Document e790JGrxDj1Lj28eR79kGOZg

/: ,.ym - :*r tV: A ft*!.; 1*1 ^iag-3Ttfi^fc?raAMSSfWiTii i Tlic EfTecl of DDT and PCB on Lipid Metabolism in E. coli and B. fragilis - i' by Douglas E. Greek and Julian E, Keil f'rcicnn'i'c Medicine Section Department of Medicine Lewis W. Stillway Department of Biochemistry and Samuel H. Sandifer Preventive Medicine Section Department of Medicine Medical University of South Carolina Charleston, S.C. 29401 DDT and PCB, chlorinated hydrocarbons similar in structure and physiological effects (LICHTENSTEIN, et al, 1969), are of environmental concern because of their ubiquity, magnification and effects in natural food chains (COX, 1970; MACEK and KORN, 1970; GOULD, 1972; McLANE and HALL, 1972). Recent work has also suggested that DDT could be unrecognized source of PCB (MAUGH, 1973). These chemicals have been of concern in human health because of their accumulation in the fatty tissues ' of man (DEICHMAN, RADOMSKI, 1968). A number of workers have shown a statistical association between DDT and its metabolites to risk factors of heart disease (ROAN, MORGAN, 1973; SANDIFER, KEIL, 1972) and to other pathological states (HEINRICHS, et al, 1971; O'LEARY, et al, 1970a; O'LEARY, et al, 1970b). Conversely, DDT and its metabo... lites have been screened as chemotherapeutic agents for neoplastic diseases (WALKER, et al, 1970; TEMPLE, et al, 1969) . In this experiment Escherichia coli and BacteroideB fragilis were selected as models to observe the effects ' of DDT and PCB on lipid metabolism in bacteria. Since these species comprise about 90% of the intestinal bac teria in areas where lipid absorption occurs (DAVIES, et al, 1970) , it was also of interest to study the meta bolism of DDT and PCB in these organisms. Methods E. coli was cultured in one liter Erlenmeyer flasks contaTning"300 ml Trypticase Soy Broth, B. fragilis was grown in 60 ml Scott Anaerobic Broth Bottles" Toxi cant treatment solutions were prepared with Geigy 99.9% pp'DDT and PCB (Aroclor 1242 ) dissolved in acetone, (Nanograde, Mallinckrodt Chemical Works). Each species of bacteria was treated with .01 ppm DDT, .1 ppm DDT, .01 ppm PCB, .1 ppm PCB and acetone (solvent control). Each treatment consisted of four replicates. E. coli and B. fragilis cultures were in cubated at 35C for 24 and~"49~hours, respectively. Cul ture flasks were agitated every four hours during the incubation period. Upon completion of incubation, each 295 Hvtlello of LnxJri-iinirm-l CcnjriJnii<m & Toxkolofy. Yul. J2, No. SC 1974 by Sjutrjci-VtjUj New York Inc. DSW 025334 STLCOPCB4009289 replicate culture was centrifuged at 2000 xg and the pellet was lyophilized and weighed. Total lipids were extracted from the lyophilized samples (VORBECK, MARINETTI, 1965) and the solvent was evaporated under a stream of nitrogen in tared vials. The residue was weighed, brought up to a volume of 5 ml in chloroform-methanol (2:1) and stored at 4C in Teflon-lined screw-cap vials. Total lipids were determined from aliquots containing approximately 100 ug of total lipid by chromic acid oxidation (PANDE, et al, 1963) using a standard consisting of a known con centration of total lipids extracted from a large culture of E. coli. All solvents were distilled. DDT and its metabolites were quantitated by gas-liquid chromatography on two different columns. Aliquots of the samples in chloroform-methanol were evaporated to dryness under a stream of nitrogen and were'made up to volume in hexane (Matheson, Coleman and Bell,. hanograde). Determina tions were obtained on a Tracor 220 gas chromatograph fit ted with two 6 ft x 2 mm ID glass columns and tritium-foil electron capture detectors. Primary determinations were obtained with a column packed with a mixed stationary phase consisting of 1,5% OV-17 and 1.95% QF-1 on 100/120 Chromasorb WHP. Determinations were confirmed on another column packed with a mixed stationary phase consisting of 4% SE-3Q and 6% QF-1 on 60/100 Chromosorb WHP. Operating parameters were: inlet 235C, column oven 200C, and detec tor 205C. The flow-rate of the nitrogen carrier gas was 70 cm3/min for the OV-17, QF-1 column and 80 cm. /min for the SE-30, QF-1 column. Results One way analysis of variance allowed calculation of least significant difference at the 95% probability level, for different numbers of replicate comparisons. As shown in Table 1, only the .01 ppm DDT treatment group showed a significant increase in dry weight in the E. coli series. This parallels the findings of Keil, et aTTKEIL, et, al,, 1972). There was no significant difference in total lipid yields of any treatment grou~^ as compared to the acetone control. PCB had no detect.. .le effect on coli growth or lipid yield. Z96 DSW 025335 STLCOPCB4009290 :-*, ''' (' '*'<i^^>tt^'piw^4^wjrl.ii&l.vi;A,.-?itfeii^.~.jiLig|:`^^' . -A ^<-*-'->;>.v?-:--\ ' ' ;. - *t>**iJum -...... - ...........- ... ....................... . : ^V^*L>i'4Vf;cA '*> ss.- ^VvV A TABLE 1 Harvest weights, toxicant residues, and lipid yields of Escherichia coli cultures treated with DDT and PCB Treatment No. Replicates Mean Harvest Weight (mg) Mean Toxicant Residues (ug) Mean Lipid Yields (ug/mg dry wt) .01 ppm DDT . 1, ppm DDT .01 ppm PCB .1 ppm PCB Control Acetone Control 4 4 4 3 3 4 311.3 219.5 257.3 268.9 235.9 220.1 2.05** 14:97** N.D. N.D. 0 0 38.9 36.6 36.8 37.7 31.9 35.3 i1I'/. - **+*>v*'' few# ffitllS fertf jfe ~/f iT'># A v u"', 'J'k'JL (** *v;Vr. fesr* , -K--5P LSD05* 4/4 51.6 4.8 3/4 31.8 9.5 ''LSD *= Least significant difference at 55% probability level calculated from one way variance analysis. **Total DDT DDT + 1.114 (DDE + DDD) N.D. = Not Determined fe ->> nr sfe 7 V-i-fV-..- ' As shown in Table 2, .01 ppm DDT significantly depressed the growth of fragilis and .1 ppm PCB significantly stimulated growth. Neither dosage of DDT had any effect on B. fragilis lipid synthesis, but PCB caused a significant and apparently dose-related depression of lipid synthesis. `Sr, 297 Zb.-pxJ:. sk ^ WWmiM. far; ..;h'V-V> ':-.5vr':a;..-- v .- STLCOPCB4009291 V v ;4 ` ' "*. , ' jtVAf-Ol TABLE 2 Harvest weights, toxicant residue, and lipid yields of Bacteriods fragilis cultures treated with DDT and PCB Treatment No. ;Replicates Mean Harvest Weight (mg) .01 ppm DDT 4 93.0 .1 ppm DDT 4 99.0 .01 ppm PCB 4 137.4 .1 ppm PCB 2 210.4 Control 4 119.7 Acetone Control 4 109.9 Mean Toxicant Residues (ug) Mean Lipid Yields (ug/mg dry wt 4.2** 36.9 68.38** 36.7 N.D. 23.2 N.D. 16.2 0 34.9 0 35.7 LSD05* 4/4 26.0 2/4 31.8 7.8 9.5 Level Calculated from one way analysis of variance **Total DDT <= DDT + 1.114 (DDE + DDD) N.D. Not Determined TABLE 3 Comparison of DDT metabolites in coli and B^ fragilis DDT Media Concentration Escherichia coll Bacteroides fragilis- 01 ppm DDT 1 ppm DDT DDD DDE DDD 95% 5% 100% 98% --1 2% 93% -- ..................... ................ . 298 DDE 0% 7% w,-n i i i 1 STLCOPCB4009292 In Table 3 it is shown that of the DDT metabolites, DDD was the principal metabolite in both species, although DDE was present in small to trace quantities. Discussion An important finding was that conversion of DDT by E_. coli and B_^ fragilis was primarily to DDD, a metabolite easily hydroxylated and excreted in urine, thus demon strating that these key aerobic and anaerobic human bac teria are capable of participating in the degradation of this insecticide. Although neither DDT nor PCB significantly altered total lipid levels in these organisms, there remains the possibility of changes in ratios of lipid classes. There appears to be no relationship between DDT residues and lipid levels although both PCB treatments significantly depressed lipid production by B. fragilis. While these findings are not conclusive, it does suggest that if there is a relationship between DDT exposure and blood pressure and cholesterol, it may be via a mechanism other than stimulation of lipid synthesis. Values approaching the extreme limits of LSD-- were discounted because of the inaccuracies arising from the small number of treatments. Summary Escherichia coli and Bacteroides fragilis, princi pal Human enteric aerobic and anaerobic bacteria, were cultured with suitable controls in the presence of .01 and .1 ppm of DDT and PCB. The bacteria were assayed for dry weight and total lipids. DDT increased the growth of coli at the .01 ppm level but had no detectable effect at .1 ppm. Total lipid content was not significantly altered by either DDT or PCB. DDT at .01 ppm depressed B. fragilis growth, but had no significant effect on lipid levels. PCB in creased the growth of B. fragilis significantly at the .1 ppm level, but significantly depressed lipid synthe sis at both concentrations. The effect was apparently dose dependent. All cultures metabolized DDT to DDD. Trace amounts of DDE were also found in several repli cates. DDT/PCB are statistically associated with cardiovas cular disease risk factors, but the mechanism does not appear to be that of increased lipid anabolism by enteric bacteria. `k*' '*,-.1. rA -A; . ... .>. 299 y-'y v.T v - > .. .*, ..f * V' , t''i*' .. ft -VV. i. - V - DSW 025338 'iC-**'**'* STLCOPCB4009293 Acknowledgements This research was funded in part by Environmental ;; Protection Agency contract #NEG-68-02-05?7. We acknow ledge technical assistance from Drs. C.D. Graber and C.B. 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