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Detection of carbonyl functions in phospholipids of liver microsomes in CCl4- and BrCCl3-poisoned rats
628
Bwchimica
et Bwphysica Acta, 7 12 (1982) 628-638 Elsevier Biomedical Press
BBA 51182
DETECTION OF CARRONYL FUNCTIONS IN CCI A- AND BrCCl 3-POISONED BATS
IN PHOSPHOLIPIDS
ANGELO BENEDETTI a, ROSELLA FULCERI a, MARCO FERRALI HERMANN ESTERBAUER b and MARIO COMPORT1 a,*
a, LUCIA
OF LIVER MICROSOMES
CICCOLI a,
u Istituto di Patologia Generale dell’Universit~ di Siena, Via de1 Laterino 8, 53100 Siena (Italy) and b Institut fiir Biochemie, Uniuersitiit Graz, Schubertstrasse 1, A-8010 Grar (Austria)
(Received June 3rd, 1982)
Key words: Lipid peroxidation;
Poisoning; Phospholipid;
Carbonyl function;
(Liver microsome)
Since the peroxidative cleavage of unsaturated fatty acids can result in either the release of carbonyl compounds or the formation of carbonyl functions in the acyl residues, evidence for the presence of carbonyl groups in liver microsomal phospholipids was searched for in in vivo conditions (Ccl, and BrCCl, intoxications) in which peroxidation of lipids of hepatic endoplasmic reticulum had been previously demonstrated. The spectrophotometric examination of 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes from the intoxicated animals showed absorption spectra similar to those observed for the dinitrophenylhydrazones of various carbonyls. Similar spectra, although magnified from a quantitative point of view, were also observed with 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes peroxidized in the NADPH-Fe-dependent system. A time-course study of microsomal lipid peroxidation showed that the amount of 2,4-dinitrophenylhydrazine-reacting groups (carbonyl functions) in phospholipids of liver microsomes increases with the incubation time and is correlated to the amount of malonic dialdehyde formed in the incubation mixture. The kinetics of the production of 4-hydroxynonenal was somewhat similar to that of malonic dialdehyde formation. In both the in vivo conditions (Ccl, and BrCCl, intoxications) the amount of carbonyl functions in microsomal phospholipids, which was higher in the BrCCl,-intoxicated animals as compared to the Ccl,-poisoned ones, was close to that found in the vitro condition in which lipid peroxidation is induced by 6 PM Fe*+. The possible p athological significance of formation of carbonyl functions in membrane phospholipids is discussed.
Introduction It is widely known that peroxidation of unsaturated lipids in biological membranes is highly detrimental to the membrane structure and function. Also, lipid peroxidation is believed to play an important role in many conditions of cellular damage, including the liver injury produced by some hepatotoxins such as Ccl, and BrCCl, [l-4]. Recent studies on the mechanisms by which lipid *To whom correspondence
should be addressed.
OOOS-2760/82/0000-0000/$02.7501982
Elsevier Biomedical
Press
peroxidation brings about its effects have led to the knowledge that products evolved from lipid peroxidation can produce damaging effects on biological structures [5-71. Some of the most active products have been separated [8] and identified as 4-hydroxyalkenals, the major part of them (more than 95%) being represented by 4-hydroxynonenal, with minor amounts of 4-hydroxyoctenal, 4-hydroxydecenal and 4-hydroxyundecenal[9]. Studies concerned with the mechanism of the formation of 4-hydroxynonenal during NADPHFe-dependent microsomal lipid peroxidation have
629
demonstrated [lo] that the aldehyde originates from phospholipid-bound arachidonic acid. The chemical pathways leading to the formation of various carbonyls from unsaturated fatty acids have been extensively studied. A main reaction is the dismutation of monohydroperoxides which procedes, most probably, via the formation of an alkoxy radical which then decomposes by a chain-cleavage reaction to an aldehyde and to an alkyl radical [ 111: R’ + R’CHO R-CH-R’I OOH
R-CH-R’ I 0
/ \
RCHO+R’
As shown in the scheme, the aldehyde group can be, after chain cleavage, on either side of the carbon chain. Therefore, in membrane phospholipids, where most of polyunsaturated fatty acids are bound at the /3 position, not only are aldehydes produced, but also /I acyl residues bearing carbonyl functions are formed. This occurrence has been demonstrated by Tam and McCay [ 121 in phospholipids of liver microsomes peroxidized in the NADPH-Fe-dependent system. Thus, in addition to the release of products provided with cytopathological activities such as 4-hydroxyalkenals, an alteration of the membrane structure is produced directly by lipid peroxidation. The latter fact, which is complementary to the former, is expected to have important pathological consequences since the formation of polar groups at sites normally containing nonpolar hydrocarbon chains could result in perturbations in molecular orientation (see Refs. 13 and 14). Since, in a previous investigation [15], positive evidence has been presented for aldehydes (presumably alkenals) being bound to liver microsomal protein of rats intoxicated with Ccl, or BrCCl,, in the present study evidence for the presence of carbonyl groups in phospholipids of the membranes of hepatic endoplasmic reticulum was searched for in the same in vivo conditions, in which the occurrence of lipid peroxidation has been demonstrated. An in vitro study on the time course appearance of carbonyl functions in phospholipids of peroxidizing microsomes is also reported.
Materials and Methods Male Sprague-Dawley rats (200-250 g) maintained on a pellet diet (Nossan, Correzzana, Milan, Italy) free of preservative compounds were used. Female rats were used for ethionine intoxication. The animals were starved overnight before being used. Ccl, and BrCCl, poisonings were accomplished by oral administration of the toxins in equimolar amounts (2600 ~mol/lOO g body wt.). DL-Ethionine (Sigma Chemical Co., St. Louis, MO, U.S.A.), as a 2.5% (w/v) solution in saline, was administered orally at a dosage of 1 mg per g body wt., in two equally divided doses at 0 time and 2 h. Ethionine-treated rats were killed 6 h after the initial dose. 33% (w/v) liver homogenates in ice-cold 0.25 M sucrose/6 mM EDTA, pH 7.0, were prepared. The homogenization medium contained 0.2% (w/v) butylated hydroxytoluene which was added immediately before use and suspended with a Potter-Elvehjem homogenizer. Butylated hydroxytoluene was omitted in the in vitro lipid peroxidation experiments. The homogenate was centrifuged at 9000 X g for 15 min and the supernatant fraction was recentrifuged as above. The final supernatant fraction was centrifuged at 100000 X g for 60 min. In vivo experiments. The microsomal pellet was resuspended in 6 mM EDTA (brought to pH 7.0 with Tris and containing 0.2% butylated hydroxytoluene) and recentrifuged at 100000 X g for 30 min. The washed microsomal pellet derived from 5 g of liver was resuspended with a small amount of 6 mM EDTA and the suspension was extracted with 24 vol. of chloroform/methanol (2: 1, v/v) containing 0.1% butylated hydroxytoluene. After filtration on a Whatman no. 41 filter, the chloroform/methanol extract was acidified by the addition of glacial acetic acid (0.6 ml per 100 ml of extract). Carbonyl functions were detected after derivativisation with 2,4-dinitrophenylhydrazine essentially as done by Tam and McCay [ 121. To this end, 2,4_dinitrophenylhydrazine was added to the sample (14 mg per 100 ml). A duplicate sample, to which no 2,4-dinitrophenylhydrazine was added, served as blank sample (see below). The samples were kept at room temperature for 1 h and then washed with 0.2 vol. of 0.5% NaCl. After
630 separation of the two phases at 0-4°C the upper (methanol/water) phase was removed and the lower phase was dried down in a rotatory evaporator. The residue was subjected to silicic acid (SilicAr CC-7, 100-200 mesh, Mallinckrodt) column chromatography. Unreacted 2,4-dinitrophenylhydrazine and neutral lipids were eluted with chloroform containing 0.0005% butylated hydroxytoluene. Phospholipids were eluted with methanol containing the same amount of butylated hydroxytoluene. The methanol eluate exhibited a yellow color (due to 2,4_dinitrophenylhydrazine-derivatives of phospholipids) in the samples deriving from Ccl,or BrCCl,-intoxicated rats, while appeared almost normal in color (or only slightly yellowish) in the samples deriving from control or ethionine-treated rats. Additional traces of unreacted 2,4_dinitrophenylhydrazine and other contaminants were removed from the methanol eluate by thin-layer chromatography (TLC). The chromatoplate (20 X 20 cm, silica gel H, precoated, Merck) was developed three times with methylene chloride containing 0.0005% butylated hydroxytoluene. The origin (containing phospholipids and 2,4-dinitrophenylhydrazine-derivatives of phospholipids) was scraped off and eluted first with 5 ml of chloroform/methanol (2 : 1, v/v) and then with 5 ml of methanol. The recovery with respect to the amount applied to the chromatoplate was 80-90% (as calculated on the base of lipid Pi determination) in the samples deriving from both control and intoxicated animals. The eluate was dried down and the residue was dissolved in chloroform to give a concentration of 2.5 mg of phospholipids per ml. The phospholipid concentration was determined according to the method of Shin [16], assuming a factor of 25 to convert lipid Pi to phospholipids. The absorbance spectrum over the range 280-500 nm was then recorded for both 2,4-dinitrophenylhydrazine-treated samples and blank samples, using chloroform in the reference cuvette. Subsequently, for each 2,4_dinitrophenylhydrazine-treated sample (i.e., control, Ccl,, BrCCl,) the absorption spectrum was recorded over the same nm range, using the corresponding blank sample (i.e., control, Ccl,, BrCCl, sample not treated with 2,4_dinitrophenylhydrazine) in the reference cuvette. It was necessary to use the ap-
propriate blank sample as reference since the phospholipids of liver microsomes showed (expecially in the case of intoxicated rats) an intrinsic yellow-brownish color with a relatively high absorbance in the region where hydrazones absorb. By subtracting the control spectrum (sample from non-intoxicated rats) from the spectra of the samples of the intoxicated rats, the difference spectra (hereafter referred to as Ccl, minus control or BrCCl, minus control) were calculated. In vitro experiments. The microsomal pellet was resuspended in 0.15 M KC1/0.05 M Tris-maleate buffer, pH 7.0, and recentrifuged at 100000 X g for 30 min. The washed microsomal pellet was resuspended in the same medium. The composition of the incubation mixture was microsomes derived from 250 mg liver/ml, 0.15 M KCl, 0.05 M Tris-maleate buffer, pH 7.4, 60 or 6 PM FeSO, and an NADPH-generating system (0.1 mM 2.5 mM nicotinamide, 3 NADP, 5mM MgCl,, mM DL-isocitrate and 0.1 PM units/ml isocitrate dehydrogenase (Boehringer)). The incubation was carried out aerobically at 37°C. A sample of the microsomal suspension added to the buffer alone was kept at 0-4°C and represents the non-peroxidized microsomes (0 time) sample. At the end of each incubation time aliquots were drawn and the peroxidation was stopped by the addition of EDTA (final concentration 6 mM). One part was used to measure [ 171 the amount of malonic dialdehyde formed. Another part was extracted with 24 vol. of chloroform/methanol (2 : 1, v/v) according to the method of Folch et al. [ 181. Fatty acid methyl esters were prepared and purified [19] as previously reported. They were analyzed by gasliquid chromatography in a Fractovap apparatus Model GI (Carlo Erba, Milan, Italy) using a spiral glass column (2 m X 2 mm internal diameter) packed with 20% diethyleneglycol succinate on Chromosorb W. Other conditions were as previously reported [20]. The absolute amount of individual fatty acid was determined by adding a known amount of an internal standard (margaric acid) to the lipid extract, before transmethylation. The remainder of each aliquot drawn from the incubation mixture was centrifuged at 80000 X R for 45 min and the supernatant fraction was separated from the microsomal pellet. The microsomal sediment (containing the microsomes equiv-
631
alent to 5 g of liver) was resuspended with a small amount of 6 mM EDTA, and processed as for the in vivo experiments to detect the presence of carbonyl functions in phospholipids after derivativisation with 2,4_dinitrophenylhydrazine. In the in vitro lipid peroxidation experiments, in which 60 PM FeSO, was used, some yellow or brownish material remained in the silicic acid column after methanol elution of the 2,4_dinitrophenylhydrazine-reacted samples or blank-reacted samples, respectively. This material, which increased with the incubation time, could not be eluted with methanol or with methanol/water in various proportions and probably consisted of very altered phospholipids with greatly increased polarity. In the TLC of the methanol eluates, a broad yellow or brownish material moving from the origin was seen in the same samples. Only the areas corresponding to the origins were scraped off to have samples comparable as far as possible to those obtained in the in vivo experiments. The elution procedure, carried out as for the in vivo experiments, resulted, however, in a low recovery (50-608) with respect to the amount applied to the chromatoplate. Therefore, the quantitative figure for carbonyl functions in phospholipids of liver microsomes peroxidized in the presence of 60 PM Fe’+ could be underestimated. The phospholipid concentration of the sample dissolved in chloroform was 0.1 mg/ml or 2.5 mg/ml in the case of 60 or 6 PM Fe’+-induced lipid peroxidation, respectively. The absorbance spectra were recorded and calculated, respectively, as described for the in vivo experiments. The supernatant fraction, separated as mentioned above from the microsomal sediment, was used to measure the amount of 4-hydroxynonenal formed. To this end the supernatant fraction derived from 20 ml of incubation mixture was extracted thrice with 1 vol. of chloroform. In preliminary experiments it was ascertained that 4-hydroxynonenal (and other lipid peroxidation products is extracted almost completely by this procedure. The chloroform phases were collected and the separation of the residual water content from the chloroform extract was achieved by cold (-20°C) filtration. The chloroform extract was concentrated down to 20 ml by evaporation. Aliquots (3 or 15 ml in the case of 60 or 6 I_LM Fe2+ -induced lipid peroxidation, respectively)
were dried down, redissolved in small amounts of chloroform and applied to TLC plates (precoated with silica gel 60, Merck). TLC was performed by using a solvent system of n-heptane/ethyl acetate/acetic acid (75 : 25 : 1, v/v) as previously done [S]. The spots were detected with a spray solution of 10% (w/v) phosphomolybdic acid in 95% ethanol. The area corresponding to 4-hydroxyalkenals, identified by using synthetic 4-hydroxynonenal as a standard, was scraped off and the products were eluted three times with chloroform. After evaporation of the solvent, the eluted material, redissolved in a small amount of acetonitrile/water (1: 1, v/v) was analyzed by high-pressure liquid chromatography. The peak corresponding to 4-hydroxynonenal was identified by means of a standard of synthetic 4-hydroxynonenal. Together with the peak of 4-hydroxynonenal, another very small peak, probably corresponding to 4-hydroxyoctenal, was seen in the chromatogram. The absolute amount of 4-hydroxynonenal was calculated by means of a calibration curve of known amounts of synthetic 4-hydroxynonenal (0.2-30 nmol/ml or 0.05-0.2 nmol/ml in the case of 60 or 6 PM Fe2+ -induced lipid peroxidation, respectively). The curves were obtained by adding synthetic 4-hydroxynonenal to the complete medium in which microsomes were omitted, and by processing these ‘standard’ samples as the experimental samples. High-pressure liquid chromatography was performed with a Du Pont Liquid Chromatograph, Model 830, equipped with a variable wave length detector, Model 837. Operating conditions were: reversed-phase column ZorbaxTM ODS (4.6 mm X 25 cm); column tem’perature, 50°C; mobile phase, acetonitrile/water (1 : 1, v/v); flow rate 0.95 ml/min; ultraviolet photometer, 220 nm. Synthetic 4-hydroxynonenal was prepared as previously reported [21]. Results The ultraviolet-visible spectra of 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes from control, Ccl,and BrCCl,-poisoned rats are shown in Fig. 1A. It seems evident from the spectra that 2,4-dinitrophenylhydrazine-reacting groups are present
632
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Fig. 1. (A) Solid lines: absorption spectra of 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes derived from control, Ccl,-poisoned and BrCCl,-poisoned rats. Dashed lines: absorption spectra of blank samples, See text for additional explanations. The rats were killed I h after poisoning. A typical experiment is reported in the figure (ABC). The phospholipid concentration of the sample in chloroform was 2.5 mg/ml. The spectra were recorded against chloroform in the reference cuvette. (B) Spectra for control, Ccl,- and BrCCl,-poisoned rats. The spectra for 2,4-dinitrophenylhydrazine-treated samples were recorded against the corresponding blank samples in the reference cuvette. (C) Difference spectra calculated from the spectra of Fig. 1B by subtracting the spectrum of the control sample from the spectra of the samples of the intoxicated rats. (D) Absorption spectrum obtained with a mixture of 2,4-dinitrophenylhydra~nes of various aldehydes (4-hydroxynonenal, hcxadecanal and decyl aldehyde, 10 nmol/ml,
for each aldehyde).
in phospholipids of liver microsomes from the intoxicated animals. An absorption characteristic of hydrazones, although much lower in intensity, occurs even in the microsomal phospholipids of the control group. Fig. IA also shows the absorption spectra of the blank samples (samples to which no 2,4-dinitrophenylhydrazine was added) from both control and intoxicated animals. The nature of this absorption, which is sometimes higher in the intoxicated rats than in the control ones, was not explored in this study. Because of this intrinsic absorption of phospholipids, the proper hydrazone spectra were obtained by recording the spectra of the 2,4-dinitrophenylhydra~ne-treated samples (from control, CC1 4- and BrCCl ,-poisoned rats) against their respective blank samples (Fig. 1B). These spectra, which closely resemble the spectum of a reference mixture of dinitrophenylhydr~ones of standard
aldehydes (Fig. lD), show that the relatively low amount of 2,4-dinitrophenylhydrazine-reacting groups present in the microsomal phospholipids of the non-intoxicated rats is dramatically increased in the case of Ccl, and especially in the case of BrCCl, poisoning. A small shift of the absorption maximum toward longer wavelengths was also seen in the spectra from the intoxicated animals with respect to those from controls (from 360-365 nm in the controls to 365-370 nm in the intoxicated animals). The intoxicated minus control difference spectra (Fig. lC), calculated by substracting the spectrum of the control from the spectra of the intoxicated animals, show a maximum in the 365-370 nm range, which can be considered as an intermediate value among the wave length maxima (in chloroform) of aliphatic saturated aldehydes = 358 nm), alkan-2-ones (X,,, = 363 nm) (L,,
633
TABLE
I
DATA FOR SPECTROPHOTOMETRIC ANALYSES ACTED PHOSPHOLIPIDS OF LIVER MICROSOMES
OF 2,4-DINITROPHENYLHYDRAZINE-TREATED OR BLANK REFROM CONTROL, AND Ccl,-, BrCCl,- OR ETHIONINE-POISONED
RATS Ccl, or BrCCl, was administered orally at a dose of 2600 pmol per 100 g wt. DL-Ethionine, as 2.5% (w/v) solution in saline, was administered orally at a dosage of 1 mg per g body wt., in two equally divided doses (0 time and 2 h). Figures represent the mean (-t S.E.) of the absorption or difference absorption values at 365 nm. The number of experiments is reported in brackets. For each experiment three pooled livers were used. The phospholipid concentration of the sample dissolved in chloroform was 2.5 mg/ml. Time after poisoning
2,4-Dinitrophenylhydrazinereacted samples
Blank reacted samples (Es&
(Es& Control ccl, ccl, ccl, Control BrCCl s BrCCl, BrCCl, Control Ethionine
(4) (3) (3) (4) (4) (1) (4) (1) (2) (2)
15 min
1h 2 h 15 min
1h 2h 6 h
0.230%0.016 0.357* 0.039 0.49O-cO.023 0.5OO-cO.028 0.221’0.011 0.548 0.968*0.057 0.685 0.350 0.322
and n-alkan-Zenals (X max= 373-376 nm) [22,23]. Such difference spectra are, therefore, indicative for a multiclass mixture of carbonyls present in the phospholipid molecules and consisting most likely of both saturated and c+3 unsaturated aldehydes. The results (absorption or difference absorption values at 365 nm) of the in vivo studies are reported in full in Table I (Fig. 1 shows only the results of a typical experiment). It can be seen that in both the intoxications the amount of 2,4-dinitrophenylhydrazine-reacting groups in liver microsomal phospholipids increases from 15 min up to l-2 h of intoxication. The finding of a higher level of 2,4_dinitrophenylhydrazine_reacting groups in microsomal phospholipids from BrCCl,-poisoned rats as compared to the Ccl,-poisoned ones at all the times examined is consistent with the different levels of microsomal lipid peroxidation and toxicity found after the in vivo intoxication or the in vitro treatment with these two toxins [24-261. A higher level of protein-bound carbonyls (measured as 2,4-dinitrophenylhydrazine-reacting groups) has also been found [ 151 in liver microsomes from BrCCl,-poisoned rats as compared to the CC1 ,-poisoned ones.
2,CDinitrophenyl hydrazine minus blank
Intoxicated minus control (A&,,)
(A&s) 0.112 i 0.009 0.102~0.004 0.128”0.021 0.1201-0.005 0.107~0.018 0.084 0.120*0.017 0.100 0.155 0.115
0.118’0.017 0.255 “0.028 0.363 iO.032 0.380*0.031 0.114~0.012 0.464 0.848 kO.054 0.585 0.195 0.207
0.137 0.245 0.262 _ 0.350 0.734 0.471 _ 0.012
Results similar to those of the in vivo experiments, although magnified from a quantitative point of view, were also obtained in in vitro lipid peroxidation experiments. Fig. 2 shows the absorption spectra obtained with 2,4_dinitrophenylhydrazine-treated phospholipids from liver microsomes peroxidized in the NADPH-Fe-dependent (60 PM Fe’+) system for 10 min. These spectra, which are similar to those obtained in the in vivo experiments, show that a high amount of 2,4-dinitrophenylhydrazine-derivatives of phospholipids are present in the sample and, therefore, that relatively high levels of carbonyl functions are formed in the phospholipids of peroxidizing microsomes. Fig. 3 shows the results obtained in a timecourse study of microsomal lipid peroxidation induced by NADPH and two different concentration of Fe2+ (60 and 6 PM in panels A and B, respectively). As can be seen, in each instance the amount of 2,4_dinitrophenylhydrazine_reacting groups (expressed as absorbance units, see legend to Fig. 3) in microsomal phospholipids increases with the incubation time and is correlated (r = 0.993 and 0.981 in panel A and B, respectively) to
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Fig. 2. (A) Solid lines: absorption spectra of 2,4-dinitropheny~ydr~ne-treats phospholipids of either liver microsomes peroxidized in the NADPH-Fe-dependent system or non-peroxidized (control) microsomes. Dashed lines: absorption spectra of blank samples. See text for additional explanations. The incubation conditions are described in Materials and Methods. The concentration of FeSO, was 60 PM. The incubation time was 10 min. A typical experiment is reported in the figure (A,B,C,). The phospholipid concentration of the sample in chloroform was 0.1 mg/ml. The spectra were recorded against chloroform in the reference cuvette. The means (two experiments) of the absorptions at 365-370 nm of the 2,4-dinitropheny~ydr~ne-treaty samples were 0.010 and 0.616 for non-peroxidixed and peroxidized microsomes, respectively. The means of the absorptions at 365-370 nm of the blank reacted samples were 0.005 and 0.010 for non-peroxidized and peroxidized microsomes, respectively. (B) Spectra for either liver microsomes peroxidized in the NADPH-Fe-dependent system or non-peroxidized (control) microsomes. The spectra for 2,4-dinitrophenylhydrazine-treated samples were recorded against the corresponding blank samples in the reference cuvette. The means of the 2,4-dinitropheny~ydr~ne minus blank difference absorptions at 365-370 nm were 0.005 and 0.606 for non-peroxidized and peroxidized microsomes, respectively. (C) Difference spectrum peroxidized microsomes minus non-peroxidized microsomes calculated from the spectra of Fig. 2B by subtracting the spectrum of the sample for non-peroxidized (control) microsomes from the spectrum of the sample for peroxidized microsomes.
the amount of malonic dialdehyde formed in the incubation mixture. Since in previous work [ 151 a similar correlation has been found between the amount of protein-bound carbonyls and the amount of malonic dialdehyde formed, it can be stated that the appearance of 2,4-dinitrophenylhydrazine-reacting groups in both protein (protein-bound carbonyls) and phospholipids (phospholipid-bound fatty acid residues bearing carbonyl functions) of liver microsomes can be assumed as a good index of lipid peroxidation. From the difference spectra obtained (see the
results reported in Fig. 1, Table I and Fig. 3), the content of carbonyl functions in microsomal phospholipids in the case of in vitro lipid peroxidation (in the presence of 60 or 6 PM Fe2+) and in the case of Ccl, or BrCCl, into~cations can be calculated roughly (Table II> by using an average molar extinction coefficient of 25 500. This value is a mean for the dinitrophenylhydrazones of carbonyls (in chloroform) which can theoretically be expected as major lipid peroxidation products and which show wavelength maxima in the 355375 nm range, i.e., alkanals (Ed = 22200), alken-2-
635
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Fig. 3. Time-course appearance of 2,4-dinitrophenyfhydrazinereacting groups in phospholipids of liver microsomes peroxidizing in the NADPH-Fe-dependent system and concomitant evolution of malonic dialdehyde. See text for technical details. Result are the means of two experiments. 2,4-dinitrophenylhydrazine-reacting groups are expressed as absorbance units for a phospholipid concentration of 2.5 mg/ml. The absorbance units were calculated from the value at 365 nm of the difference spectra peroxidized microsomes minus non-peroxidized microsomes. (A) 60 PM FeSO,. (B) 6 nM FeSO,. 0, malonic dialdehyde concentration; A, 2,4-dinitrophenylhydrazine-reacting groups.
als (Ed = 28 700), alkan-2-ones (Ed = 22 500) and 4-hydroxyalken-2-als (Ed = 28 500) [22,23,27]. As can be seen (Table II), 25% or more (a possible underestimation of the figure must be considered, see Materials and Methods) mol of phospholipids contain carbonyl functions when liver microsomes are peroxidized in the presence of the higher amount (60 pM) of Fe *+ . The value is much lower and close to those of the in vivo experiments (expecially in the case of BrCCl, poisoning) when
lipid peroxidation is induced by the lower amount (6 PM) of Fe*+. As mentioned above, the peroxidative cleavage of phospholipid-bound polyunsaturated fatty acids can result in both the release of carbonyl compounds and the formation of carbonyl functions in phospholipid-bound acyl residues. Since it has been demonstrated [lo] that in the peroxidation of liver microsomal lipids 4-hydroxynonenal derives specifically from phospholipid-bound arachidonic acid, the time-course release of 4-hydroxynonenal and decrease of phospholipid-bound arachidonic acid was followed (Fig.4) in the same microsomal lipid peroxidation system (i.e., 60 and 6 PM Fe2+induced lipid peroxidation). In comparison to data given in Fig. 3, the results show that the release of 4-hydroxynonenal is correlated to both the formation of malonic dialdehyde (r = 0.973 and 0.998 in panels A and B, respectively) and the production of 2,4-dinitrophenylhydrazine-reacting groups in microsomal phospholipids (r = 0.942 and 0.995 in panels A and B, respectively). The amount of 4-hydroxynonenal that can be recovered free in the incubation medium accounts, on a molar base, for only 2-3% of the decrease of phospholipidbound arachidonic acid. Even if a consistent part of 4-hydroxynonenal formed during lipid peroxidation binds to microsomal protein, as it has been demonstrated in a previous paper [ 151, it can nevertheless be concluded that 4-hydroxynonenal represents only a small portion of the total amount of aldehydes and other products derived from the peroxidative breakdown of phospholipid-bound arachidonic acid. In order to ascertain with which phospholipid fraction the 2,4-dinitrophenylhydrazine derivatives of microsomal phospholipids were associated, the samples used for the spectrophotometric examination in both the in vivo and the in vitro experiments were analyzed by TLC (technical details are reported in Materials and Methods). As shown in Fig. 5, phospholipid dinitrophenylhydrazones, visible as a yellowish spot, were associated with the phosphatidylethanolamine and phosphatidylcholine fractions, although beeing somewhat displaced towards the solvent front. The feature was similar in the case of both in vivo intoxications and in vitro lipid peroxidation induced by 6 PM Fe*+. When lipid peroxidation was induced by 60 PM
636
TABLE
II
CARBONYL FUNCTIONS IN PHOSPHOLIPIDS CALCULATED NITROPHENYLHYDRAZINE-TREATED SAMPLES ASSUMING [22,23,27]
Microsomes Microsomes Ccl, BrCCl,
peroxidized peroxidized
intoxication
in vitro, 20 min (60 pM Fe2+ ) in vitro, 20 min (6 pM Fez+ )
(2600 pmol/lOO
intoxication
HYDRAZONE EXTINCTION
Carbonyl
functions
nmol/mg
phospholipids
g body wt.), 1 h
Fe2+, on the other hand, the amount of dinitrophenylhydrazones was dramatically increased, phosphatidylethanolamine was decreased
SPECTRA OF THE 2,4-DICOEFFICENT OF 25 500
(mol/mol
315.7 13.0
g body wt.), 1 h
(2600 pmol/lOO
FROM THE A MOLAR
3.8
24.43 1.00 0.29
11.5
0.96
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.I00
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1 0
(min)
Fig. 4. Time-course decrease of arachidonic acid in phospholipids of liver microsomes peroxidizing in the NADPH-Fe-dependent system and concomitant release of 4-hydroxynonenal into the incubation medium. The incubation system was the same as that reported in Fig. 3. Results are the means of two experiments. (A) 60 pM FeSO,. (B) 6 CM FeSO,. 0, 4-hydroxynonenal concentration; A, arachidonic acid.
Fig. 5. Thin-layer chromatography of 2,4_dinitrophenylhydrazine-treated phospholipids of liver microsomes either derived from Ccl.,- and BrCCl,-poisoned rats or peroxidized in the NADPH-Fe-dependent system. Solvent system, chloroform/ methanol/H,0 (65: 30:4, v/v). In the in vivo experiments, rats were killed 1 h after poisoning. 1, Phosphatidylcholine; 2, phosphatidylethanolamine; 3, butylated hydroxytoluene; 4, 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes derived from control rats; 5, 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes derived from Ccl,-poisoned rats; 6, 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes derived from BrCCl,-poisoned rats; 7, 2,4-dinitrophenylhydrazine-treated phospholipids of non-peroxidized (control) microsomes; 8, 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes peroxidized for 40 min in the NADPH-Fe-dependent system (6 PM Fe2+ ); 9, 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes peroxidized for 40 min in the NADPH-Fe-dependent system (60 pM Fez+). Crosshatching indicates yellowish spots corresponding to phospholipid dinitrophenylhydrazones.
637
Discussion The present results indicate that carbonyl functions are formed in phospholipids of liver microsomes after in vivo intoxication with carbon tetrachloride or monobromotrichloromethane. Such carbonyl functions are conceivably formed as a consequence of the peroxidative cleavage of phospholipid-bound unsaturated fatty acids according to the scheme given in the Introduction. This possibility is strongly supported by the results of the in vitro experiments, which show that similar carbonyl functions are formed in phospholipids of liver microsomes peroxidizing in the NADPH-Fedependent system. Since the amount of the aldehyde groups increases with the incubation time and strictly parallels the amount of malonic dialdehyde formed in the incubation mixture, the measurement of carbonyl functions in membrane phospholipids represents a simple and reproducible method to detect lipid peroxidation in biological systems and may be of great usefulness, expecially in in vivo conditions in which determination of various parameters (diene conjugation, hepatic concentration of thiobarbituric acid-reacting products, ethane or penthane evolution, etc.) sometimes gives not univocal results in the hands of different investigators. The results of ethionine intoxication confirm the specificity of the detection of carbonyl functions in membrane phospholipids with respect to the in vivo conditions in which lipid peroxidation occurs. As shown in Table I, no 2,4-dinitrophenylhydrazine-reacting groups could be detected in liver microsomal phospholipids from ethionine-treated rats. Unpublished results from one of our laboratories (Benedetti, A. and Comporti, M., unpublished data) have in fact shown that lipid peroxidation cannot be detected by all the other methods in liver cell membranes following ethionine poisoning. As shown in Fig. 1, 2,4_dinitrophenylhydrazinereacting groups, although in relatively low amounts, were detected by the method used in this experimental work even in the microsomal phospholipids of the control rats. This fact is hard to explain at present, since a physiological route for lipid peroxidation has not been demonstrated with certainty up to now. It must be considered, however, that the presence of 2,4_dinitrophenylhydra-
zinc-reacting groups in control samples has also been observed in spectrophotometric examination of 2,Cdinitrophenylhydrazine-treated microsomal protein in our previous work [15] concerned with the detection of protein-bound carbonyls in liver microsomes from Ccl,- or BrCCl,-poisoned rats. The possibility must be considered that some of the carbonyl functions detected by the present method are formed through the cleavage of phospholipid-bound fatty acid hydroperoxides during technical procedures, and primarily during the reaction with 2,4_dinitrophenylhydrazine. This possibility, however, is ruled out by the following arguments: (i) the decomposition of peroxides is slow in an acidic medium such as that in which the reaction with 2,4_dinitrophenylhydrazine takes place; and (ii) the kinetics reported [12] for the formation of fatty acid hydroperoxides in phospholipids of liver microsomes peroxidizing under conditions similar to those used in the present work is quite different from that of the formation of the carbonyl functions. In fact the time-course of the formation peroxides (see Ref. 12, Fig. 1) shows a transient increase during the early periods of incubation and a sharp decrease thereafter; the formation of carbonyl functions (this paper, Fig. 3), on the contrary, shows an exponential shape. The present study, together with our previous one [ 151, indicates that lipid peroxidation produces, either in vitro or in vivo, marked damages to cellular membranes. The damage can be produced directly through the breakdown of phospholipid-bound unsaturated fatty acids and the resultant disorganization of the hydrophobic core of the membrane in which polar groups appear where unpolar hydrocarbon chains normally occur. Also, indirect damage can be produced through the binding of products originating from the peroxidative breakdown of fatty acids to membrane molecular structures. Thus, both the appearance of altered phospholipids and the binding of lipid peroxidation products represent two complementary aspects of the same phenomenon. As shown in Table II, the amount of carbonyl functions in microsomal phospholipids found in the in vivo intoxications is similar to or not so different from the amount of carbonyl functions found in the in vitro condition in which lipid peroxidation is induced by the lower amount (6
638
PM) of Fe2+. A strict similiarity between the results of Ccl, or BrCCl, intoxication and those of the in vitro microsomal lipid peroxidation induced by 6 PM Fe 2+ has also been observed when the amount of carbonyls bound to the microsomal protein was determined [ 151. It must be added that in this in vitro condition a significant 30% decrease of microsomal glucose-6-phosphatase activity was observed. Therefore, it is reasonable to assume that the alterations of membrane phospholipids and/or the binding of aldehydes to the microsomal protein are significant to the development of functional alterations of cellular membranes as those observed after the CCI, or BrCCl, intoxication. Furthermore, the possibility must be considered that in the in vivo situation sufficient amounts of altered phospholipids and/or toxic aldehydes are formed at discrete sites in the membrane where the homolytic cleavage of the halogenated hydrocarbons occurs, so as to produce important functional alterations. In the in vitro lipid peroxidation on the other hand, the extensive alterations of the membrane can be envisaged as occurring both at molecular sites essential for functional activities and at nonessential ones. Whatever the molecular pathway leading to functional changes may be, it is reasonable to assume that the formation of phospholipid-bound fatty acyl residues bearing carbonyl functions in cellular membranes has important pathological consequences. Acknowledgements This work was supported by a grant from the National Foundation for Cancer Research (U.S.A.). Additional funds were derived from a grant of the Consiglio Nazionale delle Ricerche, Roma, Italy.
References C. and Dianzani, M.U. (1965) Enzymologia 29, 185-204 2 Recknagel, R.O. and Ghoshal, A.K. (1966) Lab. Invest. 15, 132-146 3 Slater, T.F. (1966) Nature 209, 36-40 4 Slater, T.F. (1972) Free Radical Mechanisms in Tissue 1
Comporti, M., Saccocci,
Injury, Pion Ltd., London 5 Benedetti, A., Casini, A.F.
and
Ferrali,
M. (1977)
Res.
Commun. Chem. Pathol. Pharmacol. 17, 519-528 6 Roders, M.K., Glende, E.A., Jr. and Recknagel, R.O. (1978) Biochem. Pharmacol. 27, 437-443 7 Benedetti, A., Casini, A.F., Ferrali, M. and Comporti, M. (1979) Biochem. J. 180, 303-312 8 Benedetti, A., Casini, A.F., Ferrali, M. and Comporti, M. (1979) Biochem. Pharmacol. 28, 2909-2918 9 Benedetti, A., Comporti, M. and Esterbauer, H. (1980) Biochim. Biophys. Acta 620, 28 l-296 10 Benedetti, A., Esterbauer, H., Pieri, S. and Comporti, M. (1981) in Atti de1 XVI Congress0 Nazionale della Societa Italiana di Patologia, pp. 399-400, Copisteria Scientifica Universitaria, Torino 11 Bell, E.R., Raley, J.H., Rust, F.F., Senbold, F.H. and Vangham, W.E. (1951) Discuss. Faraday Sot. 10, 242-246 12 Tam, B.K. and McCay, P.B. (1970) J. Biol. Chem. 245, 2295-2300 13 Witting, L.A. (1965) Fed. Proc. 24, 912-916 14 Witting, L.A. (1965) J. Am. Oil Chem. Sot. 42, 908-913 15 Benedetti, A., Esterbauer, H., Ferrali, M., Fulceri, R. and Comporti, M. (1982) Biochim. Biophys. Acta 711, 345-356 16 Shin, Y.A. (1962) Anal. Chem. 34, 1164- 1166 17 Comporti, M., Hartman, A. and Di Luzio, N.R. (1967) Lab. Invest. 16, 616-624 18 Folch, J., Lees, M. and Sloane Stanley, G.H. (1957) J. Biol. Chem. 226,497-509 M. 19 Benedetti, A., Casini, A.F., Ferrali, M. and Comporti, (1977) Chem. -Biol. Interact. 17, 151-166 20 Benedetti, A., Casini, A.F., Ferrali, M. and Comporti, M. (1977) Chem. -Biol. Interact. 17, 167-183 H. and Weger, W. (1967) Monatsh. Chem. 98, 21 Esterbauer, 1994-2000 22 Day, E.A. (1965) Food Tech. 19, 1585-1590 23 Jones, L.A., Holmes, J.C. and Seligman, R.B. (1956) Anal. Chem. 28, 191-198 24 Slater, T.F. and Sawyer, B.C. (1971) Biochem. J. 123, 805-814 25 Koch, R.R., Glende, E.A., Jr. and Recknagel, R.O. (1974) B&hem. Pharmacol. 23, 2907-2915 26 Burdino, E., Gravela, E., Ugazio, G., Vannini, V. and Calligaro, A. (1973) Agents Actions 4, 244-253 27 Phillips, J.P. (1962) J. Org. Chem. 27, 1443-1445
Bwchimica
et Bwphysica Acta, 7 12 (1982) 628-638 Elsevier Biomedical Press
BBA 51182
DETECTION OF CARRONYL FUNCTIONS IN CCI A- AND BrCCl 3-POISONED BATS
IN PHOSPHOLIPIDS
ANGELO BENEDETTI a, ROSELLA FULCERI a, MARCO FERRALI HERMANN ESTERBAUER b and MARIO COMPORT1 a,*
a, LUCIA
OF LIVER MICROSOMES
CICCOLI a,
u Istituto di Patologia Generale dell’Universit~ di Siena, Via de1 Laterino 8, 53100 Siena (Italy) and b Institut fiir Biochemie, Uniuersitiit Graz, Schubertstrasse 1, A-8010 Grar (Austria)
(Received June 3rd, 1982)
Key words: Lipid peroxidation;
Poisoning; Phospholipid;
Carbonyl function;
(Liver microsome)
Since the peroxidative cleavage of unsaturated fatty acids can result in either the release of carbonyl compounds or the formation of carbonyl functions in the acyl residues, evidence for the presence of carbonyl groups in liver microsomal phospholipids was searched for in in vivo conditions (Ccl, and BrCCl, intoxications) in which peroxidation of lipids of hepatic endoplasmic reticulum had been previously demonstrated. The spectrophotometric examination of 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes from the intoxicated animals showed absorption spectra similar to those observed for the dinitrophenylhydrazones of various carbonyls. Similar spectra, although magnified from a quantitative point of view, were also observed with 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes peroxidized in the NADPH-Fe-dependent system. A time-course study of microsomal lipid peroxidation showed that the amount of 2,4-dinitrophenylhydrazine-reacting groups (carbonyl functions) in phospholipids of liver microsomes increases with the incubation time and is correlated to the amount of malonic dialdehyde formed in the incubation mixture. The kinetics of the production of 4-hydroxynonenal was somewhat similar to that of malonic dialdehyde formation. In both the in vivo conditions (Ccl, and BrCCl, intoxications) the amount of carbonyl functions in microsomal phospholipids, which was higher in the BrCCl,-intoxicated animals as compared to the Ccl,-poisoned ones, was close to that found in the vitro condition in which lipid peroxidation is induced by 6 PM Fe*+. The possible p athological significance of formation of carbonyl functions in membrane phospholipids is discussed.
Introduction It is widely known that peroxidation of unsaturated lipids in biological membranes is highly detrimental to the membrane structure and function. Also, lipid peroxidation is believed to play an important role in many conditions of cellular damage, including the liver injury produced by some hepatotoxins such as Ccl, and BrCCl, [l-4]. Recent studies on the mechanisms by which lipid *To whom correspondence
should be addressed.
OOOS-2760/82/0000-0000/$02.7501982
Elsevier Biomedical
Press
peroxidation brings about its effects have led to the knowledge that products evolved from lipid peroxidation can produce damaging effects on biological structures [5-71. Some of the most active products have been separated [8] and identified as 4-hydroxyalkenals, the major part of them (more than 95%) being represented by 4-hydroxynonenal, with minor amounts of 4-hydroxyoctenal, 4-hydroxydecenal and 4-hydroxyundecenal[9]. Studies concerned with the mechanism of the formation of 4-hydroxynonenal during NADPHFe-dependent microsomal lipid peroxidation have
629
demonstrated [lo] that the aldehyde originates from phospholipid-bound arachidonic acid. The chemical pathways leading to the formation of various carbonyls from unsaturated fatty acids have been extensively studied. A main reaction is the dismutation of monohydroperoxides which procedes, most probably, via the formation of an alkoxy radical which then decomposes by a chain-cleavage reaction to an aldehyde and to an alkyl radical [ 111: R’ + R’CHO R-CH-R’I OOH
R-CH-R’ I 0
/ \
RCHO+R’
As shown in the scheme, the aldehyde group can be, after chain cleavage, on either side of the carbon chain. Therefore, in membrane phospholipids, where most of polyunsaturated fatty acids are bound at the /3 position, not only are aldehydes produced, but also /I acyl residues bearing carbonyl functions are formed. This occurrence has been demonstrated by Tam and McCay [ 121 in phospholipids of liver microsomes peroxidized in the NADPH-Fe-dependent system. Thus, in addition to the release of products provided with cytopathological activities such as 4-hydroxyalkenals, an alteration of the membrane structure is produced directly by lipid peroxidation. The latter fact, which is complementary to the former, is expected to have important pathological consequences since the formation of polar groups at sites normally containing nonpolar hydrocarbon chains could result in perturbations in molecular orientation (see Refs. 13 and 14). Since, in a previous investigation [15], positive evidence has been presented for aldehydes (presumably alkenals) being bound to liver microsomal protein of rats intoxicated with Ccl, or BrCCl,, in the present study evidence for the presence of carbonyl groups in phospholipids of the membranes of hepatic endoplasmic reticulum was searched for in the same in vivo conditions, in which the occurrence of lipid peroxidation has been demonstrated. An in vitro study on the time course appearance of carbonyl functions in phospholipids of peroxidizing microsomes is also reported.
Materials and Methods Male Sprague-Dawley rats (200-250 g) maintained on a pellet diet (Nossan, Correzzana, Milan, Italy) free of preservative compounds were used. Female rats were used for ethionine intoxication. The animals were starved overnight before being used. Ccl, and BrCCl, poisonings were accomplished by oral administration of the toxins in equimolar amounts (2600 ~mol/lOO g body wt.). DL-Ethionine (Sigma Chemical Co., St. Louis, MO, U.S.A.), as a 2.5% (w/v) solution in saline, was administered orally at a dosage of 1 mg per g body wt., in two equally divided doses at 0 time and 2 h. Ethionine-treated rats were killed 6 h after the initial dose. 33% (w/v) liver homogenates in ice-cold 0.25 M sucrose/6 mM EDTA, pH 7.0, were prepared. The homogenization medium contained 0.2% (w/v) butylated hydroxytoluene which was added immediately before use and suspended with a Potter-Elvehjem homogenizer. Butylated hydroxytoluene was omitted in the in vitro lipid peroxidation experiments. The homogenate was centrifuged at 9000 X g for 15 min and the supernatant fraction was recentrifuged as above. The final supernatant fraction was centrifuged at 100000 X g for 60 min. In vivo experiments. The microsomal pellet was resuspended in 6 mM EDTA (brought to pH 7.0 with Tris and containing 0.2% butylated hydroxytoluene) and recentrifuged at 100000 X g for 30 min. The washed microsomal pellet derived from 5 g of liver was resuspended with a small amount of 6 mM EDTA and the suspension was extracted with 24 vol. of chloroform/methanol (2: 1, v/v) containing 0.1% butylated hydroxytoluene. After filtration on a Whatman no. 41 filter, the chloroform/methanol extract was acidified by the addition of glacial acetic acid (0.6 ml per 100 ml of extract). Carbonyl functions were detected after derivativisation with 2,4-dinitrophenylhydrazine essentially as done by Tam and McCay [ 121. To this end, 2,4_dinitrophenylhydrazine was added to the sample (14 mg per 100 ml). A duplicate sample, to which no 2,4-dinitrophenylhydrazine was added, served as blank sample (see below). The samples were kept at room temperature for 1 h and then washed with 0.2 vol. of 0.5% NaCl. After
630 separation of the two phases at 0-4°C the upper (methanol/water) phase was removed and the lower phase was dried down in a rotatory evaporator. The residue was subjected to silicic acid (SilicAr CC-7, 100-200 mesh, Mallinckrodt) column chromatography. Unreacted 2,4-dinitrophenylhydrazine and neutral lipids were eluted with chloroform containing 0.0005% butylated hydroxytoluene. Phospholipids were eluted with methanol containing the same amount of butylated hydroxytoluene. The methanol eluate exhibited a yellow color (due to 2,4_dinitrophenylhydrazine-derivatives of phospholipids) in the samples deriving from Ccl,or BrCCl,-intoxicated rats, while appeared almost normal in color (or only slightly yellowish) in the samples deriving from control or ethionine-treated rats. Additional traces of unreacted 2,4_dinitrophenylhydrazine and other contaminants were removed from the methanol eluate by thin-layer chromatography (TLC). The chromatoplate (20 X 20 cm, silica gel H, precoated, Merck) was developed three times with methylene chloride containing 0.0005% butylated hydroxytoluene. The origin (containing phospholipids and 2,4-dinitrophenylhydrazine-derivatives of phospholipids) was scraped off and eluted first with 5 ml of chloroform/methanol (2 : 1, v/v) and then with 5 ml of methanol. The recovery with respect to the amount applied to the chromatoplate was 80-90% (as calculated on the base of lipid Pi determination) in the samples deriving from both control and intoxicated animals. The eluate was dried down and the residue was dissolved in chloroform to give a concentration of 2.5 mg of phospholipids per ml. The phospholipid concentration was determined according to the method of Shin [16], assuming a factor of 25 to convert lipid Pi to phospholipids. The absorbance spectrum over the range 280-500 nm was then recorded for both 2,4-dinitrophenylhydrazine-treated samples and blank samples, using chloroform in the reference cuvette. Subsequently, for each 2,4_dinitrophenylhydrazine-treated sample (i.e., control, Ccl,, BrCCl,) the absorption spectrum was recorded over the same nm range, using the corresponding blank sample (i.e., control, Ccl,, BrCCl, sample not treated with 2,4_dinitrophenylhydrazine) in the reference cuvette. It was necessary to use the ap-
propriate blank sample as reference since the phospholipids of liver microsomes showed (expecially in the case of intoxicated rats) an intrinsic yellow-brownish color with a relatively high absorbance in the region where hydrazones absorb. By subtracting the control spectrum (sample from non-intoxicated rats) from the spectra of the samples of the intoxicated rats, the difference spectra (hereafter referred to as Ccl, minus control or BrCCl, minus control) were calculated. In vitro experiments. The microsomal pellet was resuspended in 0.15 M KC1/0.05 M Tris-maleate buffer, pH 7.0, and recentrifuged at 100000 X g for 30 min. The washed microsomal pellet was resuspended in the same medium. The composition of the incubation mixture was microsomes derived from 250 mg liver/ml, 0.15 M KCl, 0.05 M Tris-maleate buffer, pH 7.4, 60 or 6 PM FeSO, and an NADPH-generating system (0.1 mM 2.5 mM nicotinamide, 3 NADP, 5mM MgCl,, mM DL-isocitrate and 0.1 PM units/ml isocitrate dehydrogenase (Boehringer)). The incubation was carried out aerobically at 37°C. A sample of the microsomal suspension added to the buffer alone was kept at 0-4°C and represents the non-peroxidized microsomes (0 time) sample. At the end of each incubation time aliquots were drawn and the peroxidation was stopped by the addition of EDTA (final concentration 6 mM). One part was used to measure [ 171 the amount of malonic dialdehyde formed. Another part was extracted with 24 vol. of chloroform/methanol (2 : 1, v/v) according to the method of Folch et al. [ 181. Fatty acid methyl esters were prepared and purified [19] as previously reported. They were analyzed by gasliquid chromatography in a Fractovap apparatus Model GI (Carlo Erba, Milan, Italy) using a spiral glass column (2 m X 2 mm internal diameter) packed with 20% diethyleneglycol succinate on Chromosorb W. Other conditions were as previously reported [20]. The absolute amount of individual fatty acid was determined by adding a known amount of an internal standard (margaric acid) to the lipid extract, before transmethylation. The remainder of each aliquot drawn from the incubation mixture was centrifuged at 80000 X R for 45 min and the supernatant fraction was separated from the microsomal pellet. The microsomal sediment (containing the microsomes equiv-
631
alent to 5 g of liver) was resuspended with a small amount of 6 mM EDTA, and processed as for the in vivo experiments to detect the presence of carbonyl functions in phospholipids after derivativisation with 2,4_dinitrophenylhydrazine. In the in vitro lipid peroxidation experiments, in which 60 PM FeSO, was used, some yellow or brownish material remained in the silicic acid column after methanol elution of the 2,4_dinitrophenylhydrazine-reacted samples or blank-reacted samples, respectively. This material, which increased with the incubation time, could not be eluted with methanol or with methanol/water in various proportions and probably consisted of very altered phospholipids with greatly increased polarity. In the TLC of the methanol eluates, a broad yellow or brownish material moving from the origin was seen in the same samples. Only the areas corresponding to the origins were scraped off to have samples comparable as far as possible to those obtained in the in vivo experiments. The elution procedure, carried out as for the in vivo experiments, resulted, however, in a low recovery (50-608) with respect to the amount applied to the chromatoplate. Therefore, the quantitative figure for carbonyl functions in phospholipids of liver microsomes peroxidized in the presence of 60 PM Fe’+ could be underestimated. The phospholipid concentration of the sample dissolved in chloroform was 0.1 mg/ml or 2.5 mg/ml in the case of 60 or 6 PM Fe’+-induced lipid peroxidation, respectively. The absorbance spectra were recorded and calculated, respectively, as described for the in vivo experiments. The supernatant fraction, separated as mentioned above from the microsomal sediment, was used to measure the amount of 4-hydroxynonenal formed. To this end the supernatant fraction derived from 20 ml of incubation mixture was extracted thrice with 1 vol. of chloroform. In preliminary experiments it was ascertained that 4-hydroxynonenal (and other lipid peroxidation products is extracted almost completely by this procedure. The chloroform phases were collected and the separation of the residual water content from the chloroform extract was achieved by cold (-20°C) filtration. The chloroform extract was concentrated down to 20 ml by evaporation. Aliquots (3 or 15 ml in the case of 60 or 6 I_LM Fe2+ -induced lipid peroxidation, respectively)
were dried down, redissolved in small amounts of chloroform and applied to TLC plates (precoated with silica gel 60, Merck). TLC was performed by using a solvent system of n-heptane/ethyl acetate/acetic acid (75 : 25 : 1, v/v) as previously done [S]. The spots were detected with a spray solution of 10% (w/v) phosphomolybdic acid in 95% ethanol. The area corresponding to 4-hydroxyalkenals, identified by using synthetic 4-hydroxynonenal as a standard, was scraped off and the products were eluted three times with chloroform. After evaporation of the solvent, the eluted material, redissolved in a small amount of acetonitrile/water (1: 1, v/v) was analyzed by high-pressure liquid chromatography. The peak corresponding to 4-hydroxynonenal was identified by means of a standard of synthetic 4-hydroxynonenal. Together with the peak of 4-hydroxynonenal, another very small peak, probably corresponding to 4-hydroxyoctenal, was seen in the chromatogram. The absolute amount of 4-hydroxynonenal was calculated by means of a calibration curve of known amounts of synthetic 4-hydroxynonenal (0.2-30 nmol/ml or 0.05-0.2 nmol/ml in the case of 60 or 6 PM Fe2+ -induced lipid peroxidation, respectively). The curves were obtained by adding synthetic 4-hydroxynonenal to the complete medium in which microsomes were omitted, and by processing these ‘standard’ samples as the experimental samples. High-pressure liquid chromatography was performed with a Du Pont Liquid Chromatograph, Model 830, equipped with a variable wave length detector, Model 837. Operating conditions were: reversed-phase column ZorbaxTM ODS (4.6 mm X 25 cm); column tem’perature, 50°C; mobile phase, acetonitrile/water (1 : 1, v/v); flow rate 0.95 ml/min; ultraviolet photometer, 220 nm. Synthetic 4-hydroxynonenal was prepared as previously reported [21]. Results The ultraviolet-visible spectra of 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes from control, Ccl,and BrCCl,-poisoned rats are shown in Fig. 1A. It seems evident from the spectra that 2,4-dinitrophenylhydrazine-reacting groups are present
632
A
0.6 -
B
C
D
w
\
0 z
0 .6 .
4 m a 0
0.
4$ i
v) m
\ 0.2-
4
o-
, 350
I
I
400
1
I
450
I
,
350
WA
400
v
450
E
350
LENGTH
400
450
350
400
450
(am)
Fig. 1. (A) Solid lines: absorption spectra of 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes derived from control, Ccl,-poisoned and BrCCl,-poisoned rats. Dashed lines: absorption spectra of blank samples, See text for additional explanations. The rats were killed I h after poisoning. A typical experiment is reported in the figure (ABC). The phospholipid concentration of the sample in chloroform was 2.5 mg/ml. The spectra were recorded against chloroform in the reference cuvette. (B) Spectra for control, Ccl,- and BrCCl,-poisoned rats. The spectra for 2,4-dinitrophenylhydrazine-treated samples were recorded against the corresponding blank samples in the reference cuvette. (C) Difference spectra calculated from the spectra of Fig. 1B by subtracting the spectrum of the control sample from the spectra of the samples of the intoxicated rats. (D) Absorption spectrum obtained with a mixture of 2,4-dinitrophenylhydra~nes of various aldehydes (4-hydroxynonenal, hcxadecanal and decyl aldehyde, 10 nmol/ml,
for each aldehyde).
in phospholipids of liver microsomes from the intoxicated animals. An absorption characteristic of hydrazones, although much lower in intensity, occurs even in the microsomal phospholipids of the control group. Fig. IA also shows the absorption spectra of the blank samples (samples to which no 2,4-dinitrophenylhydrazine was added) from both control and intoxicated animals. The nature of this absorption, which is sometimes higher in the intoxicated rats than in the control ones, was not explored in this study. Because of this intrinsic absorption of phospholipids, the proper hydrazone spectra were obtained by recording the spectra of the 2,4-dinitrophenylhydra~ne-treated samples (from control, CC1 4- and BrCCl ,-poisoned rats) against their respective blank samples (Fig. 1B). These spectra, which closely resemble the spectum of a reference mixture of dinitrophenylhydr~ones of standard
aldehydes (Fig. lD), show that the relatively low amount of 2,4-dinitrophenylhydrazine-reacting groups present in the microsomal phospholipids of the non-intoxicated rats is dramatically increased in the case of Ccl, and especially in the case of BrCCl, poisoning. A small shift of the absorption maximum toward longer wavelengths was also seen in the spectra from the intoxicated animals with respect to those from controls (from 360-365 nm in the controls to 365-370 nm in the intoxicated animals). The intoxicated minus control difference spectra (Fig. lC), calculated by substracting the spectrum of the control from the spectra of the intoxicated animals, show a maximum in the 365-370 nm range, which can be considered as an intermediate value among the wave length maxima (in chloroform) of aliphatic saturated aldehydes = 358 nm), alkan-2-ones (X,,, = 363 nm) (L,,
633
TABLE
I
DATA FOR SPECTROPHOTOMETRIC ANALYSES ACTED PHOSPHOLIPIDS OF LIVER MICROSOMES
OF 2,4-DINITROPHENYLHYDRAZINE-TREATED OR BLANK REFROM CONTROL, AND Ccl,-, BrCCl,- OR ETHIONINE-POISONED
RATS Ccl, or BrCCl, was administered orally at a dose of 2600 pmol per 100 g wt. DL-Ethionine, as 2.5% (w/v) solution in saline, was administered orally at a dosage of 1 mg per g body wt., in two equally divided doses (0 time and 2 h). Figures represent the mean (-t S.E.) of the absorption or difference absorption values at 365 nm. The number of experiments is reported in brackets. For each experiment three pooled livers were used. The phospholipid concentration of the sample dissolved in chloroform was 2.5 mg/ml. Time after poisoning
2,4-Dinitrophenylhydrazinereacted samples
Blank reacted samples (Es&
(Es& Control ccl, ccl, ccl, Control BrCCl s BrCCl, BrCCl, Control Ethionine
(4) (3) (3) (4) (4) (1) (4) (1) (2) (2)
15 min
1h 2 h 15 min
1h 2h 6 h
0.230%0.016 0.357* 0.039 0.49O-cO.023 0.5OO-cO.028 0.221’0.011 0.548 0.968*0.057 0.685 0.350 0.322
and n-alkan-Zenals (X max= 373-376 nm) [22,23]. Such difference spectra are, therefore, indicative for a multiclass mixture of carbonyls present in the phospholipid molecules and consisting most likely of both saturated and c+3 unsaturated aldehydes. The results (absorption or difference absorption values at 365 nm) of the in vivo studies are reported in full in Table I (Fig. 1 shows only the results of a typical experiment). It can be seen that in both the intoxications the amount of 2,4-dinitrophenylhydrazine-reacting groups in liver microsomal phospholipids increases from 15 min up to l-2 h of intoxication. The finding of a higher level of 2,4_dinitrophenylhydrazine_reacting groups in microsomal phospholipids from BrCCl,-poisoned rats as compared to the Ccl,-poisoned ones at all the times examined is consistent with the different levels of microsomal lipid peroxidation and toxicity found after the in vivo intoxication or the in vitro treatment with these two toxins [24-261. A higher level of protein-bound carbonyls (measured as 2,4-dinitrophenylhydrazine-reacting groups) has also been found [ 151 in liver microsomes from BrCCl,-poisoned rats as compared to the CC1 ,-poisoned ones.
2,CDinitrophenyl hydrazine minus blank
Intoxicated minus control (A&,,)
(A&s) 0.112 i 0.009 0.102~0.004 0.128”0.021 0.1201-0.005 0.107~0.018 0.084 0.120*0.017 0.100 0.155 0.115
0.118’0.017 0.255 “0.028 0.363 iO.032 0.380*0.031 0.114~0.012 0.464 0.848 kO.054 0.585 0.195 0.207
0.137 0.245 0.262 _ 0.350 0.734 0.471 _ 0.012
Results similar to those of the in vivo experiments, although magnified from a quantitative point of view, were also obtained in in vitro lipid peroxidation experiments. Fig. 2 shows the absorption spectra obtained with 2,4_dinitrophenylhydrazine-treated phospholipids from liver microsomes peroxidized in the NADPH-Fe-dependent (60 PM Fe’+) system for 10 min. These spectra, which are similar to those obtained in the in vivo experiments, show that a high amount of 2,4-dinitrophenylhydrazine-derivatives of phospholipids are present in the sample and, therefore, that relatively high levels of carbonyl functions are formed in the phospholipids of peroxidizing microsomes. Fig. 3 shows the results obtained in a timecourse study of microsomal lipid peroxidation induced by NADPH and two different concentration of Fe2+ (60 and 6 PM in panels A and B, respectively). As can be seen, in each instance the amount of 2,4_dinitrophenylhydrazine_reacting groups (expressed as absorbance units, see legend to Fig. 3) in microsomal phospholipids increases with the incubation time and is correlated (r = 0.993 and 0.981 in panel A and B, respectively) to
1.6 -
PeroxIdized
Pororldizad
i WA
V
’
’
350 E
’
’
’
400
LENGTH
1
450
’
L
I
’
’
350
’
’
400
1
’
’
‘.
450
(n ml
Fig. 2. (A) Solid lines: absorption spectra of 2,4-dinitropheny~ydr~ne-treats phospholipids of either liver microsomes peroxidized in the NADPH-Fe-dependent system or non-peroxidized (control) microsomes. Dashed lines: absorption spectra of blank samples. See text for additional explanations. The incubation conditions are described in Materials and Methods. The concentration of FeSO, was 60 PM. The incubation time was 10 min. A typical experiment is reported in the figure (A,B,C,). The phospholipid concentration of the sample in chloroform was 0.1 mg/ml. The spectra were recorded against chloroform in the reference cuvette. The means (two experiments) of the absorptions at 365-370 nm of the 2,4-dinitropheny~ydr~ne-treaty samples were 0.010 and 0.616 for non-peroxidixed and peroxidized microsomes, respectively. The means of the absorptions at 365-370 nm of the blank reacted samples were 0.005 and 0.010 for non-peroxidized and peroxidized microsomes, respectively. (B) Spectra for either liver microsomes peroxidized in the NADPH-Fe-dependent system or non-peroxidized (control) microsomes. The spectra for 2,4-dinitrophenylhydrazine-treated samples were recorded against the corresponding blank samples in the reference cuvette. The means of the 2,4-dinitropheny~ydr~ne minus blank difference absorptions at 365-370 nm were 0.005 and 0.606 for non-peroxidized and peroxidized microsomes, respectively. (C) Difference spectrum peroxidized microsomes minus non-peroxidized microsomes calculated from the spectra of Fig. 2B by subtracting the spectrum of the sample for non-peroxidized (control) microsomes from the spectrum of the sample for peroxidized microsomes.
the amount of malonic dialdehyde formed in the incubation mixture. Since in previous work [ 151 a similar correlation has been found between the amount of protein-bound carbonyls and the amount of malonic dialdehyde formed, it can be stated that the appearance of 2,4-dinitrophenylhydrazine-reacting groups in both protein (protein-bound carbonyls) and phospholipids (phospholipid-bound fatty acid residues bearing carbonyl functions) of liver microsomes can be assumed as a good index of lipid peroxidation. From the difference spectra obtained (see the
results reported in Fig. 1, Table I and Fig. 3), the content of carbonyl functions in microsomal phospholipids in the case of in vitro lipid peroxidation (in the presence of 60 or 6 PM Fe2+) and in the case of Ccl, or BrCCl, into~cations can be calculated roughly (Table II> by using an average molar extinction coefficient of 25 500. This value is a mean for the dinitrophenylhydrazones of carbonyls (in chloroform) which can theoretically be expected as major lipid peroxidation products and which show wavelength maxima in the 355375 nm range, i.e., alkanals (Ed = 22200), alken-2-
635
A /.20 ,,’ - 15
- 10
,' : I'
[,
,, -5
0 6 -1
- 0.5
5
10
INCUBATION
' 40
20
TIME
0
(mln)
Fig. 3. Time-course appearance of 2,4-dinitrophenyfhydrazinereacting groups in phospholipids of liver microsomes peroxidizing in the NADPH-Fe-dependent system and concomitant evolution of malonic dialdehyde. See text for technical details. Result are the means of two experiments. 2,4-dinitrophenylhydrazine-reacting groups are expressed as absorbance units for a phospholipid concentration of 2.5 mg/ml. The absorbance units were calculated from the value at 365 nm of the difference spectra peroxidized microsomes minus non-peroxidized microsomes. (A) 60 PM FeSO,. (B) 6 nM FeSO,. 0, malonic dialdehyde concentration; A, 2,4-dinitrophenylhydrazine-reacting groups.
als (Ed = 28 700), alkan-2-ones (Ed = 22 500) and 4-hydroxyalken-2-als (Ed = 28 500) [22,23,27]. As can be seen (Table II), 25% or more (a possible underestimation of the figure must be considered, see Materials and Methods) mol of phospholipids contain carbonyl functions when liver microsomes are peroxidized in the presence of the higher amount (60 pM) of Fe *+ . The value is much lower and close to those of the in vivo experiments (expecially in the case of BrCCl, poisoning) when
lipid peroxidation is induced by the lower amount (6 PM) of Fe*+. As mentioned above, the peroxidative cleavage of phospholipid-bound polyunsaturated fatty acids can result in both the release of carbonyl compounds and the formation of carbonyl functions in phospholipid-bound acyl residues. Since it has been demonstrated [lo] that in the peroxidation of liver microsomal lipids 4-hydroxynonenal derives specifically from phospholipid-bound arachidonic acid, the time-course release of 4-hydroxynonenal and decrease of phospholipid-bound arachidonic acid was followed (Fig.4) in the same microsomal lipid peroxidation system (i.e., 60 and 6 PM Fe2+induced lipid peroxidation). In comparison to data given in Fig. 3, the results show that the release of 4-hydroxynonenal is correlated to both the formation of malonic dialdehyde (r = 0.973 and 0.998 in panels A and B, respectively) and the production of 2,4-dinitrophenylhydrazine-reacting groups in microsomal phospholipids (r = 0.942 and 0.995 in panels A and B, respectively). The amount of 4-hydroxynonenal that can be recovered free in the incubation medium accounts, on a molar base, for only 2-3% of the decrease of phospholipidbound arachidonic acid. Even if a consistent part of 4-hydroxynonenal formed during lipid peroxidation binds to microsomal protein, as it has been demonstrated in a previous paper [ 151, it can nevertheless be concluded that 4-hydroxynonenal represents only a small portion of the total amount of aldehydes and other products derived from the peroxidative breakdown of phospholipid-bound arachidonic acid. In order to ascertain with which phospholipid fraction the 2,4-dinitrophenylhydrazine derivatives of microsomal phospholipids were associated, the samples used for the spectrophotometric examination in both the in vivo and the in vitro experiments were analyzed by TLC (technical details are reported in Materials and Methods). As shown in Fig. 5, phospholipid dinitrophenylhydrazones, visible as a yellowish spot, were associated with the phosphatidylethanolamine and phosphatidylcholine fractions, although beeing somewhat displaced towards the solvent front. The feature was similar in the case of both in vivo intoxications and in vitro lipid peroxidation induced by 6 PM Fe*+. When lipid peroxidation was induced by 60 PM
636
TABLE
II
CARBONYL FUNCTIONS IN PHOSPHOLIPIDS CALCULATED NITROPHENYLHYDRAZINE-TREATED SAMPLES ASSUMING [22,23,27]
Microsomes Microsomes Ccl, BrCCl,
peroxidized peroxidized
intoxication
in vitro, 20 min (60 pM Fe2+ ) in vitro, 20 min (6 pM Fez+ )
(2600 pmol/lOO
intoxication
HYDRAZONE EXTINCTION
Carbonyl
functions
nmol/mg
phospholipids
g body wt.), 1 h
Fe2+, on the other hand, the amount of dinitrophenylhydrazones was dramatically increased, phosphatidylethanolamine was decreased
SPECTRA OF THE 2,4-DICOEFFICENT OF 25 500
(mol/mol
315.7 13.0
g body wt.), 1 h
(2600 pmol/lOO
FROM THE A MOLAR
3.8
24.43 1.00 0.29
11.5
0.96
phospholipid)
.I00
and the yellowish-coloured components appeared as a group of tailing spots, from the origin to the fast-running butylated hydroxytoluene spot. 0
0 J iO0 E
0
u
0
0
5 2
3
4
5
6
8
J ,
1000
*
(r
a
750
5
10
20
INCUBATION
40
TIME
1 0
(min)
Fig. 4. Time-course decrease of arachidonic acid in phospholipids of liver microsomes peroxidizing in the NADPH-Fe-dependent system and concomitant release of 4-hydroxynonenal into the incubation medium. The incubation system was the same as that reported in Fig. 3. Results are the means of two experiments. (A) 60 pM FeSO,. (B) 6 CM FeSO,. 0, 4-hydroxynonenal concentration; A, arachidonic acid.
Fig. 5. Thin-layer chromatography of 2,4_dinitrophenylhydrazine-treated phospholipids of liver microsomes either derived from Ccl.,- and BrCCl,-poisoned rats or peroxidized in the NADPH-Fe-dependent system. Solvent system, chloroform/ methanol/H,0 (65: 30:4, v/v). In the in vivo experiments, rats were killed 1 h after poisoning. 1, Phosphatidylcholine; 2, phosphatidylethanolamine; 3, butylated hydroxytoluene; 4, 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes derived from control rats; 5, 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes derived from Ccl,-poisoned rats; 6, 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes derived from BrCCl,-poisoned rats; 7, 2,4-dinitrophenylhydrazine-treated phospholipids of non-peroxidized (control) microsomes; 8, 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes peroxidized for 40 min in the NADPH-Fe-dependent system (6 PM Fe2+ ); 9, 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes peroxidized for 40 min in the NADPH-Fe-dependent system (60 pM Fez+). Crosshatching indicates yellowish spots corresponding to phospholipid dinitrophenylhydrazones.
637
Discussion The present results indicate that carbonyl functions are formed in phospholipids of liver microsomes after in vivo intoxication with carbon tetrachloride or monobromotrichloromethane. Such carbonyl functions are conceivably formed as a consequence of the peroxidative cleavage of phospholipid-bound unsaturated fatty acids according to the scheme given in the Introduction. This possibility is strongly supported by the results of the in vitro experiments, which show that similar carbonyl functions are formed in phospholipids of liver microsomes peroxidizing in the NADPH-Fedependent system. Since the amount of the aldehyde groups increases with the incubation time and strictly parallels the amount of malonic dialdehyde formed in the incubation mixture, the measurement of carbonyl functions in membrane phospholipids represents a simple and reproducible method to detect lipid peroxidation in biological systems and may be of great usefulness, expecially in in vivo conditions in which determination of various parameters (diene conjugation, hepatic concentration of thiobarbituric acid-reacting products, ethane or penthane evolution, etc.) sometimes gives not univocal results in the hands of different investigators. The results of ethionine intoxication confirm the specificity of the detection of carbonyl functions in membrane phospholipids with respect to the in vivo conditions in which lipid peroxidation occurs. As shown in Table I, no 2,4-dinitrophenylhydrazine-reacting groups could be detected in liver microsomal phospholipids from ethionine-treated rats. Unpublished results from one of our laboratories (Benedetti, A. and Comporti, M., unpublished data) have in fact shown that lipid peroxidation cannot be detected by all the other methods in liver cell membranes following ethionine poisoning. As shown in Fig. 1, 2,4_dinitrophenylhydrazinereacting groups, although in relatively low amounts, were detected by the method used in this experimental work even in the microsomal phospholipids of the control rats. This fact is hard to explain at present, since a physiological route for lipid peroxidation has not been demonstrated with certainty up to now. It must be considered, however, that the presence of 2,4_dinitrophenylhydra-
zinc-reacting groups in control samples has also been observed in spectrophotometric examination of 2,Cdinitrophenylhydrazine-treated microsomal protein in our previous work [15] concerned with the detection of protein-bound carbonyls in liver microsomes from Ccl,- or BrCCl,-poisoned rats. The possibility must be considered that some of the carbonyl functions detected by the present method are formed through the cleavage of phospholipid-bound fatty acid hydroperoxides during technical procedures, and primarily during the reaction with 2,4_dinitrophenylhydrazine. This possibility, however, is ruled out by the following arguments: (i) the decomposition of peroxides is slow in an acidic medium such as that in which the reaction with 2,4_dinitrophenylhydrazine takes place; and (ii) the kinetics reported [12] for the formation of fatty acid hydroperoxides in phospholipids of liver microsomes peroxidizing under conditions similar to those used in the present work is quite different from that of the formation of the carbonyl functions. In fact the time-course of the formation peroxides (see Ref. 12, Fig. 1) shows a transient increase during the early periods of incubation and a sharp decrease thereafter; the formation of carbonyl functions (this paper, Fig. 3), on the contrary, shows an exponential shape. The present study, together with our previous one [ 151, indicates that lipid peroxidation produces, either in vitro or in vivo, marked damages to cellular membranes. The damage can be produced directly through the breakdown of phospholipid-bound unsaturated fatty acids and the resultant disorganization of the hydrophobic core of the membrane in which polar groups appear where unpolar hydrocarbon chains normally occur. Also, indirect damage can be produced through the binding of products originating from the peroxidative breakdown of fatty acids to membrane molecular structures. Thus, both the appearance of altered phospholipids and the binding of lipid peroxidation products represent two complementary aspects of the same phenomenon. As shown in Table II, the amount of carbonyl functions in microsomal phospholipids found in the in vivo intoxications is similar to or not so different from the amount of carbonyl functions found in the in vitro condition in which lipid peroxidation is induced by the lower amount (6
638
PM) of Fe2+. A strict similiarity between the results of Ccl, or BrCCl, intoxication and those of the in vitro microsomal lipid peroxidation induced by 6 PM Fe 2+ has also been observed when the amount of carbonyls bound to the microsomal protein was determined [ 151. It must be added that in this in vitro condition a significant 30% decrease of microsomal glucose-6-phosphatase activity was observed. Therefore, it is reasonable to assume that the alterations of membrane phospholipids and/or the binding of aldehydes to the microsomal protein are significant to the development of functional alterations of cellular membranes as those observed after the CCI, or BrCCl, intoxication. Furthermore, the possibility must be considered that in the in vivo situation sufficient amounts of altered phospholipids and/or toxic aldehydes are formed at discrete sites in the membrane where the homolytic cleavage of the halogenated hydrocarbons occurs, so as to produce important functional alterations. In the in vitro lipid peroxidation on the other hand, the extensive alterations of the membrane can be envisaged as occurring both at molecular sites essential for functional activities and at nonessential ones. Whatever the molecular pathway leading to functional changes may be, it is reasonable to assume that the formation of phospholipid-bound fatty acyl residues bearing carbonyl functions in cellular membranes has important pathological consequences. Acknowledgements This work was supported by a grant from the National Foundation for Cancer Research (U.S.A.). Additional funds were derived from a grant of the Consiglio Nazionale delle Ricerche, Roma, Italy.
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