C, 10% rh. In the mild heat (28 degrees C) both groups were able to work 100 min with no discomfort. At 35 and 48 degrees C tolerance time for the prepubertal subjects averaged 84.4 and 37.0 min, respectively; for adults, 100 and 75.0 min. At all temperatures the girls had higher heart rates and a lower stroke index, and finished the walks with a higher rectal temperature. There were no differences between groups in cardiac index, mean skin temperature, forearm blood flow, or percent loss in body weight. The proportion of the thermal load dissipated by the two groups was similar but the route for heat transfer was related to the BSA/wt ratio and environmental conditions. Marked circulatory instability was a primary factor in the lower tolerance of the prepubertal girls to work in the heat probably due to a shift in blood volume from the central to the peripheral circulation.

Aerobic power as a factor in women's response to work in hot environments.

Twelve young women, athletes (n = 6) and nonathletes (n = 6), walked on a treadmill at loads equivalent to approximately 30% Vo2 max for two 50-min periods in three environments: 1) 28 degrees C, 45% rh, 2) 35 degrees C, 65% rh, and 3) 48 degrees C, 10% rh. There were no differences between groups in rectal temperature, heart rate, evaporative heat loss, or mean skin temperature at 28 or 35 degrees C or during the first work period in the 48 degrees C environment. However, a significantly lower cardiac output (Q) and stroke volume (SV) observed for nonathletes by the 46th min of work at 48 degrees C may explain why no nonathletes were able to complete a 2nd h of work while four of six athletes successfully finished the period. It appears that in conditions of severe heat stress (48 degrees C) athletes were able to maintain a cardiac output sufficient to meet the metabolic requirements and the large increase in peripheral blood flow for a longer period of time than nonathletes.

Thermoregulatory response of women to intermittent work in the heat.

Seven women worked intermittently in three randomly ordered sessions at 75% Vo2max at three temperatures, 28 degrees C (45% rh) 35 degrees C (65% rh), and 48 degrees C (10% rh) and recovered in a cool environment (22 degrees C) after each 6-min work period. Although Tre was higher in each successive work period, the ambient temperature had no effect on the cardiovascular or respiratory responses or on deltaTre. In all conditions SV decreased with time with a concomitant increase in HR to maintain Q. A fall in mean blood pressure from the initial to final measurement was due entirely to a decrease in diastolic pressure. The final Tre for these women was approximately equal to that previously reported for men working continuously for 1 h under conditions equivalent to time-weighted average of the thermal and metabolic loads during work and recovery in this study.