Interaction of insecticides with human plasma lipoproteins

Chem.-Biol. Interactions, 35 (1981) 177--188 © Elsevier/North-Holland Scientific Publishers Ltd.

177

INTERACTION OF INSECTICIDES WITH HUMAN PLASMA LIPOPROTEINS

BADRI P. MALIWAL and FRANK E. GUTHRIE

Toxicology Program, Department of Entomology, North Carolina State University, Raleigh, NC 27650 (U.S.A.) (Received September 7th, 1980) (Revision received December 1 lth, 1980) (Accepted December 29th, 1980)

SUMMARY

The binding of chlorinated hydrocarbon, carbamate and organophosphate insecticides to human low density plasma lipoproteins (LDL) and high density plasma lipoproteins (HDL) was studied at pH 7.0 and 16°C and 26°C by equilibrium dialysis, difference spectra and fluorescence. The results suggest interaction to be a partitioning rather than a stoichiometric binding process. Distribution is related to lipid content and composition of the lipoproteins. The K-values vary from 3 X 10 s M -1 for 1,1,1-trichloro-2,2bis(p-chlorophenyl)ethane (DDT) to less than 10 M -1 for nicotine and aldicarb, and AG~r is in the range of 7400 cal for DDT to less than 1000 cal for aldicarb and nicotine. The K and AG~r are inversely related to the water solubility of the insecticides. A significant role of plasma lipoproteins in the transport of slightly water soluble insecticides is suggested.

INTRODUCTION

Binding to plasma proteins has physiological significance in transport, modulation and inactivation of xenobiotics and their metabolite activities [1]. Lipoproteins have been implicated in the transport of chlorinated hydrocarbon insecticides in human serum [2--5], cockroach hemolymph [6,7] and dogfish and trout serum [8,9]. These studies were based either on the solubility of the insecticides in lipoproteins or on its distribution into various serum fractions. We have undertaken a systematic investigation of the role of various plasma proteins in the transport of insecticides in the Abbreviations: DDT, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane; HDL, high desity lipoproteins; HSA, human serum albumin; LDL, low density lipoproteins; TLC, thinlayer chromatography.

178 blood. In an earlier communication, the results on binding of various insecticides with human serum albumin (HSA) were reported [10] and the present communication deals with the binding of chlorinated hydrocarbon, carbamate and organophosphate insecticides to human LDL and HDL by equilibrium dialysis, fluorescence and difference spectra. MATERIALS AND METHODS Chemicals [14C]DDT (29.7 mCi/mmol), ['4C]dieldrin (85 mCi/mmol), [14C]lindane (48 mCi/mmol), [14C]parathion (19 mCi/mmol)and [~4C]carbaryl (57 mCi/ mmol) were purchased from Amersham/Searle. [14C]Diazinon (11 mCi/ retool) was a gift from Ciba~eigy. [~4C]Nicotine (54 mCi/mmol) was obtained from New England Nuclear. [ ~4C]Carbofuran (2.85 mCi/mmol) was a gift from FMC Corporation. [ ~4C]Aldicarb (20 mCi/mmol) was a gift from Union Carbide. The radiochemical purity (~99%) of all of the above compounds was confirmed by thin-layer chromatography (TLC) with appropriate solvent systems. Non-radioactive insecticides (~99%) were purchased from Chem Service. All other chemicals of ACS grade and solvents of spectroscopy grade were purchased from Fisher Scientific Co. Cellulose dialysis tubing (16 ram) was purchased from Arthur H. Thomas Co. The agarose gel (Biogel A-5m) was obtained from BioRad Laboratories, Richmond, CA, U.S.A. Methods Dialysis tubing was boiled in an excess of distilled water containing 100 /~M EDTA to remove impurities. Insecticides were first dissolved in dioxane and then buffer added to achieve a final concentration of dioxane of 0.5% (v/v). The radioactivity of the insecticide solutions was 100 cpm//~g except in the case of lindane (380 cpm/~g), dieldrin (3750 cpm//~g) and DDT (100 000 cpm//~g). The protein and insecticide solutions were made in 10 mM Tris--HC1 buffer (pH 7.0) containing 50 mM NaC1. Aliquots (2.0 ml) of protein solution (0.5--10.0 mg/ml) were dialyzed against buffer solution (5.0 ml) containing varying amounts of insecticides for 30 h on an orbit shaker at 2 6 ° -+ 0.5°C or 16 ° +- 0.5°C. This time interval was found to be sufficient to attain equilibrium. No degradation of insecticides during equilibrium was detected as judged by appropriate TLC experiments. After completion of equilibrium, 1.0 ml each of protein solution and insecticide solution were pipetted into scintillation vials containing 10 ml of Triton X-100 scintillation liquid [11] and counted on a Packard-Tri Carb Scintillation Spectrometer. Due to limited solubility of DDT and dieldrin in buffer (~0.1 ~g/ml), binding was determined from relative solubilities in aqueous solutions with and without added protein. To obtain the solubility in buffer and protein solution, an excess of insecticide was equilibrated with buffer and protein solution (0.50 mg/ml) for 24 h at 26°C or 16°C. The suspensions were

179 centrifuged at 20 000 Xg for 30 min. The supernatants (1.0 ml) were mixed with 10 ml scintillation liquid and counted as described earlier. The LDL and HDL were prepared from human serum obtained from fasting healthy male subjects (Dr. A.L. Chasson, Rex Hospital, Raleigh, NC) by the method of Rudel et al. [12] and involved separation of lipoproteins by ultmcentrifugation after adjusting density to 1.225 g with KBr followed by gel filtration on an agarose column (Biogel A-5m) in Tris--HC1 buffer (pH 7.0), 0.01 M containing 0.15 M NaC1. Results are averages from duplicates of the same protein preparation and all experiments were repeated with at least two protein preparations.

Analysis of binding isotherm The distribution data were analyzed by the following equation: Insecticide (aqueous)

K

Insecticide (in lipoprotein)

where K is the distribution constant and the insecticide concentration is defined as mole solute per 1000 g solvent. The standard free energy change of transfer of 1 mol of insecticide from buffer to lipoprotein (AG~r) was calculated by the equation: AG~r = - R T In K Spectral investigations were conducted on a D W - 2 Aminco B o w m a n double beam spectrophotometer using the 'tandem cell' arrangement of 4.25 n m cell path length [13]. Difference spectra were obtained by placing the protein solution (1 mg/ml) in the reference beam and the protein solution containing insecticide in the same buffer in the sample beam. Spectra were measured at a pH-value of 7.0 in the presence of 0.05 M NaCI. Fluorescence measurements of protein solution (0.10 mg/ml) with appropriate insecticide concentration (as indicated in Table II) were made on a Varian 330 Recording Spectrofluorometer using triangular cuvettes. Corrections were made for internal absorption filtereffects due to insecticides wherever required [14]. Protein solutions were excited at 287 n m and 297 n m (excitation slit 3 nm), and emission was measured at 325 n m (emission slit5 nm). RESULTS

Figures 1 and 2 show the distribution of various insecticides in LDL and HDL at a pH-value of 7.0 and 26°C. The binding phenomena appear to represent partitioning rather than saturable binding as shown by a proportional increase in binding with an increase in free insecticide concentration. The distribution constants of insecticides for LDL and HDL are presented in Table I. The distribution affinities fall into three categories, one group of relatively high affinity ( K ~ 105 M -~) which include DDT and dieldrin, a

180

second group with moderate affinity (K between 103--104 M -1) which include lindane, parathion and diazinon and a third group with low affinity (K < 102 M -~) which include carbaryl, carbofuran, aldicarb, and nicotine. The affinities of DDT, dieldrin, lindane, diazinon and parathion are similar for both HDL and LDL while carbaryl, carbofuran, aldicarb and nicotine o varies from 7360 cal for show higher affinity for HDL than LDL. The AGtr DDT to 300 cal for aldicarb with LDL and from 7400 ca] for DDT to 900 cal for nicotine in the case of HDL. The large AG~r for DDT, dieldrin, lindane, parathion and diazinon indicates that at equilibrium there would be a substantial transfer of these insecticides into the lipoproteins. The relative fluorescence intensity of HDL and LDL in the presence of 14

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Fig. 1. Semflogarithmic plots of binding isotherms of LDL with various insecticides at 26°C and pH 7.0 (Tris--HCl 0.01 M with 0.05 M NaCI). ~ represents moles ligand bound per m o l e protein and [A] is equilibrium concentration of the ligand. The symbols are: o, lindane; e, diazinon; x , parathion; ~, carbaryl; =, carbofuran; A, nicotine; o, aldicarb. The details are given in Table I.

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various insecticides is presented in Table II. The results indicate quenching of both tyrosine and t r y p t o p h a n fluorescence to varying extents with different insecticides for both lipoproteins. Quenching is greater for insecticides of moderate affinity. Figure 3 shows the difference spectra of various insecticides when aqueous solutions of the insecticides are placed in the reference beam and ethanol solution of the same insecticides in the sample beam. The insecticides show a red shift in the spectral region studied (270--310 nm) in less polar environm e n t except in the case of parathion (blue shift). This blue shift for parathion in a less polar environment m a y be indicative of a decrease in dipole m o m e n t (or hydrogen bonding to a solvent molecule) in the excited state [15] and might also reflect the tendency to react covalently with proteins. Figure 4 shows the difference spectra due to solubilization of various insecticides in LDL and HDL. There is no difference spectra from

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183 T A B L E II RELATIVE FLUORESCENCE C I D E S a ,b

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OF LDL AND HDL IN THE PRESENCE OF INSECTL

Relativefluorescence mteusity(%) LDL ffc

Parathion Diazinon Carbofuran Aldicarb Nicotine

0.10 0.10 0.01 0.02 0.02

HDL ), E x c i t a t i o n ( n m ) 287

297

40 75 >95 86 87

10 60 >95 >95 65

ffc

0.05 0.05 0.02 0.04 0.04

~, E x c i t a t i o n ( n m ) 287

297

34 68 66 81 90

30 58 55 90 60

a p H 7.0, 0.01 M Tris--HCl b u f f e r c o n t a i n i n g 0 . 0 5 M NaC1. b H D L a n d L D L s o l u t i o n o f OD~s 0 = 0 . 1 0 were e x c i t e d a t 2 8 7 n m a n d 297 n m a n d t h e e m i s s i o n m e a s u r e d a t 3 2 5 n m in t r i a n g u l a r e u v e t t e s . T h e e x c i t a t i o n slit w i d t h was 3 n m a n d e m i s s i o n slit w i d t h 5 n m . c C o n c e n t r a t i o n o f insecticides u s e d t o a c h i e v e ff are c a l c u l a t e d o n t h e basis o f K in T a b l e L T h e r e s u l t s are averages o f t h r e e e s t i m a t e s w i t h t h e s a m e p r o t e i n s a m p l e a n d are e x p r e s s e d as % o f f l u o r e s c e n c e i n t e n s i t y o f p r o t e i n w i t h o u t insecticide.

+12

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290 310 Xnm Fig. 3. D i f f e r e n c e s p e c t r a o f v a r i o u s insecticides w h e n a q u e o u s s o l u t i o n o f a n i n s e c t i c i d e is p l a c e d in t h e r e f e r e n c e b e a m a n d e t h a n o l s o l u t i o n o f s a m e i n s e c t i c i d e i n t h e s a m p l e b e a m . T h e s y m b o l s are: ...... , carbaryl; , carbofuran; , aldicarb; ..... , nicotine; ..... , p a r a t h i o n ; - - , - - o --, d i a z i n o n . T h e k m a x o f t h e i n s e c t i c i d e s are c a r b a r y l 281 n m , c a r b o f u r a n 2 7 7 . 5 n m , p a r a t h i o n 2 7 4 n m , d i a z i n o n 2 4 8 n m a n d n i c o t i n e 261 n m .

184 +11

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Fig. 4. Difference spectra of LDL (1 mg/ml, ) and HDL (1 mg/ml, ) in 270--310 nm region produced by binding the indicated number of equivalents of insecticide at pH 7.0, 0.01 M Tris--HC1 buffer containing 0.05 M NaCl.

the interaction of aldicarb and nicotine with LDL and of aldicarb with HDL. All other difference spectra with the exception of parathion show a red shift either in the tyrosine residues of the protein molecule (nicotine binding to HDL) or a red shift in the spectrum of solubilized insecticide (carbaryl, carbofuran and diazinon binding to LDL and HDL) and a blue shift in the case of solubilized parathion for both the lipoproteins. Table III shows the amounts of bound insecticide at total serum insecticide concentrations of 10 ~M which one can expect to encounter at extremes of exposure. The average physiological serum protein concentrations used to calculate the distribution are albumin 4000 mg/100 ml, LDL 400 mg/100 ml and HDL 270 mg/100 ml [16,19]. The results indicate a significant role for lipoproteins in the transport of less water soluble insecticides (DDT, dieldrin, lindane, parathion and diazinon) in the blood. It also suggests that lipoproteins would be the major carrier proteins for insecticides of extremely low solubility such as DDT and dieldrin. DISCUSSION

The present investigation confirms earlier observations of the possible role of lipoproteins as carriers of insecticides in the blood [2--5]. The affinities

185 T A B L E III T H E R E L A T I V E D I S T R I B U T I O N O F I N S E C T I C I D E S I N T O A L B U M I N A N D LIPOPROTEINSa, b Ligand

Bound insecticide

% Distribution o f b o u n d insecticide

(%)

Albumin

LDL

HDL

99.9 99.9 98.0

35 12 37

35 50 37.6

30 38 25.4

98.7 96.6

67 55.5

20.5 31.0

12.5 13.5

97.4 73.6 30.0

98.7 96.8 94.3

< 1.0 1.4 1.6

< 1.0 1.8 4.1

25.0

93.8

2.5

3.7

Chlorinated hydrocarbons DDT Dieldrin Lindane

Organophosphates Parathion Diazinon

Carbamates Carbaryl Carbofuran Aldicarb

Others Nicotine

The serum c o n c e n t r a t i o n used in the calculations are: albumin 4 0 0 0 m g / 1 0 0 ml; L D L 400 m g / 1 0 0 ml; H D L 270 m g / 1 0 0 ml, insecticides 10 uM. b Calculations for L D L and H D L are based on the values o f K given in Table I. The n and K for albumin are f r o m unpublished results [ 10 ].

of various insecticides for the lipoproteins are inversely related to the water solubility of these compounds. The distribution constant K is expressed in the concentration of 1000 g solvent and would be similar to the conventional partition coefficient which is expressed on a volume basis. The partition constants for DDT, lindane, and parathion in the present study compare favorably with reported octanol-water partition coefficients [17,20]. The standard free energy change of transfer of insecticides to lipoproteins (A G~r) is comparable to that reported for transfer of various hydrocarbons from water to hydrocarbon solvents [18]. The partitioning seems to be related to total lipid content of lipoproteins. However, other factors such as protein content and lipid composition may play some role in determining distribution as indicated by relatively comparable solubflization of DDT, dieldrin, lindane, diazinon and parathion by HDL and LDL. In addition, binding of carbaryl and carbofuran is greater for HDL than LDL even though LDL has about 1.5 times the lipid content. Similar observations of a discrepancy in binding and lipid content have been reported for DDT in the case of rainbow trout serum lipoproteins [9] and for benz[o]pyrene and human plasma lipoproteins [ 19]. No attempt has been made to calculate enthalpy and entropy components of AG~r as our experimental temperatures (16°C and 26°C) are in the range

186 of phase transition temperatures of the lipid moiety in L D L and H D L , and it is not possible to differentiate the effect of temperature from that of the process of ordering of lipid molecules on K. The above results indicate the binding to be basically a transfer of insecticide molecule into the lipid moiety of the lipoproteins. However, w e cannot rule out the presence of a few discrete binding sites of moderate affinity.Our analysis of the data is a least squares fit and a few saturable sitesof moderate affinity could escape detection in the error associated with the observation. The binding of insecticidesresultsin quenching of fluorescence from both tyrosine and tryptophan residues of lipoproteins.M a n y pesticides have been shown to act as coUisional quenchers of varying efficiencies [20]. Our quenching results do not distinguish between the static and collisional interaction of insecticides with fluorophores. Both H D L and L D L have a substantial portion of tyrosine and tryptophan residues exposed to solvent [21]. The observed quenching due to binding m a y involve both collisional and staticquenching of fluorophores both in lipid and aqueous environment. The difference spectra show a red shift due to solubilization as would be expected from movement of insecticidemolecules from the aqueous environment to a hydrocarbon-like lipid phase. The lack of a red shift in the protein spectrum, except for distribution of nicotine in H D L , also supports the possibility of coUisional quenching of protein fluorophores and a possible lack of discrete binding sites.However, the lack of a red shift is also possible if binding involves the chromophores exposed to the lipid phase which are already in a less polar environment. Thus, even though w e cannot unequivocally show the absence of discrete binding siteson H D L and L D L for these insecticides, our results suggest that the transport function of lipoprotein is mainly due to the partitioning of these insecticidesinto lipoproteins. Table II! indicates that chlorinated hydrocarbon insecticidesof extremely low water solubility like D D T and dieldrin and a significantportion of other less water soluble insecticidessuch as lindane, parathion and diazinon, would be distributed with H D L and L D L in the blood during transport. Our results from equilibrium dialysis with isolated proteins support earlierobservations of higher distribution of chlorinated hydrocarbons into lipoproteins which were based on gel filtration,electrophoresis and ultracentrifugal separation of whole serum [2--5]. It also shows lack of any significantassociation to lipoproteins of highly water soluble insecticidessuch as nicotine and aldicarb. Our calculations of transport of insecticides in the blood are based on albumin and lipoproteins as earlier studies, though limited in scope, have shown a lack of any significantassociation of insecticideswith other protein fractions. These studies were based on distribution of the insecticide into various fractions of the whole serum separated by differentialcentrifugation, gel filtrationand electrophoresis [2--4,6,9]. The distribution of insecticides to lipoproteins has m a n y toxicological implications. It would result in the reduction in free insecticide concentration and affect the insecticide activity at the target site and the rate of metabolism and elimination. Also, if insecticides are transported by lipo-

187 p r o t e i n s , in vivo, t h e n t h e y m a y p l a y e i t h e r a general o r specific role in cellular u p t a k e o f insecticides. Cellular u p t a k e m a y involve simple p a r t i t i o n i n g o f insecticides b e t w e e n p l a s m a l i p o p r o t e i n s a n d cell m e m b r a n e lipids w h i c h is c o n s i s t e n t w i t h t h e o b s e r v a t i o n o f P l a c k e t al. [9] o f r a p i d e x c h a n g e o f D D T a m o n g all t h e s e r u m l i p o p r o t e i n s . A l t e r n a t i v e l y , cellular u p t a k e o f insecticides m a y p r o c e e d b y a m o r e specific p r o c e s s involving cell r e c e p t o r s . L D L has r e c e n t l y b e e n s h o w n t o b e t a k e n u p b y f i b r o b l a s t s , l y m p h o c y t e s a n d s m o o t h m u s c l e cells via specific L D L r e c e p t o r s a n d t h e e x i s t e n c e o f r e c e p t o r s in o t h e r e x t r a h e p a t i c tissues has also b e e n p o s t u l a t e d [ 2 2 ] . I t r e m a i n s t o b e seen w h e t h e r e i t h e r o f t h e s e u p t a k e p r o c e s s e s o c c u r a n d w h e t h e r t h e process o f cellular u p t a k e influences t h e s u b s e q u e n t f a t e o f intracellular insecticides. ACKNOWLEDGEMENTS This r e s e a r c h was s u p p o r t e d in p a r t b y G r a n t N o . P H S E S - 0 0 0 4 4 f r o m t h e N a t i o n a l I n s t i t u t e o f H e a l t h . This is P a p e r N o . 6 5 9 1 o f t h e J o u r n a l Series o f t h e N o r t h C a r o l i n a Agricultural R e s e a r c h Service, Raleigh, N o r t h Carolina. REFERENCES

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