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OF

Induction

BIOCHEMISTRY

AND

BIOPHYSICS

175, 262-269 (1976)

of Abnormal Fatty Acid Metabolism and Essential Deficiency in Rats by Dietary DDT

JERRY

DARSIE,

SUSAN

The Hormel Institute,

K. GOSHA,

AND RALPH

Fatty Acid

T. HOLMAN

University of Minnesota, Austin, Minnesota 55912 Received December 3, 1975

Rats maintained on lab chow diet or on an essential fatty acid (EFA)-deficient diet were dosed with 5 or 500 ppm, o,p’-DDT, 5 ppmp,p’-DDT, or 100 ppm Kelthane for 30,60, or 90 days. After 30 days all groups exhibited reduced body weights and increased liver weights, lipid contents of livers were elevated only in the group receiving 500 ppm o,p’DDT. No significant difference was found between the incorporations of tritium from T,O into liver lipids from groups fed 5 ppm o,p’-DDT, p,p’-DDT, 100 ppm Kelthane and control groups. The distribution of o,p’-DDT among subcellular fractions paralleled the distribution of lipid content. The rates of desaturation and chain elongation of palmitate and desaturation of 20:3w6 by liver microsomes were decreased significantly in the groups fed o,p’-DDT. The fatty acid composition of phospholipids from liver microsomes revealed significantly diminished contents of ~6 metabolites, increased w9 acids, and increased triene/tetraene ratios in the animals fed DDT in lab chow although the intake of linoleate was adequate (2%). Dermatitis of EFA deficiency was likewise observed in the DDT-fed rats. The desaturation and chain elongation of palmitate by normal microsomes in vitro were significantly inhibited by low concentrations ofo,p’-DDT added to the incubation medium.

Chlorinated hydrocarbons have permeated the environment to such an extent that it is unlikely one can avoid ingesting measurable quantities of these substances. A review of the literature revealed that from 1 to 7 ppm of DDT’ and its metabolites are found in most items included in a staple diet (l-3). The known physiological and metabolic effects of chronic oral doses of chlorinated hydrocarbons have been reviewed by Conney and Burns (4). Highly membranous organelles are strongly affected by DDT and its metabolites. Large doses (l-300 mg/kg body wt/ day) ofo,p’-DDT [l-(O-chlorophenyl)-l-(p’ Abbreviations used: EFA, essential fatty acids; o,p’-DDT, l-(O-chlorophenyl)-l-(p-chlorophenyl)2,2,2-trichloroethane; o,p’-DDD, l-(O-chlorophenyl)l- (p-chlorophenyl)-l,l-dichloroethane; p,p’-DDT, 2,2 - bis- (p - chlorophenyl) - l,l - dichloroethane; pp’ DDT, 2,2-bis-(p-chlorophenyl)-l,l-dichloroethylene; tic, thin-layer chromatography; glc, gas-liquid chromatography; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide.

chlorophenyl)-2,2,2-trichloroethanel and o,p’-DDD [l-(O-chlorophenyl)-l-(p-chlorophenyl)- l,l-dichloroethanel induced increased rates of detoxification of barbiturates (58), antipyrene (2,3-dimethyl-lphenyl-3-pyrazolin&one) (9), and phenylbutazene (Cbutyl-1,2-diphenyl-3,Bpyrozolidine-dione) (10) by mixed-function oxygenases of rat liver microsomes. Micromolar concentrations ofp,p’-DDT 12,2-bis-(pchlorophenyl)-l,l-dichloroethanel or p,p’DDE [2,2-bis-(p-chlorophenyl)-l,l-dichlorethylene] in the media had deleterious effects on photosynthesis in marine diatoms (11) and on isolated chloroplasts (12). Koch reported in vitro inhibition of cation-dependent ATPase in mitochondrial preparations of several tissues (13-W. Gross effects of chronic oral doses of chlorinated hydrocarbons have been observed on lipid metabolism. Compounds related to DDT, particularly the Arochlor series of chlorinated biphenyls, fed to rats at low rates (20-100 ppm) caused fatty liv262

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

CHANGES

IN LIPID

ers (16). When 30 mg/kg/day of dieldrin (1,2,3,4,10,10-hexachloro-6,7-epoxy1,4,4a,5,6,7,8,8a-octahydro-1,4-endo-exo5,gdimethano naphthalene) was fed, histopathologies appeared and lipid accumulated, mostly as triglyceride (17). Incorporation of labeled acetate into fatty acids was significantly reduced in the same rats. Geyer reported similar findings from liver biopsies of human females fed 382 mg of o,p’-DDT over 105 days (18). The biosynthesis of fatty acids by liver is mediated by membrane-bound enzyme systems whether in mitochondria or microsomes. The de nouo biosynthesis of saturated fatty acids from acetate has been shown to be adversely affected (17), but no evidence was found for aberrations in the microsomal fraction. From the abnormally large amounts of lipid material produced in livers of animals fed chlorinated biphenyls (17, 18), it seemed likely that aberrations in lipid metabolism may occur. The elucidation of these changes should explain macroscopic effects (16) as well as symptomology. MATERIALS

AND METHODS

Chemicals. The carboxyl-labeled acids, 16:0, 20:3w6, and 18:306 were obtained from New England Nuclear Corp., Boston, Mass. The radio and chemical purity of these compounds was found to be >99% by tic and glc. Nonlabeled aids were obtained from the Lipids Preparation Laboratory of The Hormel Institute, Austin, Minn., or Nu-Chek Prep, Inc., Elysian, Minn. Cofactors were purchased from Sigma Chemical Co., St. Louis, MO., and Cal-Biothem, LaJolla, Calif. The technical grade o,p’-DDT andp,p’-DDT were provided by Montrose Chemical Corp. of California, Newark, N. J. and Allied Chemical Corp., Jackson, Miss., respectively. Technical Kelthane (dicofol) [l,l-bis(4-chloropheny1)2,2,2trichloroethanoll was provided by Rohm and Haas Co., Kansas City, MO. Pure pesticides were obtained for glc and tic standards from Supelco, Inc., Bellefonte, Pa., Applied Science Labs, Inc., State College, Pa., and PolyScience Corp., Niles, Ill. All other chemicals were reagent grade and solvents were chemically purified and distilled. The petroleum ether used had a boiling point range from 30 to 60°C.

Dietary treatments. Male weanling the Sprague-Dawley strain weighing maintained on Purina Lab Chow or cient in essential fatty acids (EFA). contained 4.7-5.1% extractable lipids

albino rats of 30-40 g were on a diet detiThe lab chow of which 182

METABOLISM

BY DDT

263

was 35.18, 18:303 was 3.9%, and 18306 was 1.3% of total fatty acids. The EFA-deficient diet contained 68% sucrose, 2% hydrogenated coconut oil, 30% vitamin-free casein, and all known essential vitamins and minerals (19). Tests diets were prepared containing 5 ppm o,p’DDT, p,p’-DDT or 100 ppm Kelthane added as acetone solutions to the chow diet, and the solvent was removed by vacuum. Groups of rats were fed the test diets for 30, 60, or 90 days, with appropriate control groups. Preparation of microsomes. Rats were weighed and anesthetized with ether, and their livers were excised into cold 250 mM sucrose:5 mM MgCl, (20, 21). The chilled livers were weighed, minced, and placed into fresh cold sucrose:MgCl,. All subsequent steps were carried out in ice-cold conditions. The tissue was liquified in a blendor, strained through 20-mesh plastic window screen, and placed in a cell-disruption pressure bomb (22). The vessel was sealed and the contents were sustained under 1000 psi N, with constant agitation for 30 min. The homogenate was released 6-om the bomb into 50-ml centrifuge tubes and centrifuged at 17,300g for 30 min and the supernatant was recentrifuged in a Spinco 30 rotor at 105,OOOg for 2 h at 0-5°C. The pellet was suspended in sucrose:MgC12 solution and the suspension was stored under N, at -20°C in screw-cap tubes. Protein was determined by the method of Lowry et al. (23). Microsomes stored in this manner maintained high activity for at least 60 days. Analysis of lipid classes. Total lipids were extracted according to Bligh and Dyer (24) and lipid classes were separated on activated ITLC-SA papers (Gelman Instrument Co., Ann Arbor, Mich.) using petroleum ether:diethyl ether:glacial acetic acid @O:lO:ll as the developing solvent. Spots were made visible with 2’,7’-dichlorofluorescein and uv light. Spots representing lipid classes were cut out and placed in scintillation vials for determination of radioactivity (20) or transesterified with 14% (w/v) BFs:methanol containing 20% benzene for 1 h at 100°C. The methyl esters were extracted with petroleum ether and separated by glc on a ‘/a-in. x 6 ft 20% EGS + 2% H,PO, on Gas Chrom P (100/120) columns and FID detectors. The columns were operated at 190°C and a flow rate of 60 ml He/min. In this laboratory the methyl ester profile derived from the phospholipids is used as an indicator of EFA deficiency and a 20:3o9/20&6 ratio of >0.4 (25) indicates EFA deficiency. Tabulations of profiles include the summation of members of a fatty acid family minus the major metabolic precursor, i.e., ~6 acids - 18206. In this way the content of the precursors themselves do not obscure the variations of the metabolic products. Analysis of chlorinated hydrocarbons. Pesticide residues were extracted from intact tissue and from

264

DARSIE,

GOSHA

subcellular fractions by partitioning between acetonitrile and water followed by chromatography using florisil columns according to Coffin et al. (26) as modified by Kadoum (27) and Stanley et al. (28). Clorinated hydrocarbons were made visible with AgNO,-a- 2-phenoxyethanol chromogenic spray (29) under uv light, eluted with 7% benzene in petroleum ether, and quantified by glc on 2 mm i.d. x 6 ft glass columns packed with 10% DC200 (12,500 cs) silicone oil or 4% SE-30 + 6% QF-1 on Anakrom Q (80/90, Analabs, Inc., New Haven, Conn.) operated at 200°C. Carrier gas was 5% methane in argon at 40 ml/min. The B3Ni electron capture detector was operated at 50-ps pulse intervals and at 240°C. Samples were run on both columns and the resulting data were averaged. Nucleic acid content was determined by the method of Schneider (30). Enzymatic methods. The fatty acyl desaturase activities in the 105,OOOg microsomal pellet were determined using the procedure of Paulsrud et al. (20) except that the substrates used, 16:0 and 20:306, were Na+ salts complexed with essentially fatty acid free BSA fraction V (Sigma Chemical Co., St. Louis). Reaction products were extracted, transesterified and extracted as previously described. Separation of substrate and product was done on 10% AgNO,-Silica Gel H 0.3-mm tic plates. Petroleum ether:diethyl ether (100:5) was used to separate methyl-16:O and methyl-16:lo7 and petroleum ether:diethyl ether:glacial acetic acid (70:30:1) was used to separate methyl-20:306 and methyl-20:4o6. Spots were made visible and their radioactivity was determined as previously described. The chain elongation assay followed the method of Mohrhauer et al. (31) except Na+-BSA complex was used instead of the NH,+ salt as substrate. The incubation was terminated, and total lipids were extracted and transesterified by methods previously described. Methyl esters were separated by reversed-phase chromatography on ITLC-SA papers previously activated and developed in 5% DC200 silicone oil (10 centistoke) in petroleum ether. The developing solvent was acetonitrile:glacial acetic acid:water (70:30:10). Spots were made visible on solvent-free papers by spraying with 1% a-cyclodextrin in methanol, drying, rehumidification and then exposure to I, vapors. The in vitro inhibition experiments used microsomes obtained from rats fed an EFA-deficient diet. DDT or Kelthane was added in 1 ~1 of DMSO to the microsomes plus cofactor mixture and preincubated 15 min with constant agitation at 37°C prior to addition of the carboxyl-labeled substrate. Controls were similarly treated with 1 ~1 of DMSO, other conditions and methods being the same. Triolein (0.5 mr& in DMSO was added to one set of tubes to determine whether the inhibitions observed could be induced by lipophilic compounds in general. Rate of lipogenesis. The tissue slice method of

AND HOLMAN Eliott (321 was used to measure the incorporation of 3Hp0 into the lipid pool. The incubation mixture contained 80 mCi of 3H20 (100 mCi/g) in 37.5 ml isotonic salt solution and 15 g of freshly excised liver slices. The mixture was agitated for 2 h at 37°C. The reaction was terminated by decanting the supernatant fluid, replacing it with several volumes of icecold CHCl,:MeOH (2:l) and immediately homogenizing the sample. The homogenate was repeatedly washed on a filter with 0.88% KC1 until no activity was detected in the wash water. The lipid classes were separated and collected and radioactivity was determined by previously described methods. RESULTS

AND DISCUSSION

In vivo effects on body and liver weights.

Table I shows the body and liver weights of rats fed 5 or 500 ppm o,p’-DDT, 5 ppmp,p’DDT, or 100 ppm Kelthane, and the lipid extracted per gram of fresh liver. Rats fed chronic oral sub-LD, doses of o,p’- or p,p’DDT in a complete commercial chow diet for 30 days had body weights significantly smaller, and liver weights larger than the control animals. There was no significant difference in body and liver weights between animals fed o,p’-DDT or p,p’-DDT. Animals fed 500 ppm o,p’-DDT chow diet for 30 days were smaller, had heavier livers, and 80% more liver lipids than the animals fed 5 ppm. Rats fed the same diets for 60 days showed no significant differences between any of the treatments. All animals fed the higher dosage had statistically significant liver enlargement. All diets containing DDT caused livers to have a greater proportion of lipids, especially the animals fed 500 ppm o,p’-DDT. Rate of lipogenesis in vivo as affected by chronic oral doses of DDT. The accumula-

tion of triglycerides in livers exposed to persistent low levels of chlorinated pesticides observed in the literature and in the preceding section suggests either an increase in the anabolic or decrease in the catabolic metabolism of triglycerides. Increased triglycerides were observed in rats fed either 5 or 500 ppm o,p’-DDT for 30 or 60 days. Increased biosynthesis of triglycerides should be evidenced by a disproportionate share of labeled fatty acid synthesized de novo. Tissue slices incubated with 3H,0 should reveal any change in de novo synthesis of fatty acid and the incorporation of labeled fattv” acids into triglscer--

CHANGES

IN LIPID

METABOLISM

are shown in Table II. The inclusion of DDT in the chow diet containing adequate amounts of EFA elevated the proportions of 16:1, l&l, and 20:3o9 in a fashion parallel to the elevations known to be caused by EFA deficiency and again demonstrated by negative controls in this experiment. Conversely, the inclusion of DDT in the chow diet suppressed the proportions of all EFA components in the microsomal phospholipids, as is true in the EFA-deficient

ides. We observed no differences in the de of fatty acids (35.1% 2 0.1, 35.5% + 1.8, and 33.0% 2 0.8 of incorporated label) or triglycerides (27.2% 2 3.4, 27.6% + 5.2, and 30.1% ? 3.0 of incorporated label) in livers from rats fed 5 ppm o,p’-DDT,p,p’-DDT in a chow diet or chow alone, respectively. This suggests that the accumulation of triglyceride may be due to differences in catabolism, or that change in de novo synthesis of fatty acids, if present, was below the limits of detection by the method. novo biosynthesis

Inducton

TABLE

of EFA deficiency by dietary

Fat-free Fat-free + DDT n

16:l 18:l 20:3w9 18:206 20:206 20:3w6 20&6 Total 06 metabolites 20:3co9/20&6

Body weight (g)

Lab chow + DDT

5 1.5 9.3

5 4.0" 22.6 19.6 2.5 0.8 1.4 8.8

4 6.6 21.7 4.4 1.2 1.4 7.6

17.7 2.0 1.6 22.5

5 5.0 24.4 18.0 3.4 ndb tr” 10.2

11.0’

11.9

26.0

10.6

2.2

2.0

15.1

1.5

0.06

1.8

I

BIPHEN~L OF LIVER,

PESTICIDES IN LAB CHOW DIET AND SKIN CONDITION OF RATS

Liver weight (g)

ON BODY

SkhCIl- a

Lip;fvA;p/g

231.0 f 10.2 9.2 + 0.7 142 f 30 days 13.6 + 1.6 131 f 60 days 372.1 +- 25.0 196.0 -c 7.1” 14.6 k 0.2" 162 c 30 days 178 + 60 days 368.0 2 14.1 14.1 f 1.4 148 zt 30 days 200.3 + 8.0" 15.1 k 0.2" 367.1 + 11.9 14.7 + 1.3 172 k (5 ppm) 60 days 255 zt 30 days 182.9 k 26.7” 16.8 k 1.V o,p’-DDT 21.0 + 2.6' 279 f (500 ppm) 60 days 376.4 + 27.9 228.2 k 6.8 10.1 + OX Kelthane 30 days 148 2 14.5 + 1.5 154 * (100 ppm) 60 days 377.0 + 26.7 n 0 = no dermatitis, + = moderate dermatitis, ++ = severe dermatitis. b Mean 2 SD of seven rats except the 5 ppm p,p’-DDT which had only four, c Statistically significant from controls at

Induction of abnormal fatty acid metabolism and essential fatty acid deficiency in rats by dietary DDT.

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