Cfasa-3iol. Inteructiomt, 0 El~~er~No~h-~o~~
113
26 (1979) 113-124 Scientific Publishers Ltd.
THE INTERACTION OF CRPHALORDXNE WITH MODEL MEMRRANE SY,TRMS AND RAT KIDNEY L~S~SO~S
MITCHELL FRY * and DAVID T. PLUMIJER
Department of Biochemlistry, Chelsea College. University of London, London, SW3 6LX (United Kingdom) June 3Oth,1978) (Revision received October 25tb, 1973)
(Received f kxepted
November
3rd, 1973)
The antibiotic cephaloridine has been shown to interact with phospholipid structures, using the techniques of ultraviolet difference spectroscopy, surface pressure measurements and liposome models. The results indicate that this in~mction is at least partly hy~ophobic in nature and help explain the disruptive effects of high concentrations of cephaloridine on botb artificial and natural phospholipid stn&ures (Iysosomes). Low concentrations of cephaloridine were shown to inhibit a lysosomal membrane-bound phospholipase 2 and it is suggested that such an iuhibition may explain the cephdori~~jnduced substation of ra~~~ney lysosomes.
INTRODUCTION
The antibiotic ceph~o~~e is nephrotoxic to rats in high doses as shown by merked histological changes in the kidney [I] and an increased excretion of enzymes into the urine [2,3 1. However, the excretion of the lysosomal enzyme acid phosphatase is severely depressed in the 12-h period immediately following the administration of the drug [4 ] and this effect is also seen with low doses of cephaloridine [ 51. This led to the suggestion that cephaloridine may stabilize the lysosomal membrane and ~~~en~ carried out with isolated kidney lysosomes would seeem to support this idea [ $6). However, exactly how the antibiotic stabilizes the lysosomal membrane is not known and it was felt that further work with some model membrane systems could throw iight on this,
-4
* Present Address: Institute
of Enzyme Research, Madison, Wisconsin,
USA.
114 Ultraviolet difference spectroscopy was used to see if the drug interacts with phospholipids [ 73 and surface pressure measurements were made to see if the antibiotic penetrated a lipid monolayer. Liposomes more closely approach biological membranes and the effect of cephaloridine on their formation and stability was also investigated. Finally the effect of cephalo&line on a lysosomal membrane bound phospholipase 2 was examined as previous experiments suggested that the inhibition of this enzyme and the stabilization of lysosomes may be closely related [ 81. MATERIALS AND METHODS
Chemicals. Cephaloridine (ceporin) was supplied as a gift by Glaxo Laboratories, Greenford, Middlesex, U.K. Choline phosphoglyceride (lecithin) was obtained from egg yolk (Lipid Products, Nutfield Nurseries, Nutfield, Surrey, U.K.). Triton X-109 (scintillation grade) was supplied by Fisons, Loughborough, Leicestershire, U.K. All other biochemical reagents were obtained from the Sigma Chemical Co., London, U.K. All solutions were prepared in distilled water redistilled from an all glass still. ~Ztra~ioZetspectra Many drugs absorb strongly in the ultraviolet region of the spectrum and when such compounds interact with biological molecules a change in their ultraviolet absorption spectra is often seen. The spectral changes may be quite small and are best studied by means of ultraviolet difference spectroscopy. In this investigation the technique developed by Herskovits [7] was used in which the drug and the macromolecule are kept separate in the reference beam but combined in the test beam of a double beam spectrophotometer. If there is an interaction between the drug and the macromolecule then the spectrum of one or both components will be changed and a difference spectrum generated with the arrmgement described above. An extension of this technique is to examine the change in the difference spectrum in the presence of a range of solvents. This gives information on the location of chromophores since the spectrum of a chromophore located on the surface of a macromolecule is usually perturbed by changes in the physical properties of the solvent. In contrast to this, a chromophore that is deeply buried or sheltered by the macromolecule is protected from such changes and will not show a perturbed spectrum. In this investigation, difference spectra were obtained on a Unicam SPSOOA ultraviolet spectrophotometer and a Cecil CE50fj double beam ultraviolet spectrophotometer, both instruments having tendferncell arrangements. Silica cells of 1 cm light path were used and all measurements made at room temperature (21 + 1°C) between 210 nm and 290 nm. Unless otherwise specified, all reagents were prepared in 10 mM triethanolamine/ butyric acid buEfer (pH 7.5). Egg lecithin was present in the form of vesicles as the concentration used (25 FM) was well above the critical micelle concentration. Stock suspensions of lecithin were prepared by evaporation of a
115 chloroform solution as used for the preparation of liposomes. All recorded spectra were repeated at least five times in order to obtain the mean spectra wihich are shown in the results section. Surface pressure measurements. Monolayers of lipids were prepared by dissolving them in a volatile solvent and adding a known amount between two hydrophobic barriers pl.aced across a Teflon trough. The solution spread across the surface and the solvent evaporated leaving a monolayer. The surface pressure (?T)was measured directly by measuring the force acting on the fixed barrier [9]. The amount of material in the monolayer and the area it covers are known so ITcan be calculated as a function of the area per molecule in the monolayer. In the present studies, a stock solution of 1 mg/ml egg lecithin was prepared in a volatile solvent (chloroform/petroleum ether, 1 : 4) and 39.2 ~1 delivered onto the surface of the subphase (0.145 M NaCl, pH 7) which had pn?viously been bubbled with nitrogen gas. Isotherms were measured in duplicate at 21 + 1°C after allowing 2 min for evaporation of the solvent and were reproducible within 5 A* per molecule and +1 dyne cm-‘. The limiting area per molecule was obtained by extrapolating the linear part of the force-area curve to zero pressure. As the area per molecule is decreased the surface pressure continues to rise until further compression causes the break up of the monolayer and the surface pressure suddenly decreases at the collapse pressure. Values obtained for the parameters in the present study were comparable to those previously reported [ 10,111. Liposomes. Liposomes were prepared and their permeabilities studied by following the leakage of trapped potassium chromate or glucose after the procedure of Weissman et al. [12], For each experiment, 10 pmol of egg lecithin, 2.86 pmol of dicetyl phosphate and 1.42 pmol of cholesterol (times the number of aliquots, usually 6) were dissolved in chloroform and added to ,a round bottomed flask. The contents were evaporated to dryness on a rotary evaporator under vacuum and the last traces of chloroform removed by expelling with nitrogen gas. One millilitre of 0.145 M K,Cr04 or 0.145 M glucose per aliquot was added to the evenly spread film and suspension dispersed by shaking with glass beads. The liposomes thus formed were then allo wed to complete their swelling and sequester the marker agents by being left at 4°C for 17 h. After this time, the lipid suspensions were placed in Visking dialysis tubing (8/32”) and dialysed for 30 min at room temperature against 11 of 0.145 M NaCl/KCl salt solution adjusted to pH 7 with constant stirring. The dialysis was repeated with two more changes of dialysate which was sufficient to remove all the untrapped markers from the liquid suspension. Oine millilitre aliquots of the dialysed liposomes were placed in separate small dialysis sacs together with 50 ~1 of lysolecithin or cephaloridine dissolved in 0.145 M NaCl/KCl (pH 7) or 50 ~1 lof progesterone dissolved in ethanol. Controls were also prepared containing 50 ~1 of the salt solution or ethanol but not the test solution and 200 ~1 of Triton X-100 was added to some dialysis sacs to give a final concentration of 0.1% v/v. All of the sacs
116 were then pIaced in test tubes containing 5 ml of 0.145 M NaCl/KCl {pH 7) and shaken ti a water bath at 37°C. At suitable time intervals, the sacs were transferred to fresh tubes and the amount of leaked marker compound in the salt solution was determined. The amount of the marker compound remaining in the liposomes at the end of the experiment (usually 3 h) was determined by boiling the contents of the sacs for 20 min and analysing the resultent supematant. The concentration of chromate was determined Tom its absorbance at 380 nm by reference to a standard curve. Glucose was determined by a glucose oxidase method [ 131. Lysosomes. Rat kidney lysosomes were prepared and their integrity monitored by means of enzyme leakage and light scattering as previously described [ 141. Phospholipase 2. The membrane phospholipase 2 was purified from isolated kidney lysosomes as previously described [8] and the activity measured by following the lysis of erythrocytes in an isotonic medium. For routine assay, a pooled sample of 8-lo-week-old human blood was obtained from the South London Transfusion Centre, Sutton, U.K. and 1 ml added to 10 ml of 0.25 M sucrose buffered to pH 7.5 with 10 mM triethanolaminebutyrate. The suspension was centrifuged at 2000 g for 10 min, the supernatant removed and the red cells suspended in 10 ml of the buffered sucrose solution. Tbe suspension was centrifuged again and the packed red cells diluted to 10 ml in buffered sucrose and stored on ice until required. An aliquot of the erythrocyte suspension (10 @) was added to 3 ml of buffered sucrose containing 0.5 titi CaCl, in a glass cuvette of 1 cm light path to give an initial absorbance of about 0.5 at 520 nm. A sublytic concentration of lysolecithin (1.8 r_tM)was added to the suspension as an activator, followed by phospholipase 2 and the rate of haemolysis measured by following the fall in extinction at 520 nm and 37°C in a recording spectrophotometer. RESULTS AND DISCUSSION
Ultmviole t spectra. Cephaloridine showed a characteristic ultraviolet absorption spectrum with a maximum at 240 nm apparently attributable to the O=C-NC+chromophore of the 0 la&am ring [ 151. In contrast to this, egg lecithin exhibited a rather featureless spectrum over the same wavelength range (Fig. I). The combined absorption spectrum of these compounds differed iittle in the position of the absorption maximum compared with cephalorildine alone but did show a change in the combined intensity calculated by simply adding together the absorbance shown by the separate compounds (Fig. 1). The small spectral change was best seen in the form of a difference spectrum (Fig. 2a) which showed that some interaction between the drug and the lipid had taken place. The similarity of the difference spectrum in the presence of egg lecithin and the cationic detergent cetyltribromide (Fig. 2b) would suggest that the interaction may
117
!
I_
I.
rru
250
Wavelength (nm)
Fig. I. The ultraviolet spectrum of 60 fl$ cephaloridine and 0.25 @i 10 mM triethanolamine-butyte b’uffer (pH 7.5).
egg lecithin in
result from the burial of the antibiotic in an hydrophobic environment Information on the extent of the burial of chromophores within macromolecules can be obtained by use of the solvent perturbation technique [18]
a ’ +o OlO-
b~cetyltrim~hylammonium
lxwnide
cephaloridine in the presence of (a) 0.25 Fig. 2. Difference spectra for 60 lecithin and (b) 1.3 mM cetykrirnethyknmonium bromide.
118
(b) metbonol
(50% V,‘V)
Fig. 3. Difference spectra for 60 pM
(------)andabaencc:(-
cephaloridinein a rangeof solventsin the presence ) of 0.25 @U egglecithin.
and the degree of perturbation generally follows the series sucrose > glycerol > ethylene glycol > methanol > polyethylene-glycol > dimethylsulpbodde. Molecules at the beginning of the above series produce their effect by ~on-specitk modification of the solvent environment and the difference sp+xAzumof cephaloridine in the presence of 1 M sucrose is little affected by the lipid egg lecithin (Fig. 3a) which would suggest a small partial burial of the drug within the lipid lamelles. Perturbants at the end of this series produce &eir effects by direct interaction with the chromophore so that even a smaU partial burial of the drug within the lipid would have a large effect. This USfound in practice where lecithin exhibits a large effect on the
so-
L3-
s t?! @
Q 10 0 t z o-
\ \ 60 80 Area per molecule (A0 ’
pressure/ oridine,1
‘\ 100
)
curvesfor egglecithin(0, CaC12 ).
control;
, 2d
ceph&Gdine;
119 difference spectrum of cephaloridine in methanol (Fig. 3b) and an even greater effect in the case of polyethylene glycol (Fig. 3~). Surface pressure measurements. Confirmation that cephaloridine interacts with lipid structures is given by the next set of experiments with monolayers. Surface pressure-area curves for egg lecithin are shown in Fig. 4. The average area per molecule was found to be 72.5 A2 and a collapse pressure of about 47 dynes cm-’ was recorded for an egg lecithin monolayer which agrees closely $with the published values [ 10,111. The addition of only 2 mM cephaloridine resulted in an increase in the surface area per molecule from 72.5 A2 to 90 A2 suggesting that the antibiotic penetrates and expands the lipid monolayer. This expansion by the drug was further enhanced with 1 mM CaC12 to 98.5 ip2 which could indicate some ionic interaction of the Ca2’ with the ionic phosphate head groups. For example, Maggio and Lucy [19] have shown that bivalent cations can significantly modify the interaction of fusogenic lipids with phospholipids through interaction of the polar ionic group on the phospholipid molecules. Alternatively, the C;a’L” may interact directly with cephaloridine forming a bridge between two molecules of the drug or between the antibiotic and charged groups on the membrane. In either case the effective volume of the membrane would he increased. This second explanation is perhaps the most likely since the addition of CaC12 alone to the subphase had no effect on the average area per molecule in the monolayer (Fig. 4). Liposome studies. The penetration of cephaloridine into the lipid monolayer could affect the stability of the lipid lamellae and this possibility was examined using liposomes [20]. These structures provide a useful membrane model since their permeability characteristics for anions, cations and water are similar to biological membranes [21] and they also respond to steroids and lytic agents in a similar manner to natural phospholipid structures such as lysosomes, mitochondria and erythrocytes [ 223.
Time (hJ
Fig. 5. The effect of lytic agents on the chromate leakage from liposomes. In all cases fhe mole ratio of agent to lecithin was 0.2 : 1.
In the present study about 3!0% of potassium chromate or 54% glucose was captured and an average of 96% of the total chromate and 102% of the glucose could be accounted for. Each l-ml of liposome suspension contained an average of 16 pmol of CrOO and 81 pmol of glucose. These values agree with those obtained by other workers [ 221. The liposomes were not entirely stable and incubation at 21°C for 3 h resulted in the loss of about 18% of the trapped chromate (Fig. 5). Addition of the detergent Triton X-1190 to a final concentration of 0.1% v/v, caused the leakage of 82% of the trapped chromophore over the same time period and other surface active agents behaved in a similar although less effective manner. For example with a mole ratio of lytic agent to lecithin of 0.2 : 1, the addition of progesterone lead to the loss of 69% of the trapped chromate ion in 3 h and lysolecithin to the leakage of 32% of the chromate in the same time period. In contrast to these agents, exogenously added cephaloridine caused only a slightly higher chromate leakage (Fig. 5). The antibiotic cannot therefore be considered as a lytic ag-nt and a cephaloridine to lecithin mole ratio of 2 : 1 was needed before the drug was as effective as lysolecithin as shown in Fig. 5. Incorporation of cephaloridine into the liposomes during their formation did not greatly affect the amount of glucose trapped until quite high mole ratios were reached and even then the drug was far less effective than lysolecithin on a comparable mole ratio basis (Fig. 6). When cephaloridine and lysolecithin were preincorporated into lip.Jsomes together, the structures were able to retain less glucose than if the drug was omitted from the preparation. However the difference was not very great and a high mole ratio of CephsIoridine to lecithin (0.5 : 1) had to be used to obtain any significant effect (Fig. 7). If stearylamine was incorporated into the liposomes in place of dicetyl phosphate to give the spherules ,a net positive charge, the controls leaked more chromate (29%) than the negatively charged liposomes (18%) but otherwise the results were not proportionally different to the ones discussed (Figs. 5,6 and 7).
Male ratio of compound to Ilecithin
Fig. 6. The effect of cephdoridine (0) and lysolecithin ( ) on the alnount of glucose trapped by liposomes.
121
aryl sulphatase _-_____________ ________.______
acid phosphatase
0.1 : 1 Ratio of lysolecithin / lecithin
a
Cephaloridine
(mhJ]
Fig.7. The effect of cephaloridine on the amount of glucose trapped in liposomes of egg lecithin. Liposomes were prepared as described in the text with the drug present in a mole ratio of cephaloridine/lecithin, 0.5 : 1, S. A control group of liposomes were also prepared in which the drug was omitted O. The effect of lysolecithin on the trapping of glucose by these preparations was also investigated. Fig. 8. The effect of cephaloridine on the release of acid phosphatase and arylsulphatase from isolated lysosomes after incubation for 1 h at 37’%.
Cephaloridinc and kidney Zysosomes. The results discussed so far indicate that cephaloridine interacts with phospholipid structures. Surface pressure measurements showed that at high concentrations of the antibiotic this interaction had a disruptive effect on the lipid lamellae and caused an increased permeability (Figs. 5 and 6). The cephaloridine-phospholipid interaction also appears to accentuate the disruptive effect of lysolecithin on liposome structures (Fig. 7). This work with modlel membrane systems sc?mes to explain how high concentrations of the antibiotic (>lO m&l) increases the permeability of isolated kidney lysosomes [ 5 1. For example, incubation of isolated lysosomes for 1 h
cephaloridine
(1mMI
control
s
I_-_0
1
2
3
Tlme(min)
Fig. 9. The effect of 6.6 /.M lysolecithin on the light scattering properties of isolated kidney lysosomes. Lysoso!mes were suspended in 0.25 M sucrose buffered to pH 7.5 with 10 mM triethanolamine-butyrate and lysolecithin added at zero time to a final concentration of 6.6 @I. The change in extinction at 520 nm was then followed continuously for 3 min. No change in the extinction was seen in the absence of lysolecithin.
at NOC in an osmotically protecting medium (0.25 M sucrose, 10 mM triethanolamine-bulyrate (pII 7.5)) lead to the release of 27% of their acid phosphatase activity and 47% of their arylsulphatase whereas the presence of cephaloridine increased these values to 83% and 76% respectively 8). However, the results so far cannot explain how low concentrations of the antibiotic appear to increase the stability of lysosomes both in vitro (Fig. 8) and in vivo [5]. The present study also showed that if lysolecithin was present, less glucose was trapped during the formation of liposomes and that this effect was enhanced by the presence of high concentrations of cephaloridine (Fig. 7). This is in contrast to the results obtained with isolated lysosomes where low concentrations of cephaloridine protect the organelles against the lytic action of lysolecithin (Fig. 9). Some other explanation must therefore by sought for the protective effect of low concentrations of the drug on lysosomes. Gephaloridine and phospholipase 2. Investigations have shown that the membrane of rat kidney lysosomes contains a phospholipose 2 enzyme which catalyses the hydrolysis of phospholipid to lysophospholipids [8]. The formation of such lyso compounds could lead to local regions of micellisation in the lipid lamellae with subsequent membrane u-stability. Such a suggestion has in fact been made to explain the process of ynembrane fusion [ 221. The lytic effects of iow concentrations of lysolecithin may be partly explained by its activating effect on membrane bound phospholipases [8]. (cf. th of ‘Ikiton X-100 on the enzymic activity of phospholipase 2 phospholipase 2 will only haemolyse red blood cells if these Wm. are first treated with sublytic concentrations of detergent [ 251. Cephaloridine on&tic fashion to lysolecithin, i.e. it may alter the memone less susceptible to phospholipase 2 action. This antie of partially protecting against phospholipase 2 induced 10). There would appear to be a strong correlation between bition of the lysosom membrane phospholipase 2 by cephaloridine loll
5 8 G
550 ‘E
” .s g
%l.
r\:;:::-
0
cephaloridine
----
10
mM/
control
20
Time (min)
ted haemolysis of human described in the text by ffect of cephaloridine on ere was no haemolysis of the
128 and other lipid interacting agents, and the stabilisation of rat kidney lysosomes [8]. Clearly, cephaloridine can interact with lipid structures and be severely disruptive at high concentrations. However, this does not directly explain its ability ‘to stabilize rat kidney lysosomes at low concentrations [ 51, and other mechanisms, such as the effect on a membrane phospholipase 2, need to be invoked. ACKNOWLEDGEMENTS
The authors wis‘h to thank Glaxo Laboratories for the provision of the antibiotic cephaloridine and the Science Research Council for the provision of a scholarship to ME’. REFERENCES 1 R.M.A. Atkinson, J.P. Currie, D.A.H. Pratt, H.M. Sharpe and E.G. Tomich, Acute toxicity of cephaloridine, an antibiotic derived from cephalosporin C, Toxicol. Appl. Pharmacol., 8 (1966) 398. 2 E.O. Ngaha and D.T. Plummer, Toxic renal damage: changes in enzyme levels, Biochem. Med., 18 (1977) 71. 3 D.T. Plummer, E.G. Ngaha, P.J. Wright, P.D. Leathwood and M.E. Blake, in: U.C. Dubach and U. Schmidt, (Eds.), Biochemical Aspects of Diagnostic Significance in Urine, Hans Huber, Beme, 1979, in press. 4 D.T. Plummer and E.O. Ngahn, in: J.P. ‘Fillastre (Ed.), International Colloquium on the Interaction of Drugs with Membrane Systems, 1978, p. 175. 5 E.O. Ngaha, M. Fry and D.T. Flummer, The effect of cephaloridine on the stability of rat kidney lysosomes, Chem.-Biol. Iateract., 24 (1979) 199. 6 M. F’ry, E.O. Ngaha and D.T. Plummer, The protective effect of cephaloridine on rat kidney lysosomes in vitro, ‘Dans. Biochem. Sot., 3 (1975) 736. 7 T.T. Herskovits, in: S.P. Colowick and N.O. Kaplan (Eds.), Methods in Enzymology, Vol. XI, Academic Press, New York, 1967, p. 748. 8 M. Fry and D.T. Plummer, in: J.P. Fillastre (Ed.), International Colloquium on the Interaction of Drugs with Membrane Systems, 1978, p. 193. 9 K.F. Bromfield, MS. Wooster and J.M. Wrigglesworth, Robust attachment to film balance for surface pressure measurements, Lab. Pratt., 23 (1974) 122. 10 E.J. Rojas and J.M. Tobias, Membrane models: association of inorganic cations with phospholipid monolayers, Biochim. Biophys. Acta, 94 (1965) 394. 11 D.O. Shah, Interaction of uranyl ions with phospholipid and cholesterol monolayers, J. Coll. Inter. Sci., 29 (1969) 210. 12 G. Weissman, G. Sessa and S. Weissmann, The action of steroids and Triton X-100 upon phoapholipidlcholesterol structures, Biochem. Pharmacol., 15 (1966) 1537. 13 D.T. Plummer, Qn introduction to Practical Biochemistry, 2nd edn., McGraw-Hill, London, 1978, p. ‘185. 14 M. Fry and D.T. Ilummcr, The use of light scattering measurements to monitor the stabilisation of rat kidney lysosomes by the antibiotic cephaloridine, J. Physiol., 270 (1977) 20. 15 RR. Chauvette, E,.H. Flynn, B.G. Jackson, E.R. Lavagnino, B.G. Morin, R.A. Mueller, R.P. P&b, R.W. Roeske, C.W. Ryan, J.L. Spencer and E. Van Heyingen, Chemistry of cephalosporin sntibiotics. II. Preparation of a new class of antibiotics and the relation of structures to activity, J. Am. Chem. SOC., 84 (1962) 3401. 16 C.F. Chignell, Optical studies of drug-protein complexes. II Interactions of phenyl-
124
17 18 19 20 21 22 23 24 25
butazone and its analogues with human serum albumin, Mol. Pharmacol., 5 (1969) 244. C.F. ChLgnell, in: NJ. Harper (Ed.), Advances in Drug Research, Vol. 5, Academic Press, London, 1970, p. 55. M. Laskowski, Measurement of accessibility of protein chromophores by solvent perturbation of their ultraviolet spectra, Fed. Proc., 25 (1966) 20. B. Maggio and J. Lucy, Polar-group behaviour in mixed monolayers of phospholipids and fusogenic lipids, Biochem. J., 155 (1976) 353. A.D. Bangham, Membrane models with phospholipids, Prog. Biophys. Mol. Biol., 18 (1968) 29. AD. Bangham, M.M. 8tandii and J.C. Watkins, Diffusion of univalent ions across the lamellae of swollen phospholipids, J. Mol. Biol., 13 (1965) 238. J.A. Lucy, The fusion of biological membralws, Nature, 227 (1970) 815. M. Fry, Lysosomal stabilisation by cephaloridine: the role of a membrane-bound phospholipsse 2, Ph.D. Thesis, London, 1977. E.A. Dennis, Kinetic dependence of phospholipase A2 activity on the detergent Triton X-100, J. Lipid Res., 14 (1973) 152. C.B. Woodward and R.F.A. Zwaal, Phe lytic behaviour of pure phospholipases ,42 and C towards osmotically swolle ‘1 c-ythrocytes and reseated ghosts, Biochim. Biophys. Acta, 274 (1972) 272.
113
26 (1979) 113-124 Scientific Publishers Ltd.
THE INTERACTION OF CRPHALORDXNE WITH MODEL MEMRRANE SY,TRMS AND RAT KIDNEY L~S~SO~S
MITCHELL FRY * and DAVID T. PLUMIJER
Department of Biochemlistry, Chelsea College. University of London, London, SW3 6LX (United Kingdom) June 3Oth,1978) (Revision received October 25tb, 1973)
(Received f kxepted
November
3rd, 1973)
The antibiotic cephaloridine has been shown to interact with phospholipid structures, using the techniques of ultraviolet difference spectroscopy, surface pressure measurements and liposome models. The results indicate that this in~mction is at least partly hy~ophobic in nature and help explain the disruptive effects of high concentrations of cephaloridine on botb artificial and natural phospholipid stn&ures (Iysosomes). Low concentrations of cephaloridine were shown to inhibit a lysosomal membrane-bound phospholipase 2 and it is suggested that such an iuhibition may explain the cephdori~~jnduced substation of ra~~~ney lysosomes.
INTRODUCTION
The antibiotic ceph~o~~e is nephrotoxic to rats in high doses as shown by merked histological changes in the kidney [I] and an increased excretion of enzymes into the urine [2,3 1. However, the excretion of the lysosomal enzyme acid phosphatase is severely depressed in the 12-h period immediately following the administration of the drug [4 ] and this effect is also seen with low doses of cephaloridine [ 51. This led to the suggestion that cephaloridine may stabilize the lysosomal membrane and ~~~en~ carried out with isolated kidney lysosomes would seeem to support this idea [ $6). However, exactly how the antibiotic stabilizes the lysosomal membrane is not known and it was felt that further work with some model membrane systems could throw iight on this,
-4
* Present Address: Institute
of Enzyme Research, Madison, Wisconsin,
USA.
114 Ultraviolet difference spectroscopy was used to see if the drug interacts with phospholipids [ 73 and surface pressure measurements were made to see if the antibiotic penetrated a lipid monolayer. Liposomes more closely approach biological membranes and the effect of cephaloridine on their formation and stability was also investigated. Finally the effect of cephalo&line on a lysosomal membrane bound phospholipase 2 was examined as previous experiments suggested that the inhibition of this enzyme and the stabilization of lysosomes may be closely related [ 81. MATERIALS AND METHODS
Chemicals. Cephaloridine (ceporin) was supplied as a gift by Glaxo Laboratories, Greenford, Middlesex, U.K. Choline phosphoglyceride (lecithin) was obtained from egg yolk (Lipid Products, Nutfield Nurseries, Nutfield, Surrey, U.K.). Triton X-109 (scintillation grade) was supplied by Fisons, Loughborough, Leicestershire, U.K. All other biochemical reagents were obtained from the Sigma Chemical Co., London, U.K. All solutions were prepared in distilled water redistilled from an all glass still. ~Ztra~ioZetspectra Many drugs absorb strongly in the ultraviolet region of the spectrum and when such compounds interact with biological molecules a change in their ultraviolet absorption spectra is often seen. The spectral changes may be quite small and are best studied by means of ultraviolet difference spectroscopy. In this investigation the technique developed by Herskovits [7] was used in which the drug and the macromolecule are kept separate in the reference beam but combined in the test beam of a double beam spectrophotometer. If there is an interaction between the drug and the macromolecule then the spectrum of one or both components will be changed and a difference spectrum generated with the arrmgement described above. An extension of this technique is to examine the change in the difference spectrum in the presence of a range of solvents. This gives information on the location of chromophores since the spectrum of a chromophore located on the surface of a macromolecule is usually perturbed by changes in the physical properties of the solvent. In contrast to this, a chromophore that is deeply buried or sheltered by the macromolecule is protected from such changes and will not show a perturbed spectrum. In this investigation, difference spectra were obtained on a Unicam SPSOOA ultraviolet spectrophotometer and a Cecil CE50fj double beam ultraviolet spectrophotometer, both instruments having tendferncell arrangements. Silica cells of 1 cm light path were used and all measurements made at room temperature (21 + 1°C) between 210 nm and 290 nm. Unless otherwise specified, all reagents were prepared in 10 mM triethanolamine/ butyric acid buEfer (pH 7.5). Egg lecithin was present in the form of vesicles as the concentration used (25 FM) was well above the critical micelle concentration. Stock suspensions of lecithin were prepared by evaporation of a
115 chloroform solution as used for the preparation of liposomes. All recorded spectra were repeated at least five times in order to obtain the mean spectra wihich are shown in the results section. Surface pressure measurements. Monolayers of lipids were prepared by dissolving them in a volatile solvent and adding a known amount between two hydrophobic barriers pl.aced across a Teflon trough. The solution spread across the surface and the solvent evaporated leaving a monolayer. The surface pressure (?T)was measured directly by measuring the force acting on the fixed barrier [9]. The amount of material in the monolayer and the area it covers are known so ITcan be calculated as a function of the area per molecule in the monolayer. In the present studies, a stock solution of 1 mg/ml egg lecithin was prepared in a volatile solvent (chloroform/petroleum ether, 1 : 4) and 39.2 ~1 delivered onto the surface of the subphase (0.145 M NaCl, pH 7) which had pn?viously been bubbled with nitrogen gas. Isotherms were measured in duplicate at 21 + 1°C after allowing 2 min for evaporation of the solvent and were reproducible within 5 A* per molecule and +1 dyne cm-‘. The limiting area per molecule was obtained by extrapolating the linear part of the force-area curve to zero pressure. As the area per molecule is decreased the surface pressure continues to rise until further compression causes the break up of the monolayer and the surface pressure suddenly decreases at the collapse pressure. Values obtained for the parameters in the present study were comparable to those previously reported [ 10,111. Liposomes. Liposomes were prepared and their permeabilities studied by following the leakage of trapped potassium chromate or glucose after the procedure of Weissman et al. [12], For each experiment, 10 pmol of egg lecithin, 2.86 pmol of dicetyl phosphate and 1.42 pmol of cholesterol (times the number of aliquots, usually 6) were dissolved in chloroform and added to ,a round bottomed flask. The contents were evaporated to dryness on a rotary evaporator under vacuum and the last traces of chloroform removed by expelling with nitrogen gas. One millilitre of 0.145 M K,Cr04 or 0.145 M glucose per aliquot was added to the evenly spread film and suspension dispersed by shaking with glass beads. The liposomes thus formed were then allo wed to complete their swelling and sequester the marker agents by being left at 4°C for 17 h. After this time, the lipid suspensions were placed in Visking dialysis tubing (8/32”) and dialysed for 30 min at room temperature against 11 of 0.145 M NaCl/KCl salt solution adjusted to pH 7 with constant stirring. The dialysis was repeated with two more changes of dialysate which was sufficient to remove all the untrapped markers from the liquid suspension. Oine millilitre aliquots of the dialysed liposomes were placed in separate small dialysis sacs together with 50 ~1 of lysolecithin or cephaloridine dissolved in 0.145 M NaCl/KCl (pH 7) or 50 ~1 lof progesterone dissolved in ethanol. Controls were also prepared containing 50 ~1 of the salt solution or ethanol but not the test solution and 200 ~1 of Triton X-100 was added to some dialysis sacs to give a final concentration of 0.1% v/v. All of the sacs
116 were then pIaced in test tubes containing 5 ml of 0.145 M NaCl/KCl {pH 7) and shaken ti a water bath at 37°C. At suitable time intervals, the sacs were transferred to fresh tubes and the amount of leaked marker compound in the salt solution was determined. The amount of the marker compound remaining in the liposomes at the end of the experiment (usually 3 h) was determined by boiling the contents of the sacs for 20 min and analysing the resultent supematant. The concentration of chromate was determined Tom its absorbance at 380 nm by reference to a standard curve. Glucose was determined by a glucose oxidase method [ 131. Lysosomes. Rat kidney lysosomes were prepared and their integrity monitored by means of enzyme leakage and light scattering as previously described [ 141. Phospholipase 2. The membrane phospholipase 2 was purified from isolated kidney lysosomes as previously described [8] and the activity measured by following the lysis of erythrocytes in an isotonic medium. For routine assay, a pooled sample of 8-lo-week-old human blood was obtained from the South London Transfusion Centre, Sutton, U.K. and 1 ml added to 10 ml of 0.25 M sucrose buffered to pH 7.5 with 10 mM triethanolaminebutyrate. The suspension was centrifuged at 2000 g for 10 min, the supernatant removed and the red cells suspended in 10 ml of the buffered sucrose solution. Tbe suspension was centrifuged again and the packed red cells diluted to 10 ml in buffered sucrose and stored on ice until required. An aliquot of the erythrocyte suspension (10 @) was added to 3 ml of buffered sucrose containing 0.5 titi CaCl, in a glass cuvette of 1 cm light path to give an initial absorbance of about 0.5 at 520 nm. A sublytic concentration of lysolecithin (1.8 r_tM)was added to the suspension as an activator, followed by phospholipase 2 and the rate of haemolysis measured by following the fall in extinction at 520 nm and 37°C in a recording spectrophotometer. RESULTS AND DISCUSSION
Ultmviole t spectra. Cephaloridine showed a characteristic ultraviolet absorption spectrum with a maximum at 240 nm apparently attributable to the O=C-NC+chromophore of the 0 la&am ring [ 151. In contrast to this, egg lecithin exhibited a rather featureless spectrum over the same wavelength range (Fig. I). The combined absorption spectrum of these compounds differed iittle in the position of the absorption maximum compared with cephalorildine alone but did show a change in the combined intensity calculated by simply adding together the absorbance shown by the separate compounds (Fig. 1). The small spectral change was best seen in the form of a difference spectrum (Fig. 2a) which showed that some interaction between the drug and the lipid had taken place. The similarity of the difference spectrum in the presence of egg lecithin and the cationic detergent cetyltribromide (Fig. 2b) would suggest that the interaction may
117
!
I_
I.
rru
250
Wavelength (nm)
Fig. I. The ultraviolet spectrum of 60 fl$ cephaloridine and 0.25 @i 10 mM triethanolamine-butyte b’uffer (pH 7.5).
egg lecithin in
result from the burial of the antibiotic in an hydrophobic environment Information on the extent of the burial of chromophores within macromolecules can be obtained by use of the solvent perturbation technique [18]
a ’ +o OlO-
b~cetyltrim~hylammonium
lxwnide
cephaloridine in the presence of (a) 0.25 Fig. 2. Difference spectra for 60 lecithin and (b) 1.3 mM cetykrirnethyknmonium bromide.
118
(b) metbonol
(50% V,‘V)
Fig. 3. Difference spectra for 60 pM
(------)andabaencc:(-
cephaloridinein a rangeof solventsin the presence ) of 0.25 @U egglecithin.
and the degree of perturbation generally follows the series sucrose > glycerol > ethylene glycol > methanol > polyethylene-glycol > dimethylsulpbodde. Molecules at the beginning of the above series produce their effect by ~on-specitk modification of the solvent environment and the difference sp+xAzumof cephaloridine in the presence of 1 M sucrose is little affected by the lipid egg lecithin (Fig. 3a) which would suggest a small partial burial of the drug within the lipid lamelles. Perturbants at the end of this series produce &eir effects by direct interaction with the chromophore so that even a smaU partial burial of the drug within the lipid would have a large effect. This USfound in practice where lecithin exhibits a large effect on the
so-
L3-
s t?! @
Q 10 0 t z o-
\ \ 60 80 Area per molecule (A0 ’
pressure/ oridine,1
‘\ 100
)
curvesfor egglecithin(0, CaC12 ).
control;
, 2d
ceph&Gdine;
119 difference spectrum of cephaloridine in methanol (Fig. 3b) and an even greater effect in the case of polyethylene glycol (Fig. 3~). Surface pressure measurements. Confirmation that cephaloridine interacts with lipid structures is given by the next set of experiments with monolayers. Surface pressure-area curves for egg lecithin are shown in Fig. 4. The average area per molecule was found to be 72.5 A2 and a collapse pressure of about 47 dynes cm-’ was recorded for an egg lecithin monolayer which agrees closely $with the published values [ 10,111. The addition of only 2 mM cephaloridine resulted in an increase in the surface area per molecule from 72.5 A2 to 90 A2 suggesting that the antibiotic penetrates and expands the lipid monolayer. This expansion by the drug was further enhanced with 1 mM CaC12 to 98.5 ip2 which could indicate some ionic interaction of the Ca2’ with the ionic phosphate head groups. For example, Maggio and Lucy [19] have shown that bivalent cations can significantly modify the interaction of fusogenic lipids with phospholipids through interaction of the polar ionic group on the phospholipid molecules. Alternatively, the C;a’L” may interact directly with cephaloridine forming a bridge between two molecules of the drug or between the antibiotic and charged groups on the membrane. In either case the effective volume of the membrane would he increased. This second explanation is perhaps the most likely since the addition of CaC12 alone to the subphase had no effect on the average area per molecule in the monolayer (Fig. 4). Liposome studies. The penetration of cephaloridine into the lipid monolayer could affect the stability of the lipid lamellae and this possibility was examined using liposomes [20]. These structures provide a useful membrane model since their permeability characteristics for anions, cations and water are similar to biological membranes [21] and they also respond to steroids and lytic agents in a similar manner to natural phospholipid structures such as lysosomes, mitochondria and erythrocytes [ 223.
Time (hJ
Fig. 5. The effect of lytic agents on the chromate leakage from liposomes. In all cases fhe mole ratio of agent to lecithin was 0.2 : 1.
In the present study about 3!0% of potassium chromate or 54% glucose was captured and an average of 96% of the total chromate and 102% of the glucose could be accounted for. Each l-ml of liposome suspension contained an average of 16 pmol of CrOO and 81 pmol of glucose. These values agree with those obtained by other workers [ 221. The liposomes were not entirely stable and incubation at 21°C for 3 h resulted in the loss of about 18% of the trapped chromate (Fig. 5). Addition of the detergent Triton X-1190 to a final concentration of 0.1% v/v, caused the leakage of 82% of the trapped chromophore over the same time period and other surface active agents behaved in a similar although less effective manner. For example with a mole ratio of lytic agent to lecithin of 0.2 : 1, the addition of progesterone lead to the loss of 69% of the trapped chromate ion in 3 h and lysolecithin to the leakage of 32% of the chromate in the same time period. In contrast to these agents, exogenously added cephaloridine caused only a slightly higher chromate leakage (Fig. 5). The antibiotic cannot therefore be considered as a lytic ag-nt and a cephaloridine to lecithin mole ratio of 2 : 1 was needed before the drug was as effective as lysolecithin as shown in Fig. 5. Incorporation of cephaloridine into the liposomes during their formation did not greatly affect the amount of glucose trapped until quite high mole ratios were reached and even then the drug was far less effective than lysolecithin on a comparable mole ratio basis (Fig. 6). When cephaloridine and lysolecithin were preincorporated into lip.Jsomes together, the structures were able to retain less glucose than if the drug was omitted from the preparation. However the difference was not very great and a high mole ratio of CephsIoridine to lecithin (0.5 : 1) had to be used to obtain any significant effect (Fig. 7). If stearylamine was incorporated into the liposomes in place of dicetyl phosphate to give the spherules ,a net positive charge, the controls leaked more chromate (29%) than the negatively charged liposomes (18%) but otherwise the results were not proportionally different to the ones discussed (Figs. 5,6 and 7).
Male ratio of compound to Ilecithin
Fig. 6. The effect of cephdoridine (0) and lysolecithin ( ) on the alnount of glucose trapped by liposomes.
121
aryl sulphatase _-_____________ ________.______
acid phosphatase
0.1 : 1 Ratio of lysolecithin / lecithin
a
Cephaloridine
(mhJ]
Fig.7. The effect of cephaloridine on the amount of glucose trapped in liposomes of egg lecithin. Liposomes were prepared as described in the text with the drug present in a mole ratio of cephaloridine/lecithin, 0.5 : 1, S. A control group of liposomes were also prepared in which the drug was omitted O. The effect of lysolecithin on the trapping of glucose by these preparations was also investigated. Fig. 8. The effect of cephaloridine on the release of acid phosphatase and arylsulphatase from isolated lysosomes after incubation for 1 h at 37’%.
Cephaloridinc and kidney Zysosomes. The results discussed so far indicate that cephaloridine interacts with phospholipid structures. Surface pressure measurements showed that at high concentrations of the antibiotic this interaction had a disruptive effect on the lipid lamellae and caused an increased permeability (Figs. 5 and 6). The cephaloridine-phospholipid interaction also appears to accentuate the disruptive effect of lysolecithin on liposome structures (Fig. 7). This work with modlel membrane systems sc?mes to explain how high concentrations of the antibiotic (>lO m&l) increases the permeability of isolated kidney lysosomes [ 5 1. For example, incubation of isolated lysosomes for 1 h
cephaloridine
(1mMI
control
s
I_-_0
1
2
3
Tlme(min)
Fig. 9. The effect of 6.6 /.M lysolecithin on the light scattering properties of isolated kidney lysosomes. Lysoso!mes were suspended in 0.25 M sucrose buffered to pH 7.5 with 10 mM triethanolamine-butyrate and lysolecithin added at zero time to a final concentration of 6.6 @I. The change in extinction at 520 nm was then followed continuously for 3 min. No change in the extinction was seen in the absence of lysolecithin.
at NOC in an osmotically protecting medium (0.25 M sucrose, 10 mM triethanolamine-bulyrate (pII 7.5)) lead to the release of 27% of their acid phosphatase activity and 47% of their arylsulphatase whereas the presence of cephaloridine increased these values to 83% and 76% respectively 8). However, the results so far cannot explain how low concentrations of the antibiotic appear to increase the stability of lysosomes both in vitro (Fig. 8) and in vivo [5]. The present study also showed that if lysolecithin was present, less glucose was trapped during the formation of liposomes and that this effect was enhanced by the presence of high concentrations of cephaloridine (Fig. 7). This is in contrast to the results obtained with isolated lysosomes where low concentrations of cephaloridine protect the organelles against the lytic action of lysolecithin (Fig. 9). Some other explanation must therefore by sought for the protective effect of low concentrations of the drug on lysosomes. Gephaloridine and phospholipase 2. Investigations have shown that the membrane of rat kidney lysosomes contains a phospholipose 2 enzyme which catalyses the hydrolysis of phospholipid to lysophospholipids [8]. The formation of such lyso compounds could lead to local regions of micellisation in the lipid lamellae with subsequent membrane u-stability. Such a suggestion has in fact been made to explain the process of ynembrane fusion [ 221. The lytic effects of iow concentrations of lysolecithin may be partly explained by its activating effect on membrane bound phospholipases [8]. (cf. th of ‘Ikiton X-100 on the enzymic activity of phospholipase 2 phospholipase 2 will only haemolyse red blood cells if these Wm. are first treated with sublytic concentrations of detergent [ 251. Cephaloridine on&tic fashion to lysolecithin, i.e. it may alter the memone less susceptible to phospholipase 2 action. This antie of partially protecting against phospholipase 2 induced 10). There would appear to be a strong correlation between bition of the lysosom membrane phospholipase 2 by cephaloridine loll
5 8 G
550 ‘E
” .s g
%l.
r\:;:::-
0
cephaloridine
----
10
mM/
control
20
Time (min)
ted haemolysis of human described in the text by ffect of cephaloridine on ere was no haemolysis of the
128 and other lipid interacting agents, and the stabilisation of rat kidney lysosomes [8]. Clearly, cephaloridine can interact with lipid structures and be severely disruptive at high concentrations. However, this does not directly explain its ability ‘to stabilize rat kidney lysosomes at low concentrations [ 51, and other mechanisms, such as the effect on a membrane phospholipase 2, need to be invoked. ACKNOWLEDGEMENTS
The authors wis‘h to thank Glaxo Laboratories for the provision of the antibiotic cephaloridine and the Science Research Council for the provision of a scholarship to ME’. REFERENCES 1 R.M.A. Atkinson, J.P. Currie, D.A.H. Pratt, H.M. Sharpe and E.G. Tomich, Acute toxicity of cephaloridine, an antibiotic derived from cephalosporin C, Toxicol. Appl. Pharmacol., 8 (1966) 398. 2 E.O. Ngaha and D.T. Plummer, Toxic renal damage: changes in enzyme levels, Biochem. Med., 18 (1977) 71. 3 D.T. Plummer, E.G. Ngaha, P.J. Wright, P.D. Leathwood and M.E. Blake, in: U.C. Dubach and U. Schmidt, (Eds.), Biochemical Aspects of Diagnostic Significance in Urine, Hans Huber, Beme, 1979, in press. 4 D.T. Plummer and E.O. Ngahn, in: J.P. ‘Fillastre (Ed.), International Colloquium on the Interaction of Drugs with Membrane Systems, 1978, p. 175. 5 E.O. Ngaha, M. Fry and D.T. Flummer, The effect of cephaloridine on the stability of rat kidney lysosomes, Chem.-Biol. Iateract., 24 (1979) 199. 6 M. F’ry, E.O. Ngaha and D.T. Plummer, The protective effect of cephaloridine on rat kidney lysosomes in vitro, ‘Dans. Biochem. Sot., 3 (1975) 736. 7 T.T. Herskovits, in: S.P. Colowick and N.O. Kaplan (Eds.), Methods in Enzymology, Vol. XI, Academic Press, New York, 1967, p. 748. 8 M. Fry and D.T. Plummer, in: J.P. Fillastre (Ed.), International Colloquium on the Interaction of Drugs with Membrane Systems, 1978, p. 193. 9 K.F. Bromfield, MS. Wooster and J.M. Wrigglesworth, Robust attachment to film balance for surface pressure measurements, Lab. Pratt., 23 (1974) 122. 10 E.J. Rojas and J.M. Tobias, Membrane models: association of inorganic cations with phospholipid monolayers, Biochim. Biophys. Acta, 94 (1965) 394. 11 D.O. Shah, Interaction of uranyl ions with phospholipid and cholesterol monolayers, J. Coll. Inter. Sci., 29 (1969) 210. 12 G. Weissman, G. Sessa and S. Weissmann, The action of steroids and Triton X-100 upon phoapholipidlcholesterol structures, Biochem. Pharmacol., 15 (1966) 1537. 13 D.T. Plummer, Qn introduction to Practical Biochemistry, 2nd edn., McGraw-Hill, London, 1978, p. ‘185. 14 M. Fry and D.T. Ilummcr, The use of light scattering measurements to monitor the stabilisation of rat kidney lysosomes by the antibiotic cephaloridine, J. Physiol., 270 (1977) 20. 15 RR. Chauvette, E,.H. Flynn, B.G. Jackson, E.R. Lavagnino, B.G. Morin, R.A. Mueller, R.P. P&b, R.W. Roeske, C.W. Ryan, J.L. Spencer and E. Van Heyingen, Chemistry of cephalosporin sntibiotics. II. Preparation of a new class of antibiotics and the relation of structures to activity, J. Am. Chem. SOC., 84 (1962) 3401. 16 C.F. Chignell, Optical studies of drug-protein complexes. II Interactions of phenyl-
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17 18 19 20 21 22 23 24 25
butazone and its analogues with human serum albumin, Mol. Pharmacol., 5 (1969) 244. C.F. ChLgnell, in: NJ. Harper (Ed.), Advances in Drug Research, Vol. 5, Academic Press, London, 1970, p. 55. M. Laskowski, Measurement of accessibility of protein chromophores by solvent perturbation of their ultraviolet spectra, Fed. Proc., 25 (1966) 20. B. Maggio and J. Lucy, Polar-group behaviour in mixed monolayers of phospholipids and fusogenic lipids, Biochem. J., 155 (1976) 353. A.D. Bangham, Membrane models with phospholipids, Prog. Biophys. Mol. Biol., 18 (1968) 29. AD. Bangham, M.M. 8tandii and J.C. Watkins, Diffusion of univalent ions across the lamellae of swollen phospholipids, J. Mol. Biol., 13 (1965) 238. J.A. Lucy, The fusion of biological membralws, Nature, 227 (1970) 815. M. Fry, Lysosomal stabilisation by cephaloridine: the role of a membrane-bound phospholipsse 2, Ph.D. Thesis, London, 1977. E.A. Dennis, Kinetic dependence of phospholipase A2 activity on the detergent Triton X-100, J. Lipid Res., 14 (1973) 152. C.B. Woodward and R.F.A. Zwaal, Phe lytic behaviour of pure phospholipases ,42 and C towards osmotically swolle ‘1 c-ythrocytes and reseated ghosts, Biochim. Biophys. Acta, 274 (1972) 272.
