Prevention of agricultural injuries: an evaluation of an education-based intervention.

OBJECTIVE: To evaluate the effectiveness of an agricultural health and safety program in reducing risks of injury. DESIGN: Cross-sectional survey. SETTING: 50 rural municipalities in the Province of Saskatchewan, Canada. INTERVENTION: The Agricultural Health and Safety Network (AHSN), a mainly educational program that administered 112 farm safety interventions over 19 years. SUBJECTS: 5292 farm people associated with 2392 Saskatchewan farms. Farms and associated farm people were categorized into three groups according to years of participation in the AHSN. IMPACT: self-reported prevalence of: (1) farm safety practices; (2) physical farm hazards. OUTCOME: (1) self-reported agricultural injuries. RESULTS: After adjustment for group imbalances and clustering at the rural municipality level, the prevalence of all impact and outcome measures was not significantly different on farms grouped according to years of AHSN participation. To illustrate, the adjusted relative risk of reporting no rollover protection on tractors among farms with none (0 years) versus high (>8 years) levels of AHSN participation was 0.95 (95% CI 0.69 to 1.30). The adjusted relative risk for agricultural injuries (all types) reported for the year before the survey was 0.99 (95% CI 0.74 to 1.32). CONCLUSIONS: Educational interventions delivered via the AHSN program were not associated with observable differences in farm safety practices, physical farm hazards, or farm-related injury outcomes. There is a need for the agricultural sector to extend the scope of its injury prevention initiatives to include the full public health model of education, engineering, and regulation.

Regulation of daily locomotor activity and sleep by hypothalamic EGF receptor signaling.

The circadian clock in the suprachiasmatic nucleus (SCN) is thought to drive daily rhythms of behavior by secreting factors that act locally within the hypothalamus. In a systematic screen, we identified transforming growth factor-alpha (TGF-alpha) as a likely SCN inhibitor of locomotion. TGF-alpha is expressed rhythmically in the SCN, and when infused into the third ventricle it reversibly inhibited locomotor activity and disrupted circadian sleep-wake cycles. These actions are mediated by epidermal growth factor (EGF) receptors on neurons in the hypothalamic subparaventricular zone. Mice with a hypomorphic EGF receptor mutation exhibited excessive daytime locomotor activity and failed to suppress activity when exposed to light. These results implicate EGF receptor signaling in the daily control of locomotor activity, and identify a neural circuit in the hypothalamus that likely mediates the regulation of behavior both by the SCN and the retina.

Bradykinin modulation of tumor vasculature: I. Activation of B2 receptors increases delivery of chemotherapeutic agents into solid peripheral tumors, enhancing their efficacy.

Delivery of chemotherapeutic agents to solid peripheral tumors is compromised because the impaired microvasculature within and surrounding tumors limits diffusion and convection of agents from the vasculature to the tumor. Using a variety of rat tumor models, we show that intravenous administration of a vasoactive bradykinin B2 receptor agonist (Cereport, or labradimil; formerly RMP-7) enhances by nearly 3 times the delivery of the chemotherapeutic agent carboplatin, as well as the larger 70-kDa marker dextran, into ectopic and orthotopic solid tumors. This effect was selective for tumor tissue, with little or no increase seen in nontumor tissues and organs. Additionally, the increased carboplatin levels observed in tumors persisted for at least 90 min (the longest time point measured). In contrast to the consistent effects with hydrophilic compounds, delivery of the lipophilic, high protein-binding chemotherapeutics paclitaxel and 1,3-bis[2-chloroethyl]-1-nitrourea (BNCU) was not enhanced. Administration of Cereport with either carboplatin or another hydrophilic chemotherapeutic agent, doxorubicin, significantly increased efficacy of both agents, manifested by suppression of tumor growth and prolonged survival in tumor-bearing rats. These data demonstrate that delivery of chemotherapeutics to tumors can be pharmacologically increased (by stimulating bradykinin B2 receptors) without increasing the systemic exposure, or therefore, the toxic liability associated with higher chemotherapeutic doses.

Bradykinin modulation of tumor vasculature: II. activation of nitric oxide and phospholipase A2/prostaglandin signaling pathways synergistically modifies vascular physiology and morphology to enhance delivery of chemotherapeutic agents to tumors.

Intravenous infusions of the bradykinin agonist Cereport (labradimil, formerly RMP-7) enhance delivery of concomitantly administered hydrophilic chemotherapeutic agents to solid tumors. The enhanced delivery produces greater in vivo efficacy of chemotherapeutic agents, manifested as suppressed tumor growth and increased survival in tumor-bearing rats. Here we elucidate the mechanisms of action involved with this unique phenomenon, at both the physical and biochemical levels. At the physical level we demonstrate that Cereport modifies the tumor vasculature in several important ways, including transient 1) reductions in interstitial fluid pressure within the tumor, 2) increases in pore size of the vasculature, and 3) increases in total vascular surface area. All three of these changes modify tumor-specific characteristics of the vasculature known to impede drug delivery to the tumor interstitium. Biochemically, we demonstrate that the activation of both of bradykinin's major signaling pathways, the nitric oxide and phospholipase A2/prostaglandin E2 are necessary events. Although pharmacologically blocking either pathway greatly reduced the effects of Cereport, stimulation of either pathway alone did not enhance delivery. However, simultaneous stimulation of both pathways (without exogenous bradykinin B2 receptor stimulation) produced a nearly 2-fold increase in delivery of carboplatin to the tumor. Thus, stimulation of endogenous bradykinin B2 receptors induces at least two parallel biochemical cascades that act synergistically to uniquely modify the tumor vasculature in ways that increase delivery and efficacy of chemotherapeutic agents.

Injectable chemotherapeutic microspheres and glioma I: enhanced survival following implantation into the cavity wall of debulked tumors.

PURPOSE: Implantation of biodegradable polymers provides a powerful method to deliver high, sustained concentrations of chemotherapeutics to brain tumors. The present studies examined the ability of injectable polymeric microspheres, formulated to release carboplatin or BCNU for 2-3 weeks, to enhance survival in a rodent model of surgically-resected glioma. METHODS: Rat glioma (RG2) cells were implanted into the cortex of rats and allowed to grow for 10 days prior to surgical resection. Rats were given either surgical resection only, bolus injection (100 microg) or microspheres containing 10, 50, or 100 microg of carboplatin or BCNU. The microspheres were implanted, via hypodermic injection, either directly into the surgical cavity or into the tissue along the perimeter of the cavity. RESULTS: The order of survival among treatment groups was: no resection < resection only < bolus chemotherapy < sustained release chemotherapy. Carboplatin and BCNU did not differ in this respect and in each case, the enhanced survival achieved with sustained release was dose-related. However, the enhanced survival achieved with carboplatin was substantially greater when the microspheres were implanted into the perimeter wall of the resection cavity, compared to implantation into the cavity itself. The enhanced survival produced by carboplatin implants along the resection perimeter was associated with a significant attenuation of regrowth of the tumor. Finally, in a separate study in non-tumor brain, atomic absorption spectrophotometry revealed that while the microspheres produced significantly prolonged tissue levels of carboplatin relative to a bolus injection, carboplatin diffusion was limited to brain tissue extending primarily 0.5 mm from the injection site. CONCLUSIONS: These data demonstrate: (1) that sustained delivery of chemotherapy is superior to equipotent bolus doses following tumor resection, and (2) that direct injection of sustained release microspheres into the tissue surrounding a growing tumor mass may provide superior effects over injections into the surgical cavity. They also suggest that successful implementation of this approach in humans may require measures or circumstances that improve upon the limited spatial drug diffusion from the implantation site.

Injectable chemotherapeutic microspheres and glioma II: enhanced survival following implantation into deep inoperable tumors.

PURPOSE: Delivery of chemotherapeutics using implantable, biodegradable polymers provides a potentially powerful method of treating brain tumors. The present studies examined the ability of injectable microspheres, formulated to release carboplatin or BCNU for 2-3 weeks, to enhance survival in a rodent model of deep, inoperable glioma. METHODS: Rat glioma (RG2) cells were implanted into the striatum of rats. In a first experiment, the tumors were allowed to grow for 3 days, followed by either no treatment, bolus chemotherapy (100 microg), or implantation of microspheres containing 10, 50, or 100 microg of carboplatin. The microspheres were implanted, via hypodermic injection, directly into the center of the small, 3-day-old tumors. In a second experiment, tumors grew for 8 days prior to treatment with either carboplatin- or BCNU-loaded microspheres. The microspheres were then injected either directly into the center of these larger tumors or into three sites along the perimeter of the tumor. Separate sets of animals received bolus chemotherapy (100 microg) into either the tumor center or around the tumor perimeter. RESULTS: Injection of carboplatin-loaded microspheres into the center of the small 3 day old, tumors produced dose-related increases in survival. When injections of carboplatin- or BCNU-loaded microspheres were made into the center of the larger, 8-day-old tumors, survival was not enhanced. However, when the microspheres were injected along the perimeter of the larger tumors, sustained-release chemotherapy did significantly prolong survival. Bolus chemotherapy was less effective than sustained release chemotherapy. CONCLUSIONS: Together, these data: (1) demonstrate that sustained delivery of chemotherapy in or near the tumor site is superior to equipotent bolus doses in inoperable tumors, (2) demonstrate that injection of sustained release microspheres into the tissue surrounding a growing tumor may provide superior effects over injections directly into the tumor mass, and (3) suggest that this approach may provide a useful means of selectively delivering chemotherapeutics to tumors or portions of tumors that cannot otherwise be treated with conventional surgical approaches.

Intravenous cereport (RMP-7) modifies topographic uptake profile of carboplatin within rat glioma and brain surrounding tumor, elevates platinum levels, and enhances survival.

Several experiments studied the effects of i.v. infusions of the bradykinin agonist, Cereport (RMP-7), on permeability of the blood-brain tumor barrier in rat gliomas. First, the ability of Cereport to increase uptake of two poorly blood-brain barrier-penetrating drugs (lypophilic paclitaxel and hydrophilic carboplatin) was directly compared to provide new information regarding the scope of delivery effects achieved with Cereport. Next, the increased uptake of platinum into tumor and brain surrounding tumor was shown to closely parallel that of radiolabeled carboplatin, confirming that delivery of a biologically active moiety is increased with Cereport. This study also demonstrated that the elevated tumor levels of platinum persisted for at least 2 h. The enhanced carboplatin uptake was then examined using a novel, high spatial resolution analysis of autoradiography. This revealed that the effects of Cereport were not uniform throughout the tumor, because it especially modified those areas normally impermeable to carboplatin. Finally, a range of i.v. Cereport doses (3.0 and 9.0 microg/kg) was tested in combination with carboplatin to determine whether increased survival might be achieved and to define the relationship between Cereport dose, plasma levels, uptake of carboplatin, and enhanced survival. Survival was enhanced only by the high dose of Cereport; the high dose also produced robust increases in carboplatin uptake and plasma concentrations of Cereport estimated to achieve the K(i), whereas the low dose did not. These data offer fundamental information regarding the effects of Cereport on delivery of chemotherapeutic agents to brain tumors and provide new insight into receptor-mediated permeability of the blood-brain tumor barrier.

Evidence that Cereport's ability to increase permeability of rat gliomas is dependent upon extent of tumor growth: implications for treating newly emerging tumor colonies.

Cereport (RMP-7) enhances delivery of chemotherapeutics into brain tumors by increasing the permeability of the glioma vasculature (i.e. , the blood-brain tumor barrier; BBTB). Its effect on brain tumors has consistently been more robust than that on normal brain. The present experiments tested the hypothesis that the ability of Cereport to increase the permeability of infiltrating glioma colonies increases as the glioma colonies develop, in situ. In an initial preliminary experiment, the significant and selective effects of Cereport in tumor tissue and brain surrounding tumor were verified using [(14)C]carboplatin as a marker, 8 days after implantation of 50,000 RG2 cells. A second preliminary experiment established that the number of tumor cells initially seeded influences the growth rate of the tumor mass. Tumors seeded with 50,000 cells were larger than those seeded with 25,000 cells 3, 5, and 8 days after implantation. Next, the hypothesis that the extent of tumor growth increases Cereport's effects on the BBTB was tested by measuring the concentration of radiolabeled carboplatin in the tumor when 50,000 cells were implanted 3, 8, or 13 days prior to the experiment. While a reliable, approximately twofold increase in carboplatin concentration was seen in the 8- and 13-day-old tumors, no significant effect of Cereport was observed in the tumors that developed only 3 days, in situ. Finally, another test of the hypothesis was made by comparing Cereport's effects on 8-day-old tumors initially seeded with either 50,000 or 25,000 cells (the latter producing a smaller, more slowly developing, tumor mass). Again, significantly higher carboplatin concentrations were seen with Cereport in the 50,000 cell tumors (greater than two-fold increase), compared to the smaller, more slowly developing, 25,000 cell tumors (<30% increase). The tumor and its vasculature were characterized in additional rats implanted with RG2 cells using CD-31, laminin, and bradykinin B(2) receptor immunocytochemistry. Intense B(2) receptor staining was observed on cells within the parenchyma of normal brain and tumor but not on the vasculature of tumor or brain. An extensive network of CD-31 and laminin staining was seen within and around the tumors in all groups, indicating relatively rapid and robust changes in vascularity in response to the gliomas. However, no consistent difference in vascularity between groups was observed to account for the uptake differences seen with Cereport. Collectively, these data offer initial preclinical empirical support for the hypothesis that Cereport's effects on tumor permeability increase as the tumor grows, which we further hypothesize is likely related to features of vascular development within the tumor independent of numbers or general morphology of vessels. If a similar phenomenon is shown to occur with infiltrating colonies from spontaneously forming gliomas in humans or from newly emerging metastases in brain, these data could impact the design and conduct of future trials using approaches intended to enhance delivery of chemotherapeutics through increased permeability of the tumor vascular barrier.

Intravenous cereport (RMP-7) enhances delivery of hydrophilic chemotherapeutics and increases survival in rats with metastatic tumors in the brain.

PURPOSE: The following experiments determined whether intravenous infusions of Cereport enhance delivery of chemotherapeutics and prolong survival in rats with metastatic tumors in the brain. METHODS: Autoradiography and scintillation were used to examine uptake of the lipophilic (paclitaxel and carmustine) and the hydrophilic (carboplatin) chemotherapeutic agents, as well as the large hydrophilic marker, 70 kDa dextran. Cereport was also tested in combination with the chemotherapeutic drugs carboplatin, vinorelbine, gemcitabine and carmustine to determine if Cereport could enhance the survival benefit beyond that provided by chemotherapy alone. RESULTS: Cereport enhanced the uptake of carboplatin and dextran, but not paclitaxel or carmustine. The pattern of Cereport's uptake effect with carboplatin revealed that Cereport selectively increased the proportion of highly permeable regions. Survival was significantly enhanced when Cereport was combined with either carboplatin, vinorelbine, or gemcitabine, but not carmustine, compared to each chemotherapeutic agent alone. CONCLUSIONS: These data provide the first evidence that Cereport, or any receptor-mediated approach intended to enhance the permeability of the blood-brain tumor barrier, can increase the delivery hydrophilic drugs to metastatic tumors in the brain, increasing survival in tumor-bearing rats.

Enhanced delivery of carboplatin into brain tumours with intravenous Cereport (RMP-7): dramatic differences and insight gained from dosing parameters.

Cereport (RMP-7) is a selective bradykinin B2 receptor agonist which increases the permeability of the 'blood-brain tumour barrier' (BBTB) to increase delivery of chemotherapeutic agents to brain tumours. A series of experiments was performed in an RG2 rodent model of glioma to evaluate and refine intravenous (i.v.) parameters to optimize Cereport's clinical utility. The first experiment demonstrated that while carboplatin levels were increased by twofold when given as a bolus during the Cereport infusion, no increase in carboplatin levels were seen when Cereport and carboplatin were simultaneously co-infused for 15 min. A subsequent experiment established that a major factor responsible for the lack of an effect with the co-infusion paradigm was tachyphylaxis to Cereport during the 15 min infusion, for a progressively diminished response to Cereport occurred over that time frame, as plasma levels of carboplatin were rising. A final experiment adjusted the timing of the Cereport and carboplatin infusions so that higher plasma carboplatin levels were achieved prior to initiating the Cereport infusion. Significant uptake effects were achieved when the carboplatin infusion preceded the Cereport infusion by 10 min (i.e. 5 min overlap in the delivery of the two agents). Collectively, these data provide the first systematic evaluation of dosing parameters involving receptor-mediated changes in BBTB permeability and provide new information regarding the pharmacodynamics and potential clinical use of Cereport.

Central analgesic actions of loperamide following transient permeation of the blood brain barrier with Cereport (RMP-7).

The bradykinin analog, Cereport (RMP-7), was designed to increase permeability of the blood brain barrier (BBB). Over the past several years it has been developed primarily as a means of increasing permeability of the blood brain tumor barrier, where early evidence indicated a particularly robust and reliable effect. The present series of experiments were intended to determine whether Cereport might also be used to increase delivery of pharmacological agents across the normal (i.e., non-tumor) BBB. This was accomplished by testing the ability of Cereport to enhance delivery of the peripherally acting opiate agonist, loperamide, to the brain, as evidenced by induction of a centrally mediated analgesic effect. Intravenous administration of a combination of Cereport and loperamide produced a significant analgesic effect (2-fold increase in response times) when animals were tested on a hotplate apparatus. Loperamide alone did not produce analgesia. An analysis of the time course of analgesia revealed a graded onset of analgesia which peaked at 30 min, maintained asymptote at 60 min, and began to diminish by 120 min following Cereport and loperamide administration. Finally, the analgesic effects of combining Cereport and loperamide were completely blocked when animals were pre-treated with the opiate antagonist naloxone, demonstrating that the analgesia was mediated through opiate receptors. Collectively, these results suggest that Cereport was able to increase delivery of loperamide across the BBB, allowing it to gain access to opiate receptors in the CNS to produce a centrally mediated analgesic effect. They therefore provide clear evidence that safe and well-tolerated doses of Cereport can increase permeability of the normal (i.e., non-tumor) BBB. Moreover, they provide the first evidence of a pharmacological effect specifically enabled by controlled (i.e., receptor-mediated) modulation of the BBB.

Controlled modulation of BBB permeability using the bradykinin agonist, RMP-7.

Previous studies have shown that the bradykinin agonist, RMP-7, can safely permeabilize the blood brain barrier (BBB) by activation of constitutive B2 receptors on endothelial cells. The paper describes a series of studies using quantitative autoradiography and intracarotid infusions of RMP-7 to further elucidate the effect on BBB permeability. Because earlier studies also demonstrated that even greater effects of RMP-7 were observed in the BBB associated with brain tumors, animal models were employed so that comparisons could be made between the effects of RMP-7 within tumor, brain tissue proximal to tumor, and brain tissue distal from tumor. In the first study, the effect of RMP-7 on enhancing the BBB permeation of three compounds of different physical characteristics was directly compared ([14C]carboplatin, small, hydrophilic; [14C]dextran, large, hydrophilic; [14C]BCNU, small, lipophilic). RMP-7 increased permeability of the vascular barriers to both hydrophilic compounds, carboplatin and dextran. While the effects of RMP-7 were observed on nontumor BBB, the greatest and most consistent effects were observed on the blood brain tumor barrier. This was true for both carboplatin and dextran, with progressively less effect seen as the distance from tumor boundary increased. This topographic effect was more pronounced with the larger molecular weight compound, dextran. No effect of RMP-7 was seen in permeabilizing the BBB or the blood brain tumor barrier for the lipophilic drug, BCNU. In a second study, the generality of RMP-7's effects was established by demonstrating similar increases in permeability to carboplatin in both inbred (Fischer 344) and outbred (Wistar) rat strains, implanted with varying tumor cell lines. Finally, several additional studies were performed to gain greater insight into the dynamics involved with eventual restoration of the BBB following RMP-7 administration. In one series, it was demonstrated that the BBB begins to close nearly immediately upon withdrawal of RMP-7, with complete restoration occurring within minutes. In another series, tachyphylaxis or desensitization resulting from continuous RMP-7 infusion was studied. These studies demonstrated that 60 min of continuous RMP-7 infusion resulted in complete, spontaneous restoration of the barrier to both carboplatin and dextran. Moreover, the desensitization appears to be linked to the initial activation of the receptors in a way which suggests that obligatory desensitization may exist as part of a more complete response. These data are discussed as they relate to practical issues to enhance delivery of drugs across the BBB, as well as more fundamental issues involving the function of the BBB and its interaction with the brain.

Coordinate induction of the urea cycle enzymes by glucagon and dexamethasone is accomplished by three different mechanisms.

Induction of the mRNAs of the five urea cycle enzymes by glucagon and dexamethasone was studied in cultured rat hepatocytes to define mechanisms which coordinate the increases in the enzyme activities by these hormones. The transcription rate for arginase mRNA increased 9-fold in 7 h, the mRNA level 90-fold in 28 h, and the arginase activity 1.5-fold at 48 h, suggesting that induction is due primarily to stabilization of mRNA. Arginase mRNA induction was minimal with either hormone alone, combined hormones were synergistic, and cycloheximide pretreatment did not prevent the rise in mRNA levels. Carbamyl phosphate synthetase mRNA levels responded synergistically to the combined hormones and peaked 240-fold above controls at 24 h although activity only increased 1.4-fold at 48 h. Argininosuccinate lyase and synthetase mRNAs were induced by an increased transcriptional rate, were not induced by single hormones, responded synergistically to combined hormones, and showed a partial blockage of mRNA induction by cycloheximide. The ornithine transcarbamylase mRNA level was not increased by these hormones although activity increased 1.3-fold, suggesting stabilization of the enzyme. Thus glucagon and dexamethasone induce the urea cycle enzymes by three different mechanisms: transcriptional control of mRNA in argininosuccinate synthetase and lyase, stabilization of mRNA in carbamyl phosphate synthetase and arginase, and protein stabilization of ornithine transcarbamylase.

Dexamethasone and glucagon cause synergistic increases of urea cycle enzyme activities in livers of normal but not adrenalectomized rats.

Adrenalectomized and intact rats were given constant high-dose infusions of glucagon, 0.3 mg/kg per day for 7 days, with or without low-dose dexamethasone, 0.01 mg/kg daily, to test whether glucocorticoids potentiate glucagon induction of the 5 urea cycle enzymes as they do in cultured rat hepatocytes. Glucagon did not induce any of the urea cycle enzymes in adrenalectomized Sprague-Dawley rats and only induced argininosuccinate lyase (EC 4.3.2.1) in adrenalectomized inbred Wistar-Furth rats. Dexamethasone alone induced arginase in adrenalectomized and in intact Wistar-Furth rats and restored the other enzymes to normal levels in adrenalectomized rats. In intact Wistar-Furth rats, the combination of hormones gave synergistic increases of all 5 enzymes over the responses to each hormone alone, but in adrenalectomized rats the combination was only additive or less than additive compared with the sum of single hormone responses. The lack of synergism between the two hormones in adrenalectomized rats suggest that other factors play a role in glucagon induction of this cycle.

Urea cycle enzyme activities are normal and inducible by a high-protein diet in CCl4 cirrhosis of rats.

We produced moderately severe, inactive micronodular cirrhosis in rats using CCl4 and measured the urea cycle enzyme activities in liver after feeding a 15% casein diet for 1 week and again after a 60% casein diet for 1 week. There was no deficiency of any of the five urea cycle enzymes in cirrhotic livers of rats pair-fed the 15% casein diet. Argininosuccinate synthetase and carbamyl phosphate synthetase activities were lower than in non-pair-fed controls by some baselines. All five enzymes in cirrhotic livers were induced 1.5- to 3-fold by the high-protein diet expressed as units per 100 gm of rat. The level of carbamyl phosphate synthetase activity was lower in the livers of rats pair-fed the 60% casein diet than in control livers based on wet weight, collagen-free protein and DNA, but the activities were equal expressed as units per 100 gm of rat. This example of CCl4-induced cirrhosis in the rat does not serve as a good model for human cirrhosis, in which the urea cycle enzymes are reported to be decreased in activity.

Differing effects of arginine deficiency on the urea cycle enzymes of rat liver, cultured hepatocytes and hepatoma cells.

We have confirmed that arginine-deficient diets increase the liver activities (units per 100 g) of the first four arginine biosynthetic enzymes of the urea cycle in Wistar rats, but not the activity of arginase. In contrast, rat liver cells cultured in monolayers for 48, 72 or 96 h in arginine-free L-15 or minimum essential medium showed no changes in carbamoyl-phosphate synthase (EC 6.3.4.16), ornithine transcarbamylase (EC 2.1.3.3), argininosuccinate synthase (EC 6.3.4.5), argininosuccinase (EC 4.3.2.1) or arginase (EC 3.5.3.1) activities. The arginine content of the cells grown on deficient medium was 36% of that of cells grown on 2.9 mM arginine-sufficient L-15, yet the urea excretion rate into the medium was reduced to 7% of the rate in control cells and the excretion of orotic acid was 400% of that in control cells. A Morris rat hepatoma cell line, 7800C1, which maintains activities of all five urea cycle enzymes, showed no consistent increases in the activities of the first four enzymes when the arginine in the medium was varied between 0 and 2 mM. Thus, in spite of severe arginine deficiency, cultured rat liver cells and hepatoma cells do not show the derepression-like response seen by other investigators when nonliver cells were cultured in arginine-deficient media. The difference between in vivo and in vitro effects of arginine deficiency on urea cycle activities remains unexplained.

Effect of a maternal fast on the urea cycle enzymes of the ovine fetus.

Activities of five urea cycle enzymes were measured in maternal and fetal sheep liver during the normal fed state and following 5 days of fasting. Six ewes and 10 fetuses were studied in both the fed and fasted periods at 132 days gestation (term: 147 days) for liver protein and enzyme levels. Results indicated that protein content increased during fasting in both the maternal and fetal liver. Fetal liver weight was decreased during fasting from 108 +/- 8.5 to 71 +/- 8.2 g (mean +/- SD) (p less than 0.001). Fed state fetal enzyme activities per gram liver were 50-125% of maternal values. After fasting, four of the five fetal enzymes increased approximately twofold to fivefold (per gram tissue) (ornithine transcarbamylase did not change). Only one enzyme (argininosuccinase) increased significantly in maternal liver. Total liver activities gave similar results. These data indicate that the in vivo studies that demonstrate a doubling in fetal urea production in the fasted sheep in later gestation are associated with parallel increases in the fetal hepatic activities of several enzymes that are responsible for urea synthesis.