The Effect of Dietary o,p’-DDT on Ureagenesis from Ammonia by Isolated Rat Hepatocytes’ K. C. Department
D. L. STORY, AND R. A. FREEDLAND
Sciences, School of Veterinary Medicine, Davis, California 9.5616
University of California,
Received February 22, 1978; accepted June 13, 1978
The Effect of Dietary o,p’-DDT on Ureagenesis from Ammonia by Isolated Rat Hepatocytes. TRIEBWASSER, K. C., STORY, D. L., AND FREEDLAND, R. A. (1979). Toxicol. Appl. Pharmacol. 47, 237-244. Hepatocytes isolated from rats which had been fed 1000 ppm o,p’-DDT for 2 weeks and starved for 48 hr exhibited markedly reduced rates of urea synthesis and citrulline accumulation when incubated with 10 mM NH&I and 10 mM ornithine. The addition of 10 mM lactate to such incubations stimulated the rate of ureagenesis by both control and DDT-treated cells to nearly the same degree but did not eliminate the difference in rates of urea synthesis between control and DDT-treated cells. The activities of carbamoylphosphate synthase, ornithine carbamoyhransferase, arginine synthetase, and arginase were similar in livers from control and DDT-treated rats. The kinetics of the stimulation of urea synthesis by exogenous ornithine were similar with hepatocytes from both control and DDT-treated rats. Mitochondria isolated from the livers of control and DDT-treated rats exhibited similar rates of citrulline synthesis from ammonia and ornithine as well as similar responses to N-acetylglutamate and 2,4-dinitrophenol. These results suggest that DDT treatment may produce alterations in the kinetic behavior of urea cycle enzymes which are not detected in the standard assays of these enzymes or DDT treatment may interfere with the intermitochondrial transport processes required during ureagenesis from ammonia.
Previous reports from this laboratory have shown that hepatocytes isolated from rats fed o,p’-DDT exhibit decreased capacity for gluconeogenesis from lactate (Story et al., 1976; Story and Freedland, 1978a). Another hepatic function of critical importance to mammals, especially during periods of starvation, is the disposal of nitrogen. In mammals, this process occurs predominantly through the synthesis and excretion of urea. Urea synthesis, like gluconeogenesis, involves reactions which occur in both the cytosolic and mitochondrial compartments i Supported in part by USPHS Grant ESO054.
of the cell and therefore requires the transport of various intermediary metabolites across the mitochondrial membrane. It was of interest, therefore, to determine if the feeding of oq’-DDT to rats would result in a decrease in the capacity of isolated hepatocytes to synthesize urea from ammonia. Such a result of DDT exposure might suggest that a starving animal would be very susceptible to ammonia toxicity. This would be of partitular importance if the animal were refed a high protein diet, a treatment known to tax the nitrogen disposal system of an animal which has been starved for a period of time (Wergedal and Harper, 1963). 237 All
004-008X/79/020237-08$02.00/0 Copyright 0 1979 by Academic Press, Inc. rights of reproduction in any form reserved. Printed in Great Britain
TRIEBWASSER, STORY, AND FREEDLAND
Previous investigations (Briggs and Freedland, 1976, 1977; Triebwasser and Freedland, 1977 ; Meijer et al., 1975) have demonstrated that in hepatocytes isolated from starved rats, urea synthesis from ammonia is controlled by the level of urea cycle intermediates in the cell. Also important in the control of ureagenesis is the level of C, and C4 intermediates involved in the synthesis and transport of the aspartate which is required during urea synthesis. Only under certain conditions (i.e., the presence of adequate concentrations of ammonia, ornithine, and lactate) does the rate of urea synthesis by isolated hepatocytes approach the activity of argininosuccinate synthetase, the urea cycle enzyme having the lowest activity (Briggs and Freedland, 1977). METHODS Male Sprague-Dawley rats* were fed ground laboratory diet with or without 1000 ppm o,p’-DDT3 for 2 weeks. Rats were starved 2 days prior to isolation of the hepatocytes. Hepatocyte isolation and incubation techniques were as described by Cornell et al. (1973). Glucose was determined by the method of Krebs et al. (1964) and urea and citrulline were assayed as described by Briggs and Freedland (1976). Intact mitochondria were isolated as described by Chappell and Hansford (1972) following homogenization of the liver in 0.3 M mannitol, 3.4 mM Tris [tris(hydroxymethyl)aminomethane], and 1 mM EGTA [ethyleneglycol-bis-(8-amino-ethylether)-N,N’-tetraaceticacid]. Mitochondria were incubated as described by Triebwasser and Freedland (1977) using the incorporation of radioactivity from [%]bicarbonate into acidstable products as the measure of citrulline synthesis (McGiven et al., 1976). Radioactivity was determined by liquid scintillation using AquasoP liquid scintillation fluid. Urea cycle enzyme activities were determined as described by Brown and Cohen (1959). Urea and citrulline formed in the enzyme assays were determined by the method of Foster and Hocholzer (1971), with the exception of the arginine synthetase assay, where urea was determined as described by Brown and Cohen (1959). The data were analyzed by the paired t test or Student’s t test, as indicated (Sokal and Rohlf, 1969). z Hilltop Lab Animals, Scottsdale, Penn. “1 -(o-chlorophenyl)1 -(p-chlorophenyl)-2,2,2chloroethane, Aldrich Chemical Co. 4 New England Nuclear, Boston, Mass.
RESULTS The data in Table 1 show that hepatocytes isolated from rats which had been fed 1000 ppm o,p’-DDT for 2 weeks and starved for 48 hr exhibited a markedly reduced capacity to synthesize urea from ammonia. The rates of urea synthesis observed in hepatocytes from control rats are similar to previously reported rates of urea synthesis from ammonia by hepatocytes from starved rats (Briggs and Freedland, 1977; Triebwasser and Freedland, 1977) and are comparable to rates of urea synthesis reported for the perfused liver under similar conditions, e.g., with ammonia and ornithine present (Hems et al., 1966; Kramer and Freedland, 1972). The rate at which citrulline accumulated in the incubations was also reduced by DDT treatment, although this parameter was not as severely affected as urea synthesis. The addition of lactate resulted in a stimulation of urea synthesis in hepatocytes from both control and DDT-fed rats. The stimulation of unreagenesis by lactate was slightly greater in hepatocytes from DDT-fed rats ; however, lactate addition did not eliminate the difference in urea synthetic rates between hepatocytes from rats fed control and DDT diets. Lactate is considered to stimulate urea synthesis by increasing the intracellular levels of malate and aspartate, thus removing any limitation of the availability of aspartate in the cytosol where it is required for the conversion of citrullipe to arginine (Meijer et al., 1975; Briggs and Freedland, 1976; Treibwasser and Freedland, 1977). Urea synthesis from ammonia plus ornithine and lactate by hepatocytes from starved rats is considered to be limited by the activity of argininosuccinate synthetase (Briggs and Freedland, 1977). The response of hepatocytes from DDT-fed rats to lactate suggested that the activity of this enzyme might be depressed in these liver cells. The level of the urea cycle enzymes have been suggested to be under coordinate control in rat liver (Schimke, 1962), thus a depression in the activity of one
DDT AND LIVER UREAGENESIS
enzyme would suggest that others might also be decreased. The activities of four urea cycle enzymes were measured and reported as a function of liver weight, body weight, or liver protein content (Table 2). No value for enzyme activity was statistically significantly
different from the control values in any two of these three functions, although two enzymes were statistically different in one of the three. Since enzyme activities were not limiting urea synthesis, the possibility was con-
EFFECT OF DIETARY DDT ON UREA SYNTHESIS AND CITRULLINE ISOLATED
nmol/min/mg Substrates [lo mM] >
NH&l Lactate Ornithine
Percentage of control
Percentage of control
71.3 f 1.2
254 rt 36
709 + 55
55.5 f 12.2
a Hepatocytes from control or DDT-fed rats were incubated in duplicate with the indicated substrates. The rates of urea synthesis and citrulline accumulation were determined to be linear over a 40-min incubation period. The rates are given as the mean f SE. b Numbers in parentheses indicate the number of animals. c Statistically different from control rate by paired t statistic, p -c 0.01. * Statistically different from control rate by paired t statistic, p < 0.05. e Statistically different from control rate by paired t statistically, p-c 0.025. TABLE UREA CYCLE ENZYME
Enzyme activities”* ’ nmol/min/mg Carbamoylphosphate synthase Omithine carbamyl transferase Arginine synthetase Arginase
Control DDT Control DDT Control DDT Control DDT
36.01+ 2.8 35.9+ 1.7 1,497 + 260 1,336+223 12.2 f 0.63 13.0+0.61 39,508 f 2,308 36,171+ 2,576
4.74kO.12 4.50+0.09 268 + 1.2 221* 5.4e 1.40*0.03 1.46kO.04 522+ 16.5 459 f 40.0
g body wt
15.55kO.49 17.15f0.28” 87lk21.2 869k33.7 5.57f0.04 5.55 + 0.19 1,712.g 5 53.5 1,744.3 f 120.0
’ Rats were fed ground laboratory diet with or without 1000 ppm o.p’-DDT for 2 weeks and were starved for 48 hr prior to removal of the livers for enzyme assays. b Activities are presented as the mean+_SE, n = 4. ’ Citrulline formation was used as a measurement of carbamoylphosphate synthase and ornithine carbamyltransferase activities. Urea formation was used as a measurement of arginine synthetase and arginase activities. d Significantly different from the controls, p < 0.05 by Student’s t test. ’ Significantly different from the controls, p < 0.01 by Student’s t test.
FIG. 1. The effect of varying ornithine concentration on the rate of ureagenesis from ammonia. Hepatocytes from control (0) or DDT-fed (0) rats were incubated with 10 mM NH,Ci in the presence of 0, 2, 5, 10, 20, or 40 mM ornithine. Rates of urea synthesis were determined over a 40-min incubation period and were linear at all ornithine concentrations. The inset depicts the plot of l/[ornithine], mM versus the reciprocal of the corresponding rate of urea synthesis.
sidered that DDT could be influencing the ornithine-mediated stimulation of urea synthesis. To investigate this possibility, hepatocytes were incubated with 10 mM NH&I and increasing concentrations of ornithine and the rates of urea formation were measured. Urea synthesis by hepatocytes from rats fed control and DDT diet responded in a hyperbolic manner to increases in ornithine concentration (Fig. 1). Analysis of the data by Lineweaver-Burk plot (insert, Fig. 1) revealed that the concentration of ornithine which yielded a half-maximal rate of urea synthesis (approximately 5.7 mM) was unchanged by DDT feeding. Because the hepatocytes contained urea cycle intermediate prior to the addition of exogenous ornithine, the kinetic calculations were also examined using an adjusted value in which the rate of urea synthesis from 10 mM NH&l alone was subtracted from the rates observed when ornithine was added. Under these conditions there was also no difference in ornithine stimulation between hepatocytes from control and DDT-fed rats. DDT feeding did not significantly affect the ability of ornithine or lactate to stimulate urea synthesis nor did it influence the level of urea cycle enzyme activity as measured in vitro. When the activity of carbamoylphosphate synthase was measured in vitro,
N-acetylglutamate was added to ensure that the total enzyme activity was measured. In the intact cell, the mitochondrial content of Nacetylglutamate is postulated to play a role in the activation of carbamoylphosphate synthase (Shigesada and Tatibana, 1971; Tatibana and Shigesada, 1976; McGiven et al., 1976). It was considered that carbamoylphosphate synthase might be less activated by N-acetylglutamate in liver mitochondria from rats fed DDT, resulting in the decreased rate of ureagenesis observed. To examine this possibility, the degree of activation was estimated by comparing citrulline production in intact mitochondria with maximal carbamoylphosphate synthase activity in mitochondria homogenates. It has been suggested that the rate of citrulline formation in intact mitochondria is an indirect measure of the Nacetylglutamate content of the mitochondria and the amount of activated carbamoylphosphate synthase (McGiven et al., 1976). DDT feeding did not significantly affect the capacity of isolated liver mitochondria to synthesize citrulline from ammonia and ornithine (Table 3). When the rate of citrulline synthesis in intact mitochondria is compared to the maximal rate of citrulline synthesis in sonicated mitochondria with Nacetylglutamate present, it was found that carbamoylphosphate synthase is 10 to 20%
DDT AND LIVER UREAGENESIS TABLE CITRULLINE ISOLATED
FROM THE LIVERS OF DDT-FED RATS'
5 mM N-acetylglutamate 10 mM N-acetylglutamate 0.5 PM 2,4-dinitrophenol 1.O PM 2+dinitrophenol 2.0 ,UM2,4-dinitrophenol 5.0 PM 2,4-dinitrophenol 20 fin 2.4-dinitrophenol
MITOCHONDRIA CONTROL AND
14.8 f 3.2 18.lk1.9 Percentage of the rate in the absence of additions Control
113 &- 5.5b 126 f 11.1 84k2.5 73 + 2.2b 54$3.8b 32 0
108k8.7 12Ok8.7 82+7.3b 83+7.2b 55+3.6b 23 0
’ Mitochondria were incubated with (final concentrations) KCI, 80 mM; Tris, pH 7.4, 25 mM; KHCOS, IOmM; KHZPOI, 10 mM; NH&l, IO mM; 10 mM; succinate, 10 mM; rotenone, ornithine, 0.5 pg/ml and [14C]NaHC0,, 0.8 &i/ml. Citrulline synthesis was measured as the radioactivity in acidstable products after acidification and bubbling with CO1 to remove remaining [r4C]NaHC03. The amount of incorporation in the absence of ornithine was observed to be less than 5 % of the control rate of citrulline synthesis and this value was subtracted to correct for ornithine independent incorporation of radioactivity. b Significantly different from the rate in the absence of additions, p c 0.05 by paired t statistic.
activated by N-acetylglutamate in liver mitochondria from both control and DDT-fed rats. This observation excludes the possibility that DDT is decreasing ureagenesis by causing a decreased activation of carbamoylphosphate synthase by N-acetylglutamate. As can be seen in Table 3, the addition of N-acetylglutamate to incubations containing intact mitochondria from rats fed DDT and control diet resulted in small increases in citrulline synthesis. This indicates that the permeability of mitochondria to N-acetylglutamate was minimal and similar in both groups of rats. The two populations of mitochondria also displayed similar inhibition of citrulline synthesis by 2,4-dinitrophenol.
DISCUSSION The results presented in this study indicate that hepatocytes isolated from rats fed o,p’-DDT for 2 weeks and starved for 48 hr exhibit markedly reduced rates of urea synthesis and citrulline accumulation when incubated with 10 mM NH&l and 10 mM ornithine. The observation that the urea cycle enzyme activities were not altered by DDT feeding suggests that in hepatocytes from rats fed DDT, the rate of urea synthesis from ammonia plus ornithine and lactate does not reflect a decrease in the activity of argininosuccinate synthetase. These cells may have a different rate limiting step in urea synthesis than do hepatocytes from control animals incubated under the same conditions. Alternatively, DDT treatment may have caused changes in the kinetic characteristics of the enzyme which result in a decreased activity in the intact cell, but which were not detected under the assay conditions utilized in this study. According to a recent report by Rochovansky et al. (1977), argininosuccinate synthetase possesses complex, substrate-induced, allosteric regulatory properties and it is possible that these properties could have been altered by DDT feeding. Such an effect might explain how DDT feeding could cause the observed reduction in the urea synthetic capacity of the intact cell, without reducing the amount of enzyme activity assayable in homogenates under optimum donditions. Since urea cycle enzyme activities are known to be affected by alterations in the level of circulating glucocorticoids (Schimke, 1963; Freedland, 1964; Freedland and Sodikoff, 1962), the observation of unaltered enzyme activities also suggests that rats fed DDT have functionally normal levels of circulating glucocorticoids. This is of interest considering that o,p’-DDD, a metabolite of o,p’-DDT, has been shown to alter the metabolism of cortisol (Kupfer and Peets, 1966; Kupfer and Bulger, 1976), and suggests that feedback mechanisms may exist which
increase the rate of cortisol synthesis to maintain homeostasis if the rate of cortisol degradation is increased. When the response to ornithine stimulation was examined in hepatocytes from control and DDT fed rats, no substantial differences could be detected between the two populations of cells. Ornithine is considered to stimulate urea synthesis by increasing the intracellular level of urea cycle intermediates, thereby promoting an increased flux through the urea cycle (Hems et al., 1966; Kramer and Freedland, 1972; Briggs and Freedland, 1976). The finding that similar concentrations of ornithine produced similar half-maximal rates of urea synthesis suggests that there is no effect of DDT feeding on the ability of the cell to take up ornithine. It has been reported that the administration of DDT to rats results in changes in the structure of mitochondria which are suggestive of membrane alterations (Kimbrough et al., 1971). Lack of significant stimulation by exogenous N-acetylglutamate observed in mitochondria from both control and DDTfed rats (Table 3) suggests that the mitochondrial membranes were intact, since Nacetylglutamate does not readily penetrate the intact, inner mitochondrial membrane Charles et al., 1967; McGivan et al., 1976). Treatment of rats with p,p’-DDT has been reported to result in a decreased efficiency of oxidative phosphorylation and subsequent alteration in energy production (Byczkowski, 1976). If the electron transport chain of the mitochondria isolated from DDT-fed rats in the present study had been partially uncoupled by DDT, then one might have expected mitochondria from DDT-fed rats to be more susceptible to the effects of 2,4dinitrophenol than mitochondria from control rats. This effect was not observed (Table 3), suggesting that DDT feeding did not seriously impair the ability of mitochondria to produce energy via the oxidation of succinate. In addition to the possibility of altered enzyme kinetic behavior as an explanation for
the effects of DDT reported here, there is the possibility that DDT could be affecting anion transport, a mechanism suggested by Meijer et al. (1975) to be required during unreagenesis. This contention is supported by the observations that processes such as gluconeogenesis from fructose or glycerol and ketogenesis from oleate, which do not require intermitochondrial transport of anionic metabolites, are not affected by DDT feeding (Story and Freedland, 1978a). However, gluconeogenesis from lactate, which requires the transport of aspartate from the mitochondria (Rognstad and Clark, 1974; Smith et al., 1977) is also reduced by DDT feeding (Story and Freedland, 1978a). These observations suggest that DDT may influence the mitochondrial membrane in a manner which does not alter its permeability to N-acetylglutamate or its ability to carry out oxidative phosphorylation but which does reduce the capacity for anion transport processes required in ureagenesis from ammonia and gluconeogenesis from lactate. Singhal and Kacew (1976) have produced evidence that o,p’-DDT administration results in increase in liver adenylate cyclase and cyclic-AMP levels. Story and Freedland (1978b) have recently shown that hepatocytes isolated from rats fed o,p’-DDT exhibit a decreased stimulation of gluconeogenesis from lactate by cyclic-AMP. The effects of o,p’-DDT reported here cannot be explained by elevated levels of cyclic-AMP, as previous work indicates that an increase in intracellular cyclic-AMP would stimulate unreagenesis from ammonia rather than decrease it (Triebwasser and Freedland, 1977). It is apparent from the observations reported here that the feeding of o,p’-DDT to rats reduces the capacity of hepatocytes, subsequently isolated from those rats, to synthesize urea. The explanation for these phenomena is not yet apparent; however, the existence of such an effect may be of great importance to animals exposed to DDT and subjected to the stress of starvation.
ACKNOWLEDGMENT The authors would like to thank his technical assistance.
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