American Journal of Pathology, Vol. 129, No. 2, November 1987Copyright © American Association of Pathologists

Histochemical Detection ofLipid Peroxidation in theLiver ofBromobenzene-Poisoned Mice

ALFONSO POMPELLA, MD,EMILIA MAELLARO, PhD,

ALESSANDRO F. CASINI, MD,and MARIO COMPORTI, MD

The possibility of detecting lipid peroxidation histo-chemically was investigated in liver tissue in vivo, inconditions in which the process has been demonstratedby biochemical methods. The technique was based onthe detection ofaldehyde functions with the use oftheSchiff's reagent. The study was carried out on bromo-benzene-intoxicated mice, which generally exhibitlevels of lipid peroxidation considerably higher thanthose observed in the case ofother hepatotoxins. Liversections from control animals were unstainable by thereagent, while sections from bromobenzene-poisonedmice showed a purple stain ofvarious intensity, unho-mogeneously distributed, sometimes with a medio-lobular localization. Microphotometric measurementswere performed at 565 nm by means of a computer-

PEROXIDATION of unsaturated lipids in biologicmembranes has been implicated in a variety ofpatho-logic conditions.'-5 Consequently, many technicalprocedures have been developed to detect this processin biologic samples.6 These methods are ofbiochemi-cal nature, and therefore they do not give informationabout the reaction in individual cells.

In a recent study from our laboratory,7 a histo-chemical technique was developed to detect lipid per-oxidation in cells of liver sections exposed to pro-oxi-dants. The technique was based on the use of theSchiff's reagent to detect aldehyde functions. This wasdone because in a previous paper8 we showed thataldehydes (probably alkenals) originating from theperoxidation of liver microsomal lipids bind to themicrosomal protein. In the case of alkenals, the bind-ing occurs by a thioether linkage between the doublebond of the a, fl-unsaturated aldehyde and the -SHgroup ofthe protein.9'10 Because the aldehyde group-ing remains free in this reaction,9"0 the aldehyde func-tion can react with the fuchsin: sulfurous acid presentin the Schiff's reagent. The same reaction could also

From the Istituto di Patologia Generale dell'Universita di Siena,Siena, Italy

controlled microscope photometer. The ratios ofSchiff-positive area relative to total section area, aswell as the total extinctions referred to 100 sqj ofthesections, showed a high correlation with the corre-sponding hepatic contents ofmalonic dialdehyde, cho-sen as biochemical index oflipid peroxidation. In vitrostudies in which liver sections were incubated in thepresence of NADPH-Fe2 , showed a Schiff positivitywhich increased with the incubation time, confirmingthe reliability of the histochemical method. Anotherprocedure, based on the use of 2-OH-3-naphtoic acidhydrazide coupled with fast blue B, was also developedand proved to be possibly more sensitive than Schiff'sreagent in the detection of lipid peroxidation in livertissue. (AmJ Pathol 1987, 129:295-301)

involve the carbonyl functions present, as previouslyshown," in acyl residues ofmembrane phospholipidsand formed as a consequence of the peroxidativebreakdown of unsaturated fatty acids. In the studyquoted above7 it was observed that the liver sectionsincubated in the presence of pro-oxidants (NADPH-Fe2+, NADPH-CC14, NADPH-BrCCl3, etc.) andtherefore peroxidized were markedly stained purpleunder gross and microscopic observation after expo-sure to the Schiff's reagent. Thus, the histochemicaltechnique proved to be a good tool in detecting lipidperoxidation induced in vitro by pro-oxidants.The histochemical procedure has the advantage

Supported by Grants 85.02107.44 and 85.00753.04 fromthe Consiglio Nazionale delle Ricerche, Roma (FinalizedProgram on Oncology and Gastroenterology Group, re-spectively).Accepted for publication June 22, 1987.Address reprint requests to Prof. Mario Comporti, Isti-

tuto di Patologia Generale dell'Universita di Siena, Via La-terino 8, 53100 Siena, Italy.

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296 POMPELLA ET AL

over the biochemical methods to make it possible todetect lipid peroxidation in single cells of a tissuecomposed ofdifferent cell populations. With the pres-ently existing biochemical methods, lipid peroxida-tion in a relatively small number of cells would beobscured by normal lipids of the bulk of unaffectedcells.

In the present work we investigated whether lipidperoxidation could be detected histochemically inliver cells in vivo, in conditions in which the occur-rence of the process has been biochemically demon-strated. We particularly studied the intoxication withbromobenzene in mice, because it has been shown inour laboratory'2 that in this experimentally inducedliver injury the level of detectable peroxidized phos-pholipids can be far greater than in the case ofcarbontetrachloride or monobromotrichloromethane(BrCCI3) intoxication.

Materials and Methods

Male NMRI albino mice weighing 25-30 g andmaintained on a pellet diet (Altromin-Rieper, Bol-zano, Italy) were used. The animals were starvedovernight before being used.

In the in vivo studies the mice were given bromo-benzene (C. Erba, Milano, Italy), mixed with two vol-umes of mineral oil, by gastric intubation, at the doseof 15 mmol/kg body wt. Intoxication was carried outunder light ether anesthesia. Control animals receivedmineral oil only. After 18-20 hours, the mice weresacrificed under ether anesthesia by exsanguination,blood samples were collected for enzymatic determi-nations, and the liver was quickly removed and rinsedin ice-cold saline. Tissue samples (approximately5 X 10 mm in size) were cut from the left larger he-patic lobe, immediately frozen under a CO2 stream,and processed for histochemistry. Surrounding tissuewas employed for biochemical determinations.

Lipid peroxidation was assessed by measuring thehepatic content of malonic dialdehyde (MDA), afterhomogenization of tissue with 5% (wt/vol) trichlora-cetic acid (TCA), as previously reported.13 In fact, ithas been shown in our laboratory that the methodmeasuring the tissue content ofMDA provides valuesthat are correlated with those obtained with otherprocedures, such as the detection of the diene conju-gation absorption in cellular phospholipids'4 or thedetermination of the carbonyl functions originatingfrom the peroxidative breakdown ofunsaturated fattyacids in cellular phospholipids." Therefore, the he-patic content of MDA was taken as a reliable bio-chemical index of lipid peroxidation in liver tissue.

Hepatic glutathione content was measured in the

same TCA-homogenate, as acid-soluble SH groups,according to Sedlak and Lindsay.'" Protein determi-nation was made according to Lowry et al.'6 Serumglutamate-pyruvate transaminase (SGPT) activitywas measured (optimized ultraviolet-enzymaticmethod, Sclavo, Siena, Italy) as a parameter of liverdamage.

In the in vitro studies, liver sections (15 u thick)obtained from frozen tissue specimens were incu-bated at 37 C for 5-15 minutes, in 150 mM KCl, 50mM Tris-maleate buffer, pH 7.4, containing anNADPH-Fe system (0.8 mM NADPH, 0.1 mMFeCl3, and 4.5 mM ADP). Control sections were in-cubated in KCl Tris-maleate buffer alone, containing3 mM EDTA. The concentration ofMDA in the in-cubation medium was measured by the thiobarbituricacid test after extraction of the chromogen withn-butyl alcohol, essentially as reported previously.'7

Histochemical Procedures

Liver sections ( 15 , thick) from either the bromo-benzene-intoxicated mice or the in vitro incubationswere stained 2.5 hours in the dark at room tempera-ture with Schiff's reagent (freshly prepared afterBarger and DeLamater, as reported by Pears. 18 Afterthe reaction, the sections were rinsed in three changesofsulfite water (5 ml 10% K2S205,5 ml 1 N HCl, waterto 100 ml), dehydrated in alcohol, cleared in xylene,and mounted in balsam. Prior to dehydration andmounting, selected sections were counterstained withhematoxylin to reveal the distribution of the Schiff-positive areas within the hepatic lobule.

In some experiments, besides the Schiff's reaction,another histochemical technique was tested for de-tecting lipid peroxidation, ie, the reaction of aldehy-dic groups with 2-OH-3-naphtoic acid hydrazide(OHNAH), followed by coupling with a diazoniumsalt. 19 Liver sections were exposed 1 hour at 60 C to a0.1% (wt/vol) OHNAH solution in 50% ethanol con-taining 5% (vol/vol) acetic acid. After the reaction, thesections were washed thoroughly in 50% ethanol andstained 5-10 minutes with a 0.1% (wt/vol) fast blue Bsolution in an alcoholic buffer. The latter was pre-pared by mixing equal volumes of 100mM phosphatebuffer, pH 7.4, and 95% ethanol.'9

Microphotometric Measurements

In order to avoid bias caused by possible fading ofSchiff-stained sections, which has been reported to besometimes associated with particular batches ofSchiff's reagent,20 it was decided to admit to the mi-crophotometric study only sections belonging to ani-

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HISTOCHEMISTRY OF LIPID PEROXIDATION 297

mals all processed in the same experiment the sameday, ie, sections all stained with the same, freshlymade Schiff's reagent. In this way three experimentswere performed, of which a representative one is re-ported.

Microphotometric evaluation ofthe sections (threeper animal, cut at various depths from the same tissueblock) was performed with a computer-controlledZeiss Microscope Photometer 03 equipped with arapid scanning table, using the Zeiss APAMOS pro-gram as software for data processing. The apparatuswas set as follows: objective, lOX; magnification,1 60X; diameter ofround measuring diaphragm, 20,u;diameter ofiris diaphragm, 50 ,; step size in x-axis, 20,u; step size in y-axis, 100-300 p (depending on sec-tion's size). Approximately rectangular sections werescanned at 565 nm (predetermined absorption maxi-mum ofthe Schiff-stained areas ofthe tissue) in a waythat the total scanned area within each single sectionhad the size of the largest rectangle which could beinscribed within the section. Because the maximumnumber of single measurements per field is restrictedby the computer, it was impossible to scan the wholesection with one measuring field, even using a stepsize of 20 u in the x and y direction. Therefore, inorder to obtain a complete scanning, we set the centerof a first measuring field in the center of the largestfield that could be inscribed within the respective sec-tion. Center points of additional measuring fieldswere set at intervals of 20 , in the (-)y direction.Thus, depending on the size of each individual sec-tion, the total area of the maximum rectangle thatcould be inscribed in it could be scanned completelywith a step size of 20 pu for the movement in the xdirection, and a step size of 100-300,u (depending onsection's size) in the y direction. The total number ofmeasuring fields consequently varied from 5 to 15.Because the APAMOS program allows storage of in-formation on a maximum of9999 measurements perfield, we were forced to use a relatively large measur-ing diaphragm of20 p in diameter; with a step size of20 p, the measurement of approximately the wholesection was performed. The measurements weremade for obtaining quantitative information aboutthe percentage of Schiff-stained areas, and not aboutthe amount of carbonyls stained. Therefore, the dis-tributional error caused by the large measuring dia-phragm could be neglected.The percentage of Schiff-positive area relative to

total scanned area was calculated for each section,taking into account the extinction values per measur-ing spot which exceeded a threshold value of 0.010-0.036 AU, predetermined for each section prior toscanning. To this aim, absorption spectra over the

range of 545-585 nm were recorded from several un-stained, apparently Schiff-negative fields for the pur-pose ofruling out those exhibiting a very low degree ofpositivity, and for assessing in the remaining ones thegenuine unspecific background extinction at 565 nm(absorption maximum of the Schiff chromophore).The number and the size of such background fieldsvaried according to the extent of unstained area ineach section. The mean absorbance + 1.5 standarddeviation of each truly Schiff-negative backgroundfield was arbitrarily assumed as maximum unspecificbackground absorbance ofthe field. The mean ofsuchmaximum background absorbances was consideredas the maximum unspecific background absorbanceof the section and used as the lower threshold limitduring the scanning procedure of the whole section.In one case, unspecific background absorption couldnot be measured, because of generalized extinctionmaxima at 565 nm all over the section; in this case, thepercentage of Schiff-positive area was considered tobe 100%.

Results and DiscussionFigure 1A shows the results of a typical in vitro

experiment in which sections from a normal liverwere incubated in the presence ofNADPH-Fe2+. Thedevelopment oflipid peroxidation was ascertained bydetermining the release ofMDA into the incubationmedium (see legend to Figure 1 A). As can be seen, theintensity ofthe Schiffreaction increased progressivelywith the incubation time, confirming the ability ofthis method to detect lipid peroxidation. The sectionsincubated in the absence of NADPH-Fe 2+ (control)were absolutely unstained after exposure to theSchiff's reagent.

Therefore, it was investigated whether lipid peroxi-dation could be detected histochemically in the liverof bromobenzene-intoxicated mice. Table 1 showsthe hepatic glutathione depletion, liver necrosis (asassessed by the SGPT level), and lipid peroxidation(as measured by the hepatic MDA content) in thebromobenzene-poisoned mice whose livers wereemployed for the microphotometric measurementsreported in the present communication (one typicalexperiment out of three is reported). In agreementwith our previous findings,'2 the marked glutathionedepletion was accompanied by varying degrees ofliver necrosis and lipid peroxidation; these latter phe-nomena occurred when the glutathione depletionreached a critical level.As expected, liver sections obtained from control

animals were absolutely unstained after exposure tothe Schiff's reagent (Figure 2A), while the sections

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298 POMPELLA ET AL

Figure 1-Sections (1 5,u) obtained from normal mouse liver, incubated in anNADPH-iron dependent system and stained with the Schiff's reagent (A) orwith 2-OH-3-naphtoic acid hydrazide and fast blue B (B). (X1.5) The incuba-tion was carried out for 5, 10, and 15 minutes. The release of malonicdialdehyde in the incubation medium was as follows: 5 minutes, 2.55 umol/ml; 10 minutes, 4.22 ,mol/ml; 15 minutes, 4.59 pmol/ml. Control sectionswere incubated in the absence of the NADPH-iron system (see Materials andMethods for further details).

obtained from bromobenzene-intoxicated mice ex-hibited a purple stain of various intensity both undergross and microscopic observation (Figure 2B-E). Insections belonging to livers with lowerMDA contentsan unhomogeneous distribution ofthe stain was gen-erally appreciated, with Schiff-positive areas of var-

ious intensity alternated with wide negative areas

(Figure 2B). Moreover, positive areas appeared some-times to be concentrated in a particular region of a

section, the rest appearing negative. On the otherhand, sections belonging to livers with highest MDAcontent exhibited a diffuse positivity ofvarious inten-

Table 1 -Hepatic Glutathione (GSH) Levels, Hepatic MalonicDialdehyde (MDA) Contents, and Serum Glutamate-PyruvateTransaminase (SGPT) Levels in Mice IntoxicatedWith Bromobenzene

GSH MDA(nmol/mg protein) (nmol/g tissue)

SGPT(U/1)

Controls 15.7 (2) - 88 (2)Bromobenzene 0.7 ± 0.1 (8) 263.1 ± 68.6 (8) 2270 ± 676 (8)

Bromobenzene was given at the dose of 15 mmol/kg body wt, by mouth,and the animals were sacrificed 18 hours thereafter. Values reported are

means ± standard error of the mean. The number of animals is in parenthe-ses.

sity, involving the whole section, often with very littlesurface left unstained (Figure 2E). These aspects seemto suggest that the peroxidative process is triggered indelimited regions of hepatic parenchyma first, andthen spreads to involve adjacent tissue. In several sec-

tions of low and intermediate positivity, a medio-lobular localization of Schiff positivity was appre-

ciated, made apparent by counterstaining adjacentsections with hematoxylin in order to reveal struc-tural details of the hepatic parenchyma (Figure 2Dand F). Bromobenzene-induced liver necrosis hasbeen shown to occur mainly in centrolobular areas inrats poisoned intraperitoneally.2123 This also oc-

curred in most ofthe mice used in our study, althoughthe localization was much less defined. In any case,the Schiff-positive areas did not always coincide withthe necrotic areas, as estimated by routine histology.This should not be surprising, because, at least incarbon tetrachloride hepatotoxicity, lipid peroxida-tion as evaluated in vivo by the common biochemicaltests, is maximal during the early times after poison-ing and then declines steadily until minimal levels-ifany-are observed at later times, when liver necrosisis massive. 11,2428 This may explain why hepatic areas

in which severe necrosis has occurred are less stainedby the Schiff's reagent than the bordering areas, whereit is conceivable that lipid peroxidation is still an on-

going process.Areas of highest positivity constantly appeared

crowded with innumerable, Schiff-stained vesicularbodies, outlined against a pale to dark pink back-ground. The origin and the nature of these vesicles,also observed in our previous report,7 cannot be de-fined at present.Data concerning the microphotometric evaluation

ofSchiff-stained liver sections are reported in Table 2.The hepatic contents ofMDA (ie, the extent of liverlipid peroxidation) appeared to be significantly corre-

lated with the ratios ofSchiff-positive to total scannedarea (r = 0.836, P < 0.01); they were also correlatedwith the values E/t/ 100 sq,u, yielded by the product ofthe mean extinctions at 565 nm of positive areas bythe corresponding ratios of Schiff-positive to totalscanned area (r = 0.784, P < 0.01). The correlationbetween the hepatic MDA content and the mean ex-

tinction values at 565 nm of positive areas was notsignificant (r = 0.588, P < 0.1). This is probably dueto the vesicular appearance, characteristic of mostpositive areas, which causes a major interference witha correct measurement of extinctions. Furthermore,it must be considered that the biochemical measure-

ment ofMDA content actually provides whole tissuevalues (ie, the actual MDA content of areas affectedby lipid peroxidation is diluted in the whole tissue

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AJP * November 1987

HISTOCHEMISTRY OF LIPID PEROXIDATION

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Figure 2-Liver sections (1 5,u) obtained from control and bromobenzene-intoxicated mice, stained with the Schiff's reagent. (X40) Bromobenzene was givenat the dose of 15 mmol/kg body wt, by mouth, and the animals were sacrificed 18-20 hours thereafter. A-Control, absolutely unstained. B-Bromo-benzene. Small positive areas are scattered over the surface. Hepatic malonic dialdehyde (MDA) content: 2.4 nmol/g tissue. C-Bromobenzene. Largerpositive areas can be appreciated. Hepatic MDA content: 13.5 nmol/g tissue. D-Bromobenzene. Note the lobulelike arrangement of the positivity. HepaticMDA content: 1 18.2 nmol/g tissue. E-Bromobenzene. An intense and diffuse stain is appreciable all over the section. Hepatic MDA content: 199.5 nmol/gtissue. F-Schiff's stain plus haematoxylin. Same animal as in D, adjacent section.

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Table 2-Microphotometric Determinations in Liver Sections Stained With the Schiff's Reagent and Corresponding Hepatic MDAContent in Mice Intoxicated With Bromobenzene

Maximum unspecificbackground E/sq I R -oj1 00 sq p Hepaticextinctions (mean extinctions (ratios positive/ (total extinc- MDA contents

Animals at 565 nm (AU)* at 565 nm, AU)t total area, %)t tions, AU)§ (nmol/g tissue)

Control 1 0.011-0.013 0.035 ± 0.002 0.9 ± 0.1 .031-Control 2 0.012-0.015 0.045 ± 0.001 0.6 ± 0.1 .027 -

Bromobenzene 1 0.010-0.013 0.035 ± 0.003 1.0 ± 0.5 .035 0Bromobenzene 2 0.013-0.024 0.067 ± 0.002 66.1 ± 4.5 4.363 428.8Bromobenzene 3 0.034-0.036 0.088 ± 0.005 55.6 ± 2.8 4.948 445.0Bromobenzene 4 0.022-0.030 0.173 ± 0.002 85.1 ± 1.9 14.722 476.3Bromobenzene 5 Not determinedll 0.150 ± 0.001 10011 15.000 307.6Bromobenzene 6 0.022-0.031 0.097 ± 0.005 77.5 ± 3.5 7.517 309.1Bromobenzene 7 0.010-0.015 0.122 ± 0.015 10.4 ± 1.2 1.258 134.5Bromobenzene 8 0.011-0.012 0.051 ± 0.007 2.3 ± 0.3 .115 3.5

The bromobenzene intoxication was performed as reported in Table 1.*Arbitrarily considered as the mean + 1.5 standard deviation of the background extinctions measured in each section. Values used as lower thresholds during

scanning of the corresponding sections. Reported are the lowest and the highest of three determined values (one per section; see Materials and Methods).tMean ± SEM of three values (one per section), referring to Schiff-positive areas only.tMean ± SEM of three values (one per section).§Values yielded by the product of mean extinctions (E/sq p) by the ratios of positive/total area (R) of the corresponding sections.lPeak absorption at 565 nm was present all over the section.The correlation between the hepatic MDA content and the ratios positive/total area (%) was r = 0.836, P < 0.01. The correlation between the hepatic MDA

content and the E,J1 00 sq ju values was r = 0.784, P < 0.01.

sample during the homogenization), while the com-putation ofthe mean extinction at 565 nm takes intoaccount only the values referring to the Schiff-positiveareas (ie, the areas exhibiting extinctions higher than apreestablished threshold value). For this reason, weconsidered Etd 100 sq p values, which are calculatedfor 100 sq , of total (positive plus negative) area,taking into account the mean percentage of Schiff-positive area; and these values do in fact correlate withthe corresponding hepatic MDA contents.

Thus, the Schiffreaction proved to be a reliable toolfor the investigation of lipid peroxidation in vivo.Quantitative estimations can be drawn by the exten-sion ofareas showing positivity to the reaction. Froma qualitative point of view, the reaction appearedsometimes sharply localized, and this allowed visual-ization of the the areas that allegedly first undergolipid peroxidation in the experimental model studied.The possibility of increasing the sensitivity of the

histochemical detection of lipid peroxidation was ex-plored by various alternative procedures. In particu-lar, the use of 2-OH-3-naphtoic acid hydrazide(OHNAH) followed by staining with fast blue Bproved to be a histochemical procedure with a highsensitivity, particularly useful in detecting low levelsof lipid peroxidation.

In vitro studies carried out with this procedure (Fig-ure 1 B) revealed a higher intensity of the stain (pur-plish-violet), as compared with that obtained with theSchiff's reagent in sections incubated in the same pro-oxidant system during the same experiments (Figure

1 A). In in vivo studies, too, although preliminary, theuse of OHNAH seemed to confirm the efficiency ofthis procedure as to the detection of lipid peroxida-tion. In one experiment of bromobenzene intoxica-tion performed to date (data not shown), a strongcorrelation (r = 0.873, P < 0.001 ) was found betweenthe mean extinctions of the sections at 575 nm (ab-sorption maximum ofthe chromophore) and the cor-responding hepatic contents of MDA.

In conclusion, the development of histochemicaltechniques to detect lipid peroxidation seems to allowobservations which can be ofhigh interest in the studyof cellular damage of oxidative nature. In particular,histochemistry may prove eventually to be the ap-proach of choice for the study of tissues composed ofdifferent cell populations hardly separable with thepresently available procedures.

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AcknowledgmentThe authors wish to thank Dr. G. Nohammer (Institut fur

Biochemie, Universitat Graz, Austria) for helpful sugges-tions and discussion.