Conjugation of fatty acids to DDT in the rat: Possible mechanism for retention

Toxicology, 15 (1980) 77---82 © Elsevier/North-Holland Scientific Publishers Ltd.

CONJUGATION OF FATTY ACIDS TO DDT IN THE RAT: POSSIBLE MECHANISM FOR RETENTION

EDITH G. LEIGHTY, ALLISON F. FENTIMAN, JR. and RICHARD M. THOMPSON

Battelle, Columbus Laboratories, 505 King Avenue, Columbus, OH 43201 (U.S.A.) (Received December 10th, 1979) (Accepted January 31st, 1980)

SUMMARY

Palmitic, stearic, oleic, and linoleic f a t t y acid conjugates of DDT were retained in vivo in livers and spleens of male and female rats given chronic i.p. injections of DDT. These DDT fatty acid conjugates could also be produced in vitro in a rat liver coenzyme A fortified microsomal system using the DDT h y d r o x y l a t e d metabolite DDOH as the substrate. The p h e n o m e n o n o f fatty acid conjugation to DDT m a y be a mechanism by which it is retained in the body and ultimately exerts its toxic effect.

INTRODUCTION

DDT [2,2-bis(p-chlorophenyl)-l,l,l-trichloroethane] is a halogenated aromatic hydrocarbon insecticide which has become the classic example of an environmental micropollutant. Due to its chemical properties, the stability of its primary degradation product DDE [2,2-bis(p-chlorophenyl)1,1-dichloroethylene] and its previous world-wide production and use, DDT and its degradation products have been retained everywhere in the environm e n t including fat and fatty tissues of animals and humans [1]. The mechanism by which DDT or its metabolites is retained in the tissues of animals has not been previously shown. A possible mechanism for this retention was recently discovered in our laboratory in studies on drug metabolism. In the initial studies [ 2 ] , long-retained u n k n o w n metabolites were observed to be present in the liver, spleen, fat, and bone marrow of rats 15 days after a single i.v. or chronic i.p. injections o f ~8-[l"C]tetrahydrocannabinol (4 8_[ 14C] THC) or A 9.[ 14C] tetrahydrocannabinol (A9-[ 14C] THC). 8-THC and/~ 9-THC are psychoactive components in marihuana. The retained u n k n o w n cannabinoids comprised at least 80% of the radioactive cannabinoids present in these organs and tissues after 15 days. Later studies [3] 77

identified these retained cannabinoic metabolites as primarily conjugates o f palmitic and stearic acids, with lesser a m o u n t s of C~s-unsaturated fatty acids. The fatty acids were conjugated to a hydroxylated metabolite of the parent cannabinoid. We could also produce these in vivo f a t t y acid conjugates of AS-THC and Ag-THC in an in vitro rat liver coenzyme A fortified microsomal system [ 4 ] . The purpose of this study was to determine if fatty acid conjugates of DDT could be produced in our in vitro microsomal system from the hydroxylated DDT metabolite D D O H [2,2-bis(p-chlorophenyl)ethanol] [1] and if t h e y were also retained in vivo in certain tissues of rats given DDT. METHODS

Palmitoyl-DDOH [1-palmitoyloxy-2,2-bis(p-chlorophenyl)ethane], to be used as a reference standard was synthesized from D D O H (Aldrich) using palmitoyl chloride in pyridine according to the procedure o f Shriner [5]. F o r the in vitro studies 1 m m o l of D D O H was incubated in a 37°C metabolic shaker for 2 h in 25 ml of the rat liver coenzyme A fortified microsomal incubation mixture previously described [ 4 ] . The mixture was lyophilized and then extracted 3 times with 50 ml of chloroform. The solvent was removed on a flash evaporator and the residue resuspended in a small a m o u n t o f chloroform. The chloroform suspension was then partially purified using thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). In these studies HPLC was used only as a tool to partially clean up the sample for later separation into individual peaks and analyzing by gas chromatography-mass spectrometry (GC-MS). For TLC, aliquots of the chloroform suspension were spotted on silica gel plates (K1, Whatman) and developed in normal hexane/acetic acid (90 : 10). Reference standards of DDT, palmitoyl-DDOH, and D D O H were also spotted on the same plate. The reference standards were detected on the plates by spraying with a 0.2% solution o f 2,7
mass spectrometry (EI-MS) consisted o f a 0.6 m X 2 mm glass column packed with 1% OV-17. The column temperature was programmed from 180°C at 6°C/min. Scans were run up to mass 700. The operating parameters for CI-MS were: ionizing potential 150 eV; ion source temperature 230°C, and GC carrier gas (CH4)flow o f 17 ml/min. The reagent gas consisted of a mixture o f methane and'ammonia (approx. 8 : 1) at a total ion-source pressure of approx. 5 × 10 -s Tort. The operating parameters for EI-MS were: ionizing potential 70 eV; ion source temperature 230°C, and carrier gas (He) flow of 30 ml/min. In the in vivo studies, 6 male and 6 female Spraque--Dawley rats (approx. 250 g) were injected intraperitoneally (i.p.) for 4 days with 200 mg/kg DDT (Aldrich) in corn oil. The rats were killed by decapitation 7 days after the first injection. Their livers and spleen were removed, combined by sex and tissue, homogenized in water (3 ml/g) and lyophilized. The lyophilized residues were extracted exhaustively with chloroform, dried, resuspended in chloroform, and partially purified and analyzed as the in vitro samples. Additional in vivo studies were performed in which the livers of DDT chronically i.p. injected rats were not pooled but extracted and analyzed individually. Identical results were obtained as with the pooled liver and spleen samples. lO0.O

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RESULTS TLC analyses of-extracts of all of the in vitro and in vivo samples showed a spot close to the R F (0.81) of the palmitoyl-DDOH standard. Subsequent HPLC analyses of eluates of this TLC spot also showed peaks close to the R t (7.8 rain) of the palmitoyl-DDOH standard. Mass spectrometric analyses of the HPLC peaks identified then as DDOH conjugates of palmitic (C,6 : 0), stearic (Cls : 0), oleic (C1s : 1), and linoleic (C,s : 2) fatty acids. Figure 1 shows the CINH 3 mass spectrum of the palmitoyl-DDOH standard [1-palmitoyloxy2,2-bis(p~hlorophenyl)ethane]. The molecular ion at 522 corresponds to M(NH4) ÷ of the conjugated parent compound. The ion at m/e 249 corresponds to MH*-palmitic acid. Figure 2 shows the EI mass spectrum of the same standard. The most a b u n d a n t ion occurs at m/e 248 and corresponds to the fragment ion M%palmitic acid. Fragment ions at m/e 213 and 178 correspond to loss of chlorine. Figures 3 and 4 are representative CI and EI mass spectra of the in vivo and in vitro samples. Comparison of the spectra with that of the standard (Figs. 1, 2) show that palmitic acid is conjugated both in vitro and in vivo to a metabolite of DDT. Other CINH 3 spectra from both in vitro and in vivo samples showed high intensity molecular ions at role 550, 548, and 546 indicative of DDOH conjugation to stearic, oleic, and I00.0

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linoleic acids. Spectra to role 800 showed that these were molecular ions and n o t fragment ions of longer chain f a t t y acids. DISCUSSION

These studies show that t h e organohal~zgen DDT can be conjugated to fatty acids in a rat liver in vitro c o e n z y m e A fortified microsomal system and that these DDT conjugates are also p r o d u c e d in vivo and retained in the liver and spleen of the animals. Because of limited funds, other tissues of the rat were n o t analyzed for DDT conjugates. Previous studies in our laboratory on A9-[14C] T H C and A8-[14C] THC, however, have shown that T H C fatty acid conjugates are retained in high concentrations not only in the liverand spleen of the rat but also in the fat and bone marrow [3]. W e havealso shown that 11-palmitoyloxy-A 9-THC, a fatty acid conjugated cannabinoid synthesized in our laboratory, could be hydrolyzed by cholesterol esterase and lipase to psychoactive 11-OH-A9-THC [6]. Thus, even though a fatty acid conjugated foreign c o m p o u n d m a y not itselfbe toxic, it could be hydrolyzed to its toxic parent c o m p o u n d or a toxic metabolite. The phenomenon of fatty acid conjugation to foreign compounds such as

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DDT m a y be a mechanism b y which these c o m p o u n d s are retained in fat, lipid-containing tissues, and cells of the b o d y . By being attached in situ, the foreign c o m p o u n d m a y exert a long term low exposure direct effect on the cell or exert its effect at another site a f t e r the conjugate is hydrolyzed. REFERENCES 1 R.L. Metcalf, J. Agric. F o o d Chem., 21 (1973) 511. 2 E.G. Leighty, Biochem. Pharmacol., 22 (1973) 1613. 3 E.G. Leighty, A.F. Fentiman, Jr. and R.F. Foltz, Res. Commun. Chem. Pathol. Pharmacol., 14 (1976) 13. 4 E.G. Leighty, Res. Commun. Chem. Pathol. Pharmacol., 23 (1979) 483. 5 R.L. Shriner, R.C. Fuson and D.Y. Curtin, The Systematic Identification of Organic Compounds, 4th Edn., John Wiley and Sons, Inc., New York, 1956, p. 212. 6 E.G. Leighty, Res. Commun. Chem. Pathol. Pharmacol., 24 (1979) 393.

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