- Email: [email protected]
The effect of o,p′-DDT feeding and food deprivation on plasma and liver amino acids and liver serine dehydratase in rats
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
50, 329-335 (1979)
The Effect of o,p’-DDT Feeding and Food Deprivation on Plasma and Liver Amino Acids and Liver Serine Dehydratase in Rats DAVID L.
STORY’
AND RICHARD
A. FREEDLAND
Department of Physiological Sciences, School of Veterinary Medicine, University of Claifornia, Davis, Davis, California 95616 Received September
12, 1978; accepted May 7, 1979
The Effect of o,p’-DDT Feeding and Food Deprivation on Plasma and Liver Amino Acids and Liver Serine Dehydratase in Rats. STORY, D. L., AND FREEDLAND, R. A. (1979). Toxicol. Appl. Phurmacof. 50, 329-335. The effect of DDT feeding and food deprivation on liver and plasma amino acid concentrations was studied. DDT feeding had no effect on plasma amino acids but resulted in an increase in the concentratipn of liver isoleucine, ornithine, phenylalanine, serine, and threonine. In rats that were fed the control or DDT for 2 weeks and then deprived of food for 48 hr, the concentrations of liver isoleucine, phenylalanine, serine, and threonine were similar. The activity of liver serine dehydratase was decreased in hepatocytes from DDT-fed rats compared to fed controls. After food deprivation, the activity of serine dehydratase was similar in hepatocytes from control and DDT-fed rats, which is consistent with the response of liver serine and threonine concentrations to DDT feeding. Gluconeogenesis from serine was determined in isolated hepatocytes from DDT and control-fed rats and it was found that rates of gluconeogenesis were not affected by DDT feeding. This would indicate that the activity of serine dehydratase is not limiting for gluconeogenesis from serine in hepatocytes from fed rats.
The effects of pesticides on free amino acid concentrations have not been extensively investigated. Mehrle et al. (1971) have reported DDT and dieldrin-induced changes in serum amino acids in trout. Alterations in amino acid concentrations have also been observed in plasma (Tocci et al., 1969) and urine (Davies et al., 1969) of humans occupationally exposed to pesticides. There have been no reports as to the effects of o,p’-DDT feeding on plasma and liver-free amino acid concentrations in fed and starved rats. Starvation has been shown to influence the concentrations of specific plasma and liverfree amino acids in rats (Adibi, 1971). Furthermore, starvation results in a release of DDT from adipose tissue with a resultant r Present address: Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass. 02139.
increase in DDT concentrations in plasma, kidney, and liver of rats (Dale et al., 1962). Starvation in combination with prior DDT feeding may elicit a response in plasma or liver amino acid patterns that is different from patterns observed in DDT-fed rats that have not been starved, or in starved rats that have not been exposed to DDT. A cumulative effect could have important metabolic consequences on the amount of free amino acids available in the liver for metabolic processes. One purpose of this study is to determine if DDT feeding results in changes in plasma or liver amino acid concentrations and if subsequent food deprivation alters the response of these amino acid concentrations to DDT feeding. Studies were conducted to determine the mechanism responsible for these alterations in amino acid concentrations. 329
0041-008X/79/110329-07%02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
330
STORY
AND
METHODS Male Sprague-Dawley rats were used in all experiments. Animals were housed in a room with a 12-hr light-dark cycle beginning at ~:~OA.M. Food was withheld from some animals for 48 hr prior to sacrifice. All animals were given water ad lib&/m. Fed animals had feed available until the time of sacrifice, which was between 9:00 and 1O:OOA.M. for all animals. In experiments designed to determine the effects of DDT feeding on plasma and liver amino acid concentrations, rats weighing approximately 150 g were fed 15 % protein laboratory chow with or without 1000 ppm o,p’-DDT ad libitum for 2 weeks. Rats were anesthetized with 60 mg/kg sodium pentobarbital. Blood was taken by cardiac puncture and centrifuged in a refrigerated centrifuge at 3000g for 10 min to remove red blood cells. Livers were rapidly excised and immediately frozen with aluminium tongs prechilled in liquid nitrogen. Liver and plasma samples were stored at -70°C until analysis. For analysis, the plasma was added to an equal volume of 6% sulfosalicyclic acid (w/v in doubly distilled water) and protein was removed by centrifugation at 3000g for IO min in a refrigerated centrifuge. Protein-free liver samples were obtained by homogenizing in a Potter-Elvehjem tissue homogenizer in 9 vol of 3% sulfosalicylic acid followed by centrifugation at 3000g for 10 min under refrigeration. Amino acids were determined using a Beckman 121 amino acid analyzer. In experiments conducted to determine enzyme activities and gluconeogenic capacity, 175 to 250-g rats were fed either 15% protein laboratory chow or a 90% casein diet (Table 1) with or without IOGOppm o,p’-DDT for 2 weeks. Rats fed the laboratory chow with and without DDT were deprived of food for 48 hr prior to cell isolation. Cells were isolated by the method of Berry and Friend (1969) as modified by TABLE COMPOSTION
1 OF DIETS
90 % Casein (g/kg) Casein” Dextrose” Corn oil” Mineral premix’ Vitamin premix’
900 0 50 40 10
’ Purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. b Mazola Corn Oil, Best Foods, Englewood Cliffs, N.J. ’ Benevenga, et al. (1964).
FREEDLAND
Cornell et a/. (1973). Hyaluronidase was omitted from the perfusate. Cell preparations and incubations were conducted as per Cornell ef al. (1973). DNA content of the cells was determined by the method of Burton (1956) and glucose was measured by the method of Krebs er al. (1964). In studies using [U-Ylalanine and [U-%]serine, alanine and serine were present in flasks at a concentration of 10 mM with a specific activity of 0.01 &i/~mol. In the isotopic studies, the cell contents’were acidified with 0.3 ml 60% HCIO,, centrifuged at 5OOg for IO min and adjusted to pH1. An aliquot (3 ml) was put on a 1.2x5-cm column of AG 50W x 8 (Hi, 200400 mesh) on top of a I .2 x 5-cm column of Dowex I x 8 (acetate, 100-200 mesh). Glucose was eluted off the column with doubly distilled HZ0 until 35’ml was collected, and 1 ml of this was added to 14 ml of scintillation fluid and counted. Serine dehydratase activity was measured as described by Freedland and Avery (1964). Alanine aminotransferase activity was determined by the method of Freedland er al. (1965). Enzyme activities were measured on a 30,OOOg supernatant df a 0.14 M KC1 homogenate of 1 g of isolated hepatocytes. Statistical significance was determined by analysis of variance and Student’s t test (Sokal and Rohlf, 1969).
RESULTS DDT feeding had no effect on the concentrations of any of the plasma amino acids. Food deprivation had a statistically significant effect on the concentrations of 10 plasma amino acids (Table 2). Effects of food deprivation were observed on the concentrations of plasma aspartic acid, proline, methionine, phenylalanine, ornithine, and glutamine (p
331
DDT EFFECTS ON AMINO ACIDS TABLE EFFECTS OF
DDT FEEDING
ANR/OR
Foot
2
DEPRIVATION
ON PLASMA AMINO
ACIDS OF RATS’
(nmol/ml + SE)
Aspartic acid Threonine
Serine Proline Valine Cysteine
Methionine Isoleucine Leucine Tyrosine
Phenylalanine Tryptophane
Ornithine Lysine Histidine Arginine Hydroxyproline Glycine Alanine Glutamic acid Asparagine Glutamine
Control fed
DDT fed
14k2 188& 13 184+20 186+36 1$4+ 18
13k2 245f21 209 + 37 161+34 227 + 24 90+4 40+2 87k6
77+4
39+6 79+10 134k 18 53+9 41*3 68klO 45+4 501_+91 49+4 198+33 40+3 232k41 453+52 57+6
26+8 770 k 224
151+15 67+7
48f4 78+12
Control food deprived
DDT fooddeprived
20*1
18+3 215? 17 208+ 14 126k 15 200+14 84+6 49*5 88+9 147* 15 70+ 12 6Ok5 68+5 35+5 360+82 51+4 103 * 14 27+2 361+ 36
13 266+_ 13 133+9 197+25 91+4 207+
5Jk6
82&11 139* 17 69+9 66*7 70+7
46rt4 394 + 27 5926
31+4 386+59 54+9
217+_55 34&J
114*14 30+1
205 + 29 327+27
81+ 10 25+4 900 + 278
320+ 17 351+46 119+ 15 29+5 1244+ 143
Statistical significant@ S S S
S S S S S
274+41
111+11
S
2625 1399* 159
S
n Eighteen rats were fed a laboratory chow diet with or without 1000 ppm qp-DDT for 2 weeks; 6 rats per treatment were then deprived of food for 48 hr. Fed rats, n = 3; food-deprived rats, n = 6. b Data analyzed by analysis of variance. Statistically significant effects are designated S, food deprivation. Refer to text for level of significance.
acid, valine, isoleucine, leucine, and glutamic acid (~~0.05) and on the concentrations of liver serine, glycine, and alanine (p ~0.01). The effects of starvation on plasma and liver amino acids have been studied by many investigators, i.e. Adibi (1971). For this reason and since this study is primarily concerned with the effects of DDT and food deprivation together, the effects of food deprivation alone will not be discussed further. A statistically significant interaction (p < 0.05) between food deprivation and DDT was observed for liver serine, threonine, phenylalanine, and glutamic acid concentrations. The interaction between food deprivation and DDT was indicated by the fact that the
effects of DDT on the concentrations of these four amino acids in livers of fed rats was different than in livers of food-deprived rats. Student’s t test was used to determine the statistical significance of these differences. Despite a large interaction in liver ornithine, due to large SE values the interaction was not significant. DDT feeding resulted in a significant (p ~0.05) increase in the concentrations of threonine, serine, phenylalanine, and ornithine in livers of fed rats. There was no difference in the concentrations of serine, threonine, and phenylalanine in livers of control and DDT-fed animals after food deprivation. Thus, food deprivation mitigates the increase in liver serine, threonine,
332
STORY AND FREEDLAND TABLE EFFECU
OF DDT
FEEDING
AND/OR
Foot
3
DEPRIVATION
ON LIVER AMINO
ACIDS OF RATS~
(nmol/g k SE) Control fed Aspartic acid Threonine Serine Proline Valine Cysteine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophane Ornithine Lysine Histidine Arginine Glycine Alanine Glutamic acid Glutamine
5331 x 931 182+45 470+ 135 Trace 247 + 63 lo* 12 76+ 12 114+24 258 f 73 67+14 72+ 14 3lk6 77k8 495+ 160 441&-111 37+20 16795226 2116+ 190 1860+271 10610+1808
DDT fed 5490+ 789 418+67’ 964+ 147’ 105+65 277 f 53 29+21 53+23 199&44 324 + 34 87k 12 114+11= 29k5 212*52= 5062 162 472+_216 22k27 2125+267 213Ok 329 1864+523 95282 1696
Control food deprived
DDT fooddeprived
4437+482 263+ 14 484 rf: 30 63+23 181+_28 41*17 55k6 94* 10 197+30 54+8 73+10 11+6 136+ 14 473+ 126 318+25 17+5 2640+207 1300&350 3654f237 9606 f. 694
3639& 342 321+50 545 f 84 116k49 208+ 19 14* 10 66k8 117+ 16 202k 18 64+5 73+7 18+9 207 Ifr64 421 f 78 369+_ 35 15+6 3106+210 1175+ 150 2244+218’ 11996~1011
Statistical significanceb s D, SxD D, S, SxD S
D, S S S D, SxD D
S S D, S, SxD
4 Experiment was designed as described in Table 1. * Data analyzed by analysis of variance. Statistically significant effects are designated S, food deprivation; D, DDT; SxD, food deprivation x DDT interaction. Refer to text for level of significance. c Means of amino acids showing a starvation x DDT interaction were also analyzed by Student’s t test. Means that are statistically different from controls at p < 0.05 are indicated by a superscript c.
and phenylalanine observed in DDT-fed rats in the fed state. There was a decrease (p< 0.05) in the concentration of liver glutamic acid in DDT-fed rats that had been deprived of food when compared to the food-deprived controls. The concentration of liver ornithine remained elevated in DDT-fed rats after food deprivation, although this value is not statistically greater than the ornithine concentration in livers from control-food deprived rats due to the large SD. It has been suggested that serine and threonine are both catabolized by liver serine dehydratase (Selim and Greenberg, 1960; Goldstein et al., 1962). The possibility was considered that DDT could be influencing the concentrations of these amino
acids by inhibiting serine dehydratase. To investigate this possibility, enzyme activities were measured in hepatocytes from control and DDT-fed rats (Table 4). In hepatocytes from fed rats, DDT feeding resulted in a significant decrease in the activity of serine dehydratase (p < 0.01). The activity of alanine aminotransferase was not significantly decreased by DDT feeding, suggesting that the effect observed on serine dehydratase is not a general effect on amino acid catabolic enzymes. The activity of serine dehydratase was similar to that observed in hepatocytes from food-deprived control rats when hepatocytes were isolated from rats fed a DDT diet for 2 weeks and deprived of food for 48 hr. This effect on serine dehydratase activity in
333
DDT EFFECTS ON AMINO ACIDS TABLE
4
TABLE
EFFECTS OF o,p-DDT FEEDING ON SERINE DEHYDRATASE AND ALANINE AMINOTRANSFERASE AcnvrTIES IN LIVERS OF FED, FOOD-DEPENDENT, OR 90% CASEIN-FED RATS”
5
EFFECTS OF o,p-DDT FEEDING ON GLUCONEOGENESIS FROM AMINO ACIDS IN WLATED HEPAT~CVTES FROM FED AND 48-hr FOOD-DEPRIVED RAW
Glucose formation Control
DDT Substrate (mM)
15 % Protein fed Food-deprived 90 % Casein
Serine dehydratase (pmol/min/mg DNA) 0.71 f 0.09 (5) 0.24? 0.04b (5) 0.43 f 0.05 (5) 0.37 f 0.05 (5) 2.8OkO.40 (4) 1.59kO.13’ (4)
15 % Protein fed Food-deprived 90% Casein
Alanine Aminotransferase (pmol/min/mg DNA) 5.89 f 0.96 (5) 4.73 + 0.78 (5) 3.6551.37 (5) 3.16kO.69 (5) 10.40+ 1.20 (6) 8.77kO.80 (6)
’ Hepatocytes were isolated and enzyme activities were determined in a 30,OOOg supernatant of a 0.14 M KC1 homogenate of 1 g of hepatocytes. b Indicates values are statistically different from controls at p < 0.01 as determined by Student’s t test. c Indicates values are statistically different from controls at p < 0.05 as determined by Student’s t test.
hepatocytes from rats fed DDT correlates well with the DDT effect on serine and threonine concentrations observed (Table 3). DDT feeding also promoted a decrease in serine dehydratase activity in hepatocytes from rats fed a 90% casein diet (p <0.05), thus the DDT effect on the activity of this enzyme is apparent in hepatocytes from fed and 90% casein-fed rats but is absent in hepatocytes after food deprivation. It has been demonstrated that serine dehydratase plays an important role in the metabolism of serine in the perfused rat liver (Chan and Freedland, 1971; Bhatia et al., 1975). It is possible that a decrease in the activity of this enzyme in livers of DDT-fed rats could result in a decreased ability of these rats to synthesize glucose from serine. To determine if this DDT-induced decrease in liver serine dehydratase activity could have the effect of decreasing gluconeogenesis from these amino acids, gluconeogenic rates were measured in isolated hepatocytes. Gluconeogenesis from serine and threonine was not
Control
DDT
Serine, 10 Alanine, 10 Threonine, 10
Hepatocytes from food-deprived rats (pmol/min/mg DNA) 0.129f0.006 (8) 0.129+0.016 (6) 0.168kO.007 (8) 0.15OkO.016 (6) 0.057 f 0.006 (6) 0.043 f 0.043 (4)
Serine, 10 Alanine, 10
Hepatocytes from Fed Rats (flmol/min/mg DNA) 0.071 f. 0.004 (3) 0.073 + 0.006 (3) 0.168+0.004 0.154+0.010 (3).
’ Hepatocytes were incubated with the indicated substrate for 45 min in duplicate. In studies measuring rates of gluconeogenesis in hepatocytes from fed rats [U-Wlalanine and [U-W]serine were present at a concentration of 0.01 @/pmol. Rates have been corrected for loss of label due to randomization and decarboxylation. Values are means+ SE for the number of animals in parenthesis.
affected by DDT feeding in isolated hepatocytes from food-deprived rats (Table 5). This would be expected on the basis of the finding of similar activities of serine dehydratase in isolated hepatocytes from control and DDTfed rats that had been deprived of food (Table 4). In hepatocytes from rats that were not deprived of food prior to cell isolation, rates of gluconeogenesis were determined using [UJ4C]alanine and [UJ4C]serine. DDT feeding had no effect on glucose formation from serine in isolated hepatocytes from fed rats (Table 4) indicating that the DDTinduced decrease in liver serine dehydratase activity in livers of fed rats has no effect on gluconeogenic capacity. Gluconeogenesis from alanine was also similar in hepatocytes from control and DDT-fed rats. DISCUSSION DDT feeding has been shown to result in an increase in the concentration of numerous
334
STORY
AND
liver amino acids while plasma amino acids were not influenced by DDT. This would suggest that hepatic amino acid metabolism is affected, but overall amino acid homeostasis is apparently unimpaired by DDT feeding. These observations are in contrast with studies using other species, where p,p’DDT has been shown to alter the concentration of nine serum amino acids when administered in the diet of rainbow trout (Mehrle er al., 1971). Species differences, as well as a difference in the isomer used, could account for the different response obtained by Mehrle and co-workers and those observed in this study. Tocci et al. (I 969) reported increases in numerous plasma amino acids in humans exposed to pesticides. It is possible that a compound other than DDT was responsible for the observed changes as the subjects were occupationally exposed to pesticides, and the authors did not specify which pesticides were involved. The data presented here show that feeding o,p’-DDT to rats for a period of 2 weeks results in an increase in the concentration of specific liver amino acids and subsequent food deprivation will attenuate these DDT-induced changes. Story and Freedland (1978) have recently observed that feeding 1000 ppm p,p’-DDT for 2 weeks has no effect on the food intake of rats. Thus, the effects on both amino acid concentrations and enzyme activities reported here are not the result of differences in protein or caloric intake. The concentration of liver serine and threonine was increased by DDT feeding and this effect was abolished by a 48-hr food deprivation. Starvation has been shown to induce the activity of rat liver serine dehydratase (Freedland and Avery, 1964; Pestana, 1969; Chan and Freedland, 1971). This study was designed to determine if an induction of liver serine dehydratase by food deprivation could be responsible for the reduction of liver serine and threonine in livers of DDT-fed rats to levels observed in food deprived controls (Table 3). These data show that there is no stimulation of serine de-
FREEDLAND
hydratase activity by food deprivation in hepatocytes from DDT-fed rats (Table 4). It should be mentioned, however, that hepatocytes from fed rats contain 4.2 mg DNA/g liver while hepatocytes from starved rats contain 5.6 mg DNA/g liver (Story et al., 1976). Assuming that a similar relationship exists in rats exposed to DDT feeding, the difference in serine dehydratase activity in hepatocytes from DDT-fed rats (0.24 pmol/min/mg DNA compared to 0.37 pmol/min/mg DNA) would be exaggerated and there might actually be a significant stimulation of serine dehydratase by food deprivation in hepatocytes from DDT-fed rats. The observation of Story et al. (1976) partially explains the finding that food deprivation did not stimulate serine dehydratase in hepatocytes from control animals (Table 4), which is in contrast to the work of Freedland and Avery (1964) and others. Another explanation for the effect of food deprivation on serine and threonine concentrations in livers of DDT-fed rats could be that serine dehydratase may approach the activity of food-deprived controls during food deprivation because DDT is not present in the diet. This is a difficult interpretation since DDT would still be present in the liver during food deprivation because of the mobilization of DDT from the adipose tissue (Dale et al., 1962); but it probably would not be present at the same concentration as when DDT is ingested in the diet. Gluconeogenesis from serine is similar in isolated hepatocytes from control and DDTfed rats. This indicates that the activity of serine dehydratase in livers of DDT-fed rats does not play a limiting role in gluconeogenesis from serine. The activity of serine dehydratase in hepatocytes from DDT-fed rats would support a rate of gluconeogenesis from serine of 0.120 pmol/min/mg DNA (from Table 4). The actual rate of glucose synthesis from serine observed in hepatocytes is 0.073 pmol/min/mg DNA (Table 5). It appears that gluconeogenesis from serine is limited by some process other than serine dehydratase activity. This limitation would
DDT
EFFECTS
ON
not be at a point in the pathway beyond pyruvate since gluconeogenesis from alanine also proceeds via pyruvate and is more rapid than glucose formation from serine. This investigation demonstrates that feeding 1000 ppm o,p’-DDT for 2 weeks results in alterations in the concentrations of several liver amino acids which are alleviated by a 4%hr period of food deprivation. Although a DDT effect on liver serine dehydratase activity appears to be the cause .of the DDT effect on liver serine and threonine concentrations; this effect on serine dehydratase activity did not result in a decreased ability of hepatocytes, from DDT-fed rats, to synthesize glucose from serine. ACKNOWLEDGMENTS The authors would like to acknowledge the technical assistance of Ernest Avery and the statistical assistance of Dr. Thomas Farver.
AMINO
335
ACIDS
W. E., GAINES, T. B. AND HAYES, W. J. (1962). Storage and excretion of DDT in starved rats. Toxicol. Appl. Pharmacol. 4, 89-106. DAVIES, J. E., MANN, J. B. AND TOCCI, P. M. (1969). Renal tubular dysfunction and amino acid disturbances under conditions of pesticide exposure.
DALE,
Ann.
N. Y. Acad.
Sci. 160, 323-333.
R. A. AND AVERY, E. H. (1964). Studies on threonine and serine dehydratase. J. Biol. Chem. 239, 3357-3360. FREEDLAND, R. A., HJERPE, C. A., AND CORNELIUS, E. C. (1965). Comparative studies on plasma enzyme activities in experimental heptaic necrosis in the horse. Res. Vet. Sci. 6, 18-23. GOLDSTEIN, L., KNOX, W. E. AND BEHRMAN, E. J. (1962). Studies on the nature, inducibifity and assay of threonine and serine dehydratase activities in rat liver. J. Biol. Chem. 237, 2855-2860. KREBS, H. A., DIERKS, C. AND GASCOYNE, T. (1964). Carbohydrate synthesis from lactate in pigeon liver homogenate. Biochem. J. 93, 112-121. MEHRLE, P. M., STALKING, D. L. AND BLOOMFIELD, R. A. (1971). Serum amino acids in rainbow trout (Salmo gairdneri) as affected by DDT and diefdrin.
FREEDLAND,
Camp.
Biochem.
Physiol.
38, 373-377.
A. (1969). Dietary and hormonal control of enzymes of amino acid catabolism in liver. Ear. J.
PESTANA,
REFERENCES
Biochem.
11,40&404.
ADIBI, S. A. (1971). Interrelationships between level SELIM, A. A. AND GREENBERG,D. M. (1960). Further studies on cystathionine synthetase-serine of amino acids in plasma and tissue during starvadeaminase of rat liver. Biochem. Biophys. Acta 42, tion. Am. J. Physiol. 221, 829-838. 211-217. BENEVENGA, N. J., STIELAU, W. J., AND FREEDLAND, R. A. (1964). Factors affecting the activity of SOKAL, R. R. and ROHLF, F. J. (1969). Introduction to Statistics, pp. 186207. W. H. Freeman and Co., pentose phosphate-metabolizing enzymes. J. Nutr. San Francisco, California. 84, 345350. BERRY, M. N. AND FRIEND, D. S. (1969). High yield STORY, D. L., O’DONNELL, J. A., DONG, F. A. AND preparation of isolated rat liver parenchymaf cells. FREEDLAND, R. A. (1976). Gluconeogenesis in isolated hepatocytes from fed and forty-eight hour J. Cell Biol. 43, 506-520. BHATIA, S. C., BHATIA, S. AND Rous, S. (1975). starved rats. Biochem. Biophys. Res. Commun. 73, 799-806. Gluconeogenesis from L-serine in rat liver. Life Science, 17, 267-273. STORY, D. L. AND FREEDLAND, R. A. (1978). The BURTON, K. (1956). A study of the conditions and effect of DDT feeding on gfuconeogenesis in isolated hepatocytes from starved rats. Toxicol. mechanism of the diphenyfamine reaction for the coforimetric estimation of deoxyribonucleic acid. Appt. Pharmacol. 43, 547-557. Toccr, P. M., MANN, J. B., DAVIES, J. E. AND EDBiochem. J. 62, 315-323. MUNDSON, W. F. (1969). Biochemical differences CHAN, T. M. AND FREEDLAND, R. A. (1971). The role of L-serine dehydratase in the metabolism of Lfound in persons chronically exposed to high levels of pesticides. Ind. Med. and Sur. 58, 188-196. serine in the perfused rat liver. Biochem. Biophys. YOUNG, V. R. (1970). Skeletal and cardiac muscle Acta 237, 99- 106. CORNELL,
N.
W.,
LUND,
P.,
H. A. (1973). Acceleration lactate by fysine. Biochem.
HEMS,
R.
AND
KREBS,
of gfuconeogenesis from J. 134, 671-672.
and
regulation
in mammalian
protein
metabolism,
Vol. 4, p. 589-674. (H. N. Munro and J. B. Allison, Eds.), Academic Press, New York.
AND
APPLIED
PHARMACOLOGY
50, 329-335 (1979)
The Effect of o,p’-DDT Feeding and Food Deprivation on Plasma and Liver Amino Acids and Liver Serine Dehydratase in Rats DAVID L.
STORY’
AND RICHARD
A. FREEDLAND
Department of Physiological Sciences, School of Veterinary Medicine, University of Claifornia, Davis, Davis, California 95616 Received September
12, 1978; accepted May 7, 1979
The Effect of o,p’-DDT Feeding and Food Deprivation on Plasma and Liver Amino Acids and Liver Serine Dehydratase in Rats. STORY, D. L., AND FREEDLAND, R. A. (1979). Toxicol. Appl. Phurmacof. 50, 329-335. The effect of DDT feeding and food deprivation on liver and plasma amino acid concentrations was studied. DDT feeding had no effect on plasma amino acids but resulted in an increase in the concentratipn of liver isoleucine, ornithine, phenylalanine, serine, and threonine. In rats that were fed the control or DDT for 2 weeks and then deprived of food for 48 hr, the concentrations of liver isoleucine, phenylalanine, serine, and threonine were similar. The activity of liver serine dehydratase was decreased in hepatocytes from DDT-fed rats compared to fed controls. After food deprivation, the activity of serine dehydratase was similar in hepatocytes from control and DDT-fed rats, which is consistent with the response of liver serine and threonine concentrations to DDT feeding. Gluconeogenesis from serine was determined in isolated hepatocytes from DDT and control-fed rats and it was found that rates of gluconeogenesis were not affected by DDT feeding. This would indicate that the activity of serine dehydratase is not limiting for gluconeogenesis from serine in hepatocytes from fed rats.
The effects of pesticides on free amino acid concentrations have not been extensively investigated. Mehrle et al. (1971) have reported DDT and dieldrin-induced changes in serum amino acids in trout. Alterations in amino acid concentrations have also been observed in plasma (Tocci et al., 1969) and urine (Davies et al., 1969) of humans occupationally exposed to pesticides. There have been no reports as to the effects of o,p’-DDT feeding on plasma and liver-free amino acid concentrations in fed and starved rats. Starvation has been shown to influence the concentrations of specific plasma and liverfree amino acids in rats (Adibi, 1971). Furthermore, starvation results in a release of DDT from adipose tissue with a resultant r Present address: Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass. 02139.
increase in DDT concentrations in plasma, kidney, and liver of rats (Dale et al., 1962). Starvation in combination with prior DDT feeding may elicit a response in plasma or liver amino acid patterns that is different from patterns observed in DDT-fed rats that have not been starved, or in starved rats that have not been exposed to DDT. A cumulative effect could have important metabolic consequences on the amount of free amino acids available in the liver for metabolic processes. One purpose of this study is to determine if DDT feeding results in changes in plasma or liver amino acid concentrations and if subsequent food deprivation alters the response of these amino acid concentrations to DDT feeding. Studies were conducted to determine the mechanism responsible for these alterations in amino acid concentrations. 329
0041-008X/79/110329-07%02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
330
STORY
AND
METHODS Male Sprague-Dawley rats were used in all experiments. Animals were housed in a room with a 12-hr light-dark cycle beginning at ~:~OA.M. Food was withheld from some animals for 48 hr prior to sacrifice. All animals were given water ad lib&/m. Fed animals had feed available until the time of sacrifice, which was between 9:00 and 1O:OOA.M. for all animals. In experiments designed to determine the effects of DDT feeding on plasma and liver amino acid concentrations, rats weighing approximately 150 g were fed 15 % protein laboratory chow with or without 1000 ppm o,p’-DDT ad libitum for 2 weeks. Rats were anesthetized with 60 mg/kg sodium pentobarbital. Blood was taken by cardiac puncture and centrifuged in a refrigerated centrifuge at 3000g for 10 min to remove red blood cells. Livers were rapidly excised and immediately frozen with aluminium tongs prechilled in liquid nitrogen. Liver and plasma samples were stored at -70°C until analysis. For analysis, the plasma was added to an equal volume of 6% sulfosalicyclic acid (w/v in doubly distilled water) and protein was removed by centrifugation at 3000g for IO min in a refrigerated centrifuge. Protein-free liver samples were obtained by homogenizing in a Potter-Elvehjem tissue homogenizer in 9 vol of 3% sulfosalicylic acid followed by centrifugation at 3000g for 10 min under refrigeration. Amino acids were determined using a Beckman 121 amino acid analyzer. In experiments conducted to determine enzyme activities and gluconeogenic capacity, 175 to 250-g rats were fed either 15% protein laboratory chow or a 90% casein diet (Table 1) with or without IOGOppm o,p’-DDT for 2 weeks. Rats fed the laboratory chow with and without DDT were deprived of food for 48 hr prior to cell isolation. Cells were isolated by the method of Berry and Friend (1969) as modified by TABLE COMPOSTION
1 OF DIETS
90 % Casein (g/kg) Casein” Dextrose” Corn oil” Mineral premix’ Vitamin premix’
900 0 50 40 10
’ Purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. b Mazola Corn Oil, Best Foods, Englewood Cliffs, N.J. ’ Benevenga, et al. (1964).
FREEDLAND
Cornell et a/. (1973). Hyaluronidase was omitted from the perfusate. Cell preparations and incubations were conducted as per Cornell ef al. (1973). DNA content of the cells was determined by the method of Burton (1956) and glucose was measured by the method of Krebs er al. (1964). In studies using [U-Ylalanine and [U-%]serine, alanine and serine were present in flasks at a concentration of 10 mM with a specific activity of 0.01 &i/~mol. In the isotopic studies, the cell contents’were acidified with 0.3 ml 60% HCIO,, centrifuged at 5OOg for IO min and adjusted to pH1. An aliquot (3 ml) was put on a 1.2x5-cm column of AG 50W x 8 (Hi, 200400 mesh) on top of a I .2 x 5-cm column of Dowex I x 8 (acetate, 100-200 mesh). Glucose was eluted off the column with doubly distilled HZ0 until 35’ml was collected, and 1 ml of this was added to 14 ml of scintillation fluid and counted. Serine dehydratase activity was measured as described by Freedland and Avery (1964). Alanine aminotransferase activity was determined by the method of Freedland er al. (1965). Enzyme activities were measured on a 30,OOOg supernatant df a 0.14 M KC1 homogenate of 1 g of isolated hepatocytes. Statistical significance was determined by analysis of variance and Student’s t test (Sokal and Rohlf, 1969).
RESULTS DDT feeding had no effect on the concentrations of any of the plasma amino acids. Food deprivation had a statistically significant effect on the concentrations of 10 plasma amino acids (Table 2). Effects of food deprivation were observed on the concentrations of plasma aspartic acid, proline, methionine, phenylalanine, ornithine, and glutamine (p
331
DDT EFFECTS ON AMINO ACIDS TABLE EFFECTS OF
DDT FEEDING
ANR/OR
Foot
2
DEPRIVATION
ON PLASMA AMINO
ACIDS OF RATS’
(nmol/ml + SE)
Aspartic acid Threonine
Serine Proline Valine Cysteine
Methionine Isoleucine Leucine Tyrosine
Phenylalanine Tryptophane
Ornithine Lysine Histidine Arginine Hydroxyproline Glycine Alanine Glutamic acid Asparagine Glutamine
Control fed
DDT fed
14k2 188& 13 184+20 186+36 1$4+ 18
13k2 245f21 209 + 37 161+34 227 + 24 90+4 40+2 87k6
77+4
39+6 79+10 134k 18 53+9 41*3 68klO 45+4 501_+91 49+4 198+33 40+3 232k41 453+52 57+6
26+8 770 k 224
151+15 67+7
48f4 78+12
Control food deprived
DDT fooddeprived
20*1
18+3 215? 17 208+ 14 126k 15 200+14 84+6 49*5 88+9 147* 15 70+ 12 6Ok5 68+5 35+5 360+82 51+4 103 * 14 27+2 361+ 36
13 266+_ 13 133+9 197+25 91+4 207+
5Jk6
82&11 139* 17 69+9 66*7 70+7
46rt4 394 + 27 5926
31+4 386+59 54+9
217+_55 34&J
114*14 30+1
205 + 29 327+27
81+ 10 25+4 900 + 278
320+ 17 351+46 119+ 15 29+5 1244+ 143
Statistical significant@ S S S
S S S S S
274+41
111+11
S
2625 1399* 159
S
n Eighteen rats were fed a laboratory chow diet with or without 1000 ppm qp-DDT for 2 weeks; 6 rats per treatment were then deprived of food for 48 hr. Fed rats, n = 3; food-deprived rats, n = 6. b Data analyzed by analysis of variance. Statistically significant effects are designated S, food deprivation. Refer to text for level of significance.
acid, valine, isoleucine, leucine, and glutamic acid (~~0.05) and on the concentrations of liver serine, glycine, and alanine (p ~0.01). The effects of starvation on plasma and liver amino acids have been studied by many investigators, i.e. Adibi (1971). For this reason and since this study is primarily concerned with the effects of DDT and food deprivation together, the effects of food deprivation alone will not be discussed further. A statistically significant interaction (p < 0.05) between food deprivation and DDT was observed for liver serine, threonine, phenylalanine, and glutamic acid concentrations. The interaction between food deprivation and DDT was indicated by the fact that the
effects of DDT on the concentrations of these four amino acids in livers of fed rats was different than in livers of food-deprived rats. Student’s t test was used to determine the statistical significance of these differences. Despite a large interaction in liver ornithine, due to large SE values the interaction was not significant. DDT feeding resulted in a significant (p ~0.05) increase in the concentrations of threonine, serine, phenylalanine, and ornithine in livers of fed rats. There was no difference in the concentrations of serine, threonine, and phenylalanine in livers of control and DDT-fed animals after food deprivation. Thus, food deprivation mitigates the increase in liver serine, threonine,
332
STORY AND FREEDLAND TABLE EFFECU
OF DDT
FEEDING
AND/OR
Foot
3
DEPRIVATION
ON LIVER AMINO
ACIDS OF RATS~
(nmol/g k SE) Control fed Aspartic acid Threonine Serine Proline Valine Cysteine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophane Ornithine Lysine Histidine Arginine Glycine Alanine Glutamic acid Glutamine
5331 x 931 182+45 470+ 135 Trace 247 + 63 lo* 12 76+ 12 114+24 258 f 73 67+14 72+ 14 3lk6 77k8 495+ 160 441&-111 37+20 16795226 2116+ 190 1860+271 10610+1808
DDT fed 5490+ 789 418+67’ 964+ 147’ 105+65 277 f 53 29+21 53+23 199&44 324 + 34 87k 12 114+11= 29k5 212*52= 5062 162 472+_216 22k27 2125+267 213Ok 329 1864+523 95282 1696
Control food deprived
DDT fooddeprived
4437+482 263+ 14 484 rf: 30 63+23 181+_28 41*17 55k6 94* 10 197+30 54+8 73+10 11+6 136+ 14 473+ 126 318+25 17+5 2640+207 1300&350 3654f237 9606 f. 694
3639& 342 321+50 545 f 84 116k49 208+ 19 14* 10 66k8 117+ 16 202k 18 64+5 73+7 18+9 207 Ifr64 421 f 78 369+_ 35 15+6 3106+210 1175+ 150 2244+218’ 11996~1011
Statistical significanceb s D, SxD D, S, SxD S
D, S S S D, SxD D
S S D, S, SxD
4 Experiment was designed as described in Table 1. * Data analyzed by analysis of variance. Statistically significant effects are designated S, food deprivation; D, DDT; SxD, food deprivation x DDT interaction. Refer to text for level of significance. c Means of amino acids showing a starvation x DDT interaction were also analyzed by Student’s t test. Means that are statistically different from controls at p < 0.05 are indicated by a superscript c.
and phenylalanine observed in DDT-fed rats in the fed state. There was a decrease (p< 0.05) in the concentration of liver glutamic acid in DDT-fed rats that had been deprived of food when compared to the food-deprived controls. The concentration of liver ornithine remained elevated in DDT-fed rats after food deprivation, although this value is not statistically greater than the ornithine concentration in livers from control-food deprived rats due to the large SD. It has been suggested that serine and threonine are both catabolized by liver serine dehydratase (Selim and Greenberg, 1960; Goldstein et al., 1962). The possibility was considered that DDT could be influencing the concentrations of these amino
acids by inhibiting serine dehydratase. To investigate this possibility, enzyme activities were measured in hepatocytes from control and DDT-fed rats (Table 4). In hepatocytes from fed rats, DDT feeding resulted in a significant decrease in the activity of serine dehydratase (p < 0.01). The activity of alanine aminotransferase was not significantly decreased by DDT feeding, suggesting that the effect observed on serine dehydratase is not a general effect on amino acid catabolic enzymes. The activity of serine dehydratase was similar to that observed in hepatocytes from food-deprived control rats when hepatocytes were isolated from rats fed a DDT diet for 2 weeks and deprived of food for 48 hr. This effect on serine dehydratase activity in
333
DDT EFFECTS ON AMINO ACIDS TABLE
4
TABLE
EFFECTS OF o,p-DDT FEEDING ON SERINE DEHYDRATASE AND ALANINE AMINOTRANSFERASE AcnvrTIES IN LIVERS OF FED, FOOD-DEPENDENT, OR 90% CASEIN-FED RATS”
5
EFFECTS OF o,p-DDT FEEDING ON GLUCONEOGENESIS FROM AMINO ACIDS IN WLATED HEPAT~CVTES FROM FED AND 48-hr FOOD-DEPRIVED RAW
Glucose formation Control
DDT Substrate (mM)
15 % Protein fed Food-deprived 90 % Casein
Serine dehydratase (pmol/min/mg DNA) 0.71 f 0.09 (5) 0.24? 0.04b (5) 0.43 f 0.05 (5) 0.37 f 0.05 (5) 2.8OkO.40 (4) 1.59kO.13’ (4)
15 % Protein fed Food-deprived 90% Casein
Alanine Aminotransferase (pmol/min/mg DNA) 5.89 f 0.96 (5) 4.73 + 0.78 (5) 3.6551.37 (5) 3.16kO.69 (5) 10.40+ 1.20 (6) 8.77kO.80 (6)
’ Hepatocytes were isolated and enzyme activities were determined in a 30,OOOg supernatant of a 0.14 M KC1 homogenate of 1 g of hepatocytes. b Indicates values are statistically different from controls at p < 0.01 as determined by Student’s t test. c Indicates values are statistically different from controls at p < 0.05 as determined by Student’s t test.
hepatocytes from rats fed DDT correlates well with the DDT effect on serine and threonine concentrations observed (Table 3). DDT feeding also promoted a decrease in serine dehydratase activity in hepatocytes from rats fed a 90% casein diet (p <0.05), thus the DDT effect on the activity of this enzyme is apparent in hepatocytes from fed and 90% casein-fed rats but is absent in hepatocytes after food deprivation. It has been demonstrated that serine dehydratase plays an important role in the metabolism of serine in the perfused rat liver (Chan and Freedland, 1971; Bhatia et al., 1975). It is possible that a decrease in the activity of this enzyme in livers of DDT-fed rats could result in a decreased ability of these rats to synthesize glucose from serine. To determine if this DDT-induced decrease in liver serine dehydratase activity could have the effect of decreasing gluconeogenesis from these amino acids, gluconeogenic rates were measured in isolated hepatocytes. Gluconeogenesis from serine and threonine was not
Control
DDT
Serine, 10 Alanine, 10 Threonine, 10
Hepatocytes from food-deprived rats (pmol/min/mg DNA) 0.129f0.006 (8) 0.129+0.016 (6) 0.168kO.007 (8) 0.15OkO.016 (6) 0.057 f 0.006 (6) 0.043 f 0.043 (4)
Serine, 10 Alanine, 10
Hepatocytes from Fed Rats (flmol/min/mg DNA) 0.071 f. 0.004 (3) 0.073 + 0.006 (3) 0.168+0.004 0.154+0.010 (3).
’ Hepatocytes were incubated with the indicated substrate for 45 min in duplicate. In studies measuring rates of gluconeogenesis in hepatocytes from fed rats [U-Wlalanine and [U-W]serine were present at a concentration of 0.01 @/pmol. Rates have been corrected for loss of label due to randomization and decarboxylation. Values are means+ SE for the number of animals in parenthesis.
affected by DDT feeding in isolated hepatocytes from food-deprived rats (Table 5). This would be expected on the basis of the finding of similar activities of serine dehydratase in isolated hepatocytes from control and DDTfed rats that had been deprived of food (Table 4). In hepatocytes from rats that were not deprived of food prior to cell isolation, rates of gluconeogenesis were determined using [UJ4C]alanine and [UJ4C]serine. DDT feeding had no effect on glucose formation from serine in isolated hepatocytes from fed rats (Table 4) indicating that the DDTinduced decrease in liver serine dehydratase activity in livers of fed rats has no effect on gluconeogenic capacity. Gluconeogenesis from alanine was also similar in hepatocytes from control and DDT-fed rats. DISCUSSION DDT feeding has been shown to result in an increase in the concentration of numerous
334
STORY
AND
liver amino acids while plasma amino acids were not influenced by DDT. This would suggest that hepatic amino acid metabolism is affected, but overall amino acid homeostasis is apparently unimpaired by DDT feeding. These observations are in contrast with studies using other species, where p,p’DDT has been shown to alter the concentration of nine serum amino acids when administered in the diet of rainbow trout (Mehrle er al., 1971). Species differences, as well as a difference in the isomer used, could account for the different response obtained by Mehrle and co-workers and those observed in this study. Tocci et al. (I 969) reported increases in numerous plasma amino acids in humans exposed to pesticides. It is possible that a compound other than DDT was responsible for the observed changes as the subjects were occupationally exposed to pesticides, and the authors did not specify which pesticides were involved. The data presented here show that feeding o,p’-DDT to rats for a period of 2 weeks results in an increase in the concentration of specific liver amino acids and subsequent food deprivation will attenuate these DDT-induced changes. Story and Freedland (1978) have recently observed that feeding 1000 ppm p,p’-DDT for 2 weeks has no effect on the food intake of rats. Thus, the effects on both amino acid concentrations and enzyme activities reported here are not the result of differences in protein or caloric intake. The concentration of liver serine and threonine was increased by DDT feeding and this effect was abolished by a 48-hr food deprivation. Starvation has been shown to induce the activity of rat liver serine dehydratase (Freedland and Avery, 1964; Pestana, 1969; Chan and Freedland, 1971). This study was designed to determine if an induction of liver serine dehydratase by food deprivation could be responsible for the reduction of liver serine and threonine in livers of DDT-fed rats to levels observed in food deprived controls (Table 3). These data show that there is no stimulation of serine de-
FREEDLAND
hydratase activity by food deprivation in hepatocytes from DDT-fed rats (Table 4). It should be mentioned, however, that hepatocytes from fed rats contain 4.2 mg DNA/g liver while hepatocytes from starved rats contain 5.6 mg DNA/g liver (Story et al., 1976). Assuming that a similar relationship exists in rats exposed to DDT feeding, the difference in serine dehydratase activity in hepatocytes from DDT-fed rats (0.24 pmol/min/mg DNA compared to 0.37 pmol/min/mg DNA) would be exaggerated and there might actually be a significant stimulation of serine dehydratase by food deprivation in hepatocytes from DDT-fed rats. The observation of Story et al. (1976) partially explains the finding that food deprivation did not stimulate serine dehydratase in hepatocytes from control animals (Table 4), which is in contrast to the work of Freedland and Avery (1964) and others. Another explanation for the effect of food deprivation on serine and threonine concentrations in livers of DDT-fed rats could be that serine dehydratase may approach the activity of food-deprived controls during food deprivation because DDT is not present in the diet. This is a difficult interpretation since DDT would still be present in the liver during food deprivation because of the mobilization of DDT from the adipose tissue (Dale et al., 1962); but it probably would not be present at the same concentration as when DDT is ingested in the diet. Gluconeogenesis from serine is similar in isolated hepatocytes from control and DDTfed rats. This indicates that the activity of serine dehydratase in livers of DDT-fed rats does not play a limiting role in gluconeogenesis from serine. The activity of serine dehydratase in hepatocytes from DDT-fed rats would support a rate of gluconeogenesis from serine of 0.120 pmol/min/mg DNA (from Table 4). The actual rate of glucose synthesis from serine observed in hepatocytes is 0.073 pmol/min/mg DNA (Table 5). It appears that gluconeogenesis from serine is limited by some process other than serine dehydratase activity. This limitation would
DDT
EFFECTS
ON
not be at a point in the pathway beyond pyruvate since gluconeogenesis from alanine also proceeds via pyruvate and is more rapid than glucose formation from serine. This investigation demonstrates that feeding 1000 ppm o,p’-DDT for 2 weeks results in alterations in the concentrations of several liver amino acids which are alleviated by a 4%hr period of food deprivation. Although a DDT effect on liver serine dehydratase activity appears to be the cause .of the DDT effect on liver serine and threonine concentrations; this effect on serine dehydratase activity did not result in a decreased ability of hepatocytes, from DDT-fed rats, to synthesize glucose from serine. ACKNOWLEDGMENTS The authors would like to acknowledge the technical assistance of Ernest Avery and the statistical assistance of Dr. Thomas Farver.
AMINO
335
ACIDS
W. E., GAINES, T. B. AND HAYES, W. J. (1962). Storage and excretion of DDT in starved rats. Toxicol. Appl. Pharmacol. 4, 89-106. DAVIES, J. E., MANN, J. B. AND TOCCI, P. M. (1969). Renal tubular dysfunction and amino acid disturbances under conditions of pesticide exposure.
DALE,
Ann.
N. Y. Acad.
Sci. 160, 323-333.
R. A. AND AVERY, E. H. (1964). Studies on threonine and serine dehydratase. J. Biol. Chem. 239, 3357-3360. FREEDLAND, R. A., HJERPE, C. A., AND CORNELIUS, E. C. (1965). Comparative studies on plasma enzyme activities in experimental heptaic necrosis in the horse. Res. Vet. Sci. 6, 18-23. GOLDSTEIN, L., KNOX, W. E. AND BEHRMAN, E. J. (1962). Studies on the nature, inducibifity and assay of threonine and serine dehydratase activities in rat liver. J. Biol. Chem. 237, 2855-2860. KREBS, H. A., DIERKS, C. AND GASCOYNE, T. (1964). Carbohydrate synthesis from lactate in pigeon liver homogenate. Biochem. J. 93, 112-121. MEHRLE, P. M., STALKING, D. L. AND BLOOMFIELD, R. A. (1971). Serum amino acids in rainbow trout (Salmo gairdneri) as affected by DDT and diefdrin.
FREEDLAND,
Camp.
Biochem.
Physiol.
38, 373-377.
A. (1969). Dietary and hormonal control of enzymes of amino acid catabolism in liver. Ear. J.
PESTANA,
REFERENCES
Biochem.
11,40&404.
ADIBI, S. A. (1971). Interrelationships between level SELIM, A. A. AND GREENBERG,D. M. (1960). Further studies on cystathionine synthetase-serine of amino acids in plasma and tissue during starvadeaminase of rat liver. Biochem. Biophys. Acta 42, tion. Am. J. Physiol. 221, 829-838. 211-217. BENEVENGA, N. J., STIELAU, W. J., AND FREEDLAND, R. A. (1964). Factors affecting the activity of SOKAL, R. R. and ROHLF, F. J. (1969). Introduction to Statistics, pp. 186207. W. H. Freeman and Co., pentose phosphate-metabolizing enzymes. J. Nutr. San Francisco, California. 84, 345350. BERRY, M. N. AND FRIEND, D. S. (1969). High yield STORY, D. L., O’DONNELL, J. A., DONG, F. A. AND preparation of isolated rat liver parenchymaf cells. FREEDLAND, R. A. (1976). Gluconeogenesis in isolated hepatocytes from fed and forty-eight hour J. Cell Biol. 43, 506-520. BHATIA, S. C., BHATIA, S. AND Rous, S. (1975). starved rats. Biochem. Biophys. Res. Commun. 73, 799-806. Gluconeogenesis from L-serine in rat liver. Life Science, 17, 267-273. STORY, D. L. AND FREEDLAND, R. A. (1978). The BURTON, K. (1956). A study of the conditions and effect of DDT feeding on gfuconeogenesis in isolated hepatocytes from starved rats. Toxicol. mechanism of the diphenyfamine reaction for the coforimetric estimation of deoxyribonucleic acid. Appt. Pharmacol. 43, 547-557. Toccr, P. M., MANN, J. B., DAVIES, J. E. AND EDBiochem. J. 62, 315-323. MUNDSON, W. F. (1969). Biochemical differences CHAN, T. M. AND FREEDLAND, R. A. (1971). The role of L-serine dehydratase in the metabolism of Lfound in persons chronically exposed to high levels of pesticides. Ind. Med. and Sur. 58, 188-196. serine in the perfused rat liver. Biochem. Biophys. YOUNG, V. R. (1970). Skeletal and cardiac muscle Acta 237, 99- 106. CORNELL,
N.
W.,
LUND,
P.,
H. A. (1973). Acceleration lactate by fysine. Biochem.
HEMS,
R.
AND
KREBS,
of gfuconeogenesis from J. 134, 671-672.
and
regulation
in mammalian
protein
metabolism,
Vol. 4, p. 589-674. (H. N. Munro and J. B. Allison, Eds.), Academic Press, New York.