Immunohistochemical analysis of heme oxygenase-1 in preneoplastic and neoplastic lesions during chemical hepatocarcinogenesiss

ORIGINAL ARTICLE

Immunohistochemical analysis of heme oxygenase-1 inpreneoplastic and neoplastic lesions during chemicalhepatocarcinogenesis§

Fabiana Caballero*†, Roberto Meiss‡†, Alejandra Gimenez‡, Alcira Batlle* and Elba Vazquez*

*Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP) CONICET, Departamento de Quımica Biologica, FCEN, UBA – Ciudad

Universitaria, Buenos Aires, Argentina, and ‡Departamento de Patologıa, Instituto de Estudios Oncologicos, Academia Nacional de

Medicina, Buenos Aires, Argentina

I N T E R N AT I O N A LJOURNAL OFE X P E R I M E N TA LPAT H O L O G Y

Summary

Heme oxygenase (HO) breaks down the pro-oxidant heme into carbon monoxide, iron

and the antioxidant biliverdin. The isoform HO-1 plays an effective role to counteract

oxidative damage and to control inflammation. Prolonged cellular damage due to

chronic inflammation is one of the reasons leading to the development of tumours.

The aim of this work was to investigate HO-1 expression and localization along the

different stages of chemically induced hepatocarcinogenesis (HCC) and the occurring

morphological changes. To provoke sustained oxidative stress and chronic inflamma-

tion, CF1 mice received dietary p-dimethylaminoazobenzene (DAB, 0.5%, w/w) during

a whole period of 14 months. HO-1 expression increased along the experimental trial in

morphologically normal hepatocytes in DAB-treated animals. HO-1 expression dimin-

ished in altered hepatic foci (AHF) and oval cells and early preneoplastic lesions.

Otherwise, marked HO-1 overexpression was detected in Kupffer cells and macro-

phages surrounding necrotic and nodular areas. Adenomas showed decreased HO-1

immunostaining. In hepatocellular carcinomas, an inverse relationship was found

between the immunohistochemical expression of HO-1 and the degree of tumour

differentiation, being negative in poorly differentiated tumours. In our experimental

model, down modulation of HO-1 expression correlated with malignancy progression.

Thus, our data point to activation of HO-1 as a potential therapeutic tool.

Keywords

heme oxygenase, hepatocarcinogenesis, inflammation, oxidative stress, p-dimethyl-

aminoazobenzene

Hepatic carcinogenesis (HCC) is a complex process

that histologically progresses from benign precursor lesions

to malignant neoplasms, and it is associated with accumula-

tion of genetic and epigenetic changes, giving rise to the

stages of initiation, promotion and progression (Boone

et al. 1992).

Received for publication:

7 November 2003

Accepted for publication:

17 June 2004

Correspondence:

Prof Dr A. Batlle, Viamonte 1881 10�

‘‘A’’,1056 - Buenos Aires, Argentina.

Fax: +54 11 4811 7447;

E-mail: [email protected]

†The first two authors contributed

equally to the development of this

work.§To the memory of Cesar Polo,

deceased March 9th, 1996.

Int. J. Exp. Path. (2004), 85, 213–221

� 2004 Blackwell Publishing Ltd 213

Carcinogenesis can be experimentally induced by exposure

to exogenous agents, and chronic rodent bioassays are the best

models to predict the risks of chemical exposure in humans

(Ames & Gold 1990).

The diagnosis of HCC is based on the occurring architec-

tural and cytological changes. Numerous investigations have

been carried out to elucidate the early hepatocellular lesions

which may represent progenitor populations of HCCs (Ahn

et al. 1999).

The development of phenotypically altered hepatic foci

(AHF) has been a common feature in experimental HCC

and is believed to be caused by mutations in normal

cellular growth control genes and subsequent clonal growth

(Butterworth & Goldsworthy 1991). Subsets (1–5%) of these

foci eventually progress through a process of neoplastic trans-

formation into overt malignancy (Farber 1986; Pitot et al.

1991). Another outstanding finding in early chemical carcino-

genesis is a multipotential cell called the oval cell, described by

Farber and colleagues (Farber 1956). All of these cellular

events are accompanied by increased expression of several

growth factors that enhance cell survival of carcinogen-

activated cells (Dominguez-Malagon & Gaytan-Graham 2001).

The effect of prolonged cellular damage in carcinogenesis

such as that related to chronic inflammation has become

widely recognized (Ohshima & Bartsch 1994). The oxidants

produced by inflammation may stimulate oncogenes and cell

proliferation. Reactive oxygen species (ROS) are known to be

implicated in both the initiation and promotion of tumours.

Multiple sources of ROS may contribute to a permanent oxi-

dative stressing environment leading to pathophysiological

changes and allowing for the selective growth of preneoplastic

initiated cells (Batlle 1993; Klaunig et al. 1998).

Heme oxygenase (HO) is the rate-limiting enzyme in the

degradation of heme. It breaks down the pro-oxidant heme

into the vasodilator carbon monoxide, free iron and the anti-

oxidant biliverdin (Maines 1997; Foresti & Motterlini 1999;

Tomaro & Batlle 2002). Recently, interest has been intensively

focused on the biological effects of carbon monoxide, bili-

verdin and bilirubin. Not so long ago, these heme-derivative

metabolites were considered toxic waste products; however,

accumulating data suggest that they have antioxidative, anti-

inflammatory, antiapoptotic and signalling properties and

possibly also some immune modulatory function (Wagener

et al. 2001). It was proposed that the inducible isoform HO-1

could play an effective role to counteract the damage caused

by oxidative and nitrosative stress (Maines 1997; Naughton

et al. 2002). HO-1 is induced by host oxidative stress stimuli,

and activation of HO-1 gene expression is considered to be an

adaptive cellular response to different types of chemicals and

mediators that change the redox status of the cell (Motterlini

et al. 2002). Other suggested functions for HO-1 are related to

the regulation of blood pressure (Motterlini et al. 1998;

Abraham et al. 2002) and to neovascularization (Dulak et al.

2002). Macrophages are key participants in angiogenesis, and

its infiltration is correlated to the appearance of histologic

malignancies. Nishie et al. (1999) proposed HO-1 as a marker

for activated macrophage infiltration and for neovasculariza-

tion in human gliomas.

It was also suggested that in oral carcinoma, HO-1 expres-

sion can be used to identify patients with low risk of lymph

nodule metastases (Tsuji et al. 1999). Other authors have

detected HO-1 expression only in tumour cells, and it has

been found that intraarterial zinc protoporphyrin administra-

tion in rat hepatomas considerably suppressed tumour growth

(Doi et al. 1999).

We have developed an experimental mouse model to study

the onset of hepatocarcinogenesis induced by the administra-

tion of p-dimethylaminoazobenzene (DAB) (Gerez et al. 1997;

Caballero et al. 2001).

In our model, DAB metabolization generates free radicals

and triggers the activation of oxidative processes as indicated

by an important and sustained P450 level increase, enhanced

lipid peroxidation and diminished levels of the natural anti-

oxidant defence system (Gerez et al. 1997; Gerez et al. 1998).

We have proposed a possible cytoprotective role of HO-1

during the carcinogenic process. We have shown that HO-1

mRNA levels were greatly induced in the liver of DAB-treated

animals, and that this level of induction persisted as long as the

oxidative damage lasted. However, HO activity would be

modulated by post-transcriptional events (Vazquez et al.

2002).

Based on own previous results about the characterization of

mice with chemically induced HCC by chronic DAB adminis-

tration, we have designed the intoxication protocol used in the

present study to provoke continuous and permanent oxidative

stress, leading to inflammation and consequent liver injury, as

essential components of the carcinogenic process. The aim of

this work was to investigate HO-1 expression and localization

along the different morphological stages occurring during

experimental HCC.

Materials and methods

Reagents

Chemicals were reagent grade and were purchased from Sigma

(St Louis, MO, USA). The following primary polyclonal anti-

bodies, rabbit anti-HO-1 (Stressgen Biotech, Victoria, BC

Canada) and goat anti-actin (I-19) (Santa Cruz Biotech,

Santa Cruz, CA, USA) were used. The secondary antibodies

214 F. Caballero et al.

� 2004 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 85, 213–221

used for HO-1 Western blot analysis were antigoat IgGHRP or

antirabbit IgGHRP (Santa Cruz Biotech). The secondary

antibody used for HO-1 immunochemical detection was a

biotinylated linking antirabbit IgG followed by alkaline

phosphatase – labelled streptavidin (Biogenex, San Ramon,

CA, USA).

Animals and treatment

Male CF1 mice weighing 30 g were employed. A group of

animals (n5 100) was placed on dietary DAB (0.5%, w/w)

during a whole period of 14 months. Control animals (n5 40)

were fed with a standard laboratory diet (Purina 3, Asociacion

de Cooperativas Argentinas, San Nicolas, Buenos Aires,

Argentina) for the same period. All animals received food

and water ad libitum. Throughout the study, all animals

were inspected at least twice daily. Body weight and food

consumption were measured at intervals throughout the

study. Food was removed from animals 16 h before killing.

Similar groups of animals (at least three animals per group) on

the same laboratory standard diet, with and without the car-

cinogen, were killed under ether anaesthesia. Animals were

scheduled for killing at 54, 74, 89, 104 and 164 days and at

8, 10, 13 and 14 months.

All animals received humane care and were treated in accord-

ance with the guidelines established by the Animal Care and

Use Committee of the Argentine Association of Specialists in

Laboratory Animals (AADEALC) and in accordance with the

UKCCCR Guidelines for the Welfare of Animals in Experi-

mental Neoplasia (Workman et al. 1998).

Protein extraction and Western blotting

Expression of HO-1 protein was studied by Western blot

analysis. Protein from liver tissues (100 mg chopped in small

pieces) was extracted by using lysis buffer [50 mM Tris–HCl,

pH 6.8; 10% sodium dodecyl sulfate (SDS)] and homogenized.

After 30 min of incubation at 4 �C, the lysates were heated at

100 �C during 5 min and were centrifuged at 10 000 · g for

30 min at 4 �C. Lysates containing equal amounts of proteins

(100 mg) were resolved on 8.5% SDS-polyacrylamide gel elec-

trophoresis. Rainbow coloured protein molecular weight stand-

ards obtained from Amersham were used for the estimation of

molecular size. The proteins were blotted to a Hybond-

Enzyme Chemio Luminiscence (ECL) nitrocellulose membrane

that was probed and washed according to the instructions for

the enhanced chemiluminescence Western blotting detection

system (Amersham Pharmacia Biotech, Little Chalfont, UK),

with transfer buffer (pH 8.3) containing 20% methanol (v/v)

using an Hoefer miniEV electrotransfer unit (Amersham

Pharmacia Biotech). The membrane with transferred proteins

was blocked with 5% serum albumin in Tris-buffered saline

(TBS) containing 0.1% Tween 20 (TBST) for 1 h at room

temperature and incubated with the first antibody diluted in

TBST for 1 h at room temperature. After washing in TBST, the

membrane was incubated with horseradish peroxidase-

labelled secondary antibody for 1 h at room temperature.

For quantification of immunoblots, relative intensities of bands

were quantified by densitometry using IMAGE MASTER image

analysis software (Amersham Pharmacia Biotech). Control for

loading and transfer was obtained by probing with anti-b-actin.

Histology

For histological study, a random section, about 4 mm thick,

was removed from the median, left lateral and right lateral

lobes of the liver. This operation was repeated to examine six

samples from each liver. Additional sections were taken of

grossly visible lesions not included within the above sections.

When gross tumours were observed, sections were invariably

prepared from them for histological characterization. Sections

from other organs were also prepared.

All sections were cut 3–5 mm thick from buffered formalin-

fixed, paraffin-embedded tissue. After deparaffinization, sec-

tions were stained with haematoxylin–eosin (H&E), periodic

acid–Schiff (PAS) reagent, Gomori’s silver impregnation for

reticulin and Prussian blue for iron methods.

Liver foci as well as tumour histology were identified

according to published criteria (Frith et al. 1994).

Immunohistochemistry

Sections obtained from the paraffin blocks used for the histo-

logical diagnosis were deparaffinized with three changes of

xylene for 10 min each; they were hydrated in decreasing con-

centrations of ethanol and rinsed in water. Prior to staining,

the slides were placed in 10 mM sodium citrate buffer pH 6 in a

microwave oven (5· 2 min at 750 W, with a cooling period of

6 min after each treatment).

Sections were pretreated with horse normal serum, diluted

at 1 : 20, for 20 min. Then, they were incubated with: 1) a

rabbit polyclonal antibody against HO-1, diluted at 1 : 500,

overnight; 2) biotinylated antirabbit immunoglobulins serum

diluted at 1 : 15 for 30 min and 3) alkaline phosphatase-

labelled streptavidin diluted at 1 : 15 for 30 min. Alkaline

phosphatase activity was developed with the Fast-Red system

(DAKO). Slides were lightly counterstained with H&E. All

incubations were performed at room temperature, and all

washings were performed with phosphate-buffered saline

buffer (pH 7.5).

Immunohistochemical analysis of HO-1 in hepatocarcinogenesis 215


Control sections used for determination of antibody reac-

tion specificity included: (a) positive controls (sections of

mouse spleen) and (b) negative controls (serial sections of

each sample omitting the primary antibody).

The HO-1 immunostaining was qualitatively evaluated by

the presence of positive or negative staining, in addition to the

type of cells staining positive.

Statistics

Six to 8 animals per treatment group were used in all experi-

ments. Data were analysed using the Student’s t-test. Results

were considered statistically significant when P < 0.05.

Results

Western blot analysis

Liver samples of animals treated with DAB during a whole

period of 14 months were analysed by Western blotting. HO-1

expression increased significantly along the treatment. In

whole liver, maximal induction (sixfold) was detected after

13 months of DAB administration, whereas in nodules sam-

ples, increase was only 1.7-fold (Figure 1).

Histology – immunohistochemistry

Normal hepatocytes. In the liver of all mice treated with DAB,

HO-1-positive morphological normal hepatocytes were seen;

the immunostaining became more intense with prolonged

treatment (Figure 2a). No increased immunostaining for HO-1

was observed in control animal hepatocytes (data not shown).

Macrophages. Proliferation and enlargement of sinusoidal

lining Kupffer cells was seen after day 54 and became more

pronounced with prolonged treatment. Liver macrophages

surrounding necrotic foci and nodular lesions were seen.

Both types of cells showed golden brown granular and diffuse

brownish yellow pigments, Prussian blue staining and a PAS-

positive reaction.

A positive, intense immunostaining for HO-1, in all liver

macrophages was observed (Figure 2a,b,c,d).

Necrosis. Foci of liver necrosis was first found after 67 days of

DAB feeding; from then onwards, all animals showed foci of

hepatic necrosis of different size and shape at any time up to

14 months.

A marked decreased immunostaining for HO-1 mainly in

hepatocytes from necrotic foci, with an increased staining

in surrounding normal and regenerative hepatocytes and in

macrophages was observed in all cases (Figure 2b).

Proliferation of ductular epithelial cells (‘oval cells’). In DAB-

feeding animals, the ductular epithelial cell (DEC) prolifer-

ation was first visible in portal areas at day 89, then increasing

with time. Duct profiles with well-defined lumina were seen at

month 13.

The proliferated DECs were negative for HO-1 immuno-

staining (Figure 2c) as were biliary epithelial cells in normal

nontreated animals (data not shown).

Nodular lesions. AHF: Hepatic foci of cellular alterations

were a common histopathological finding in this study and

included acidophilic and vacuolated AHF. These lesions were

seen after day 54 up to the end of the assay. There was a

decreased HO-1 immunostaining in AHF cells when compared

with surrounding normal hepatocytes at the different stages

(Figure 2d).

[_________________]Months

Indu

ctio

n of

HO

-1 p

rote

in (

fold

)

0

2

4

6

8

*

**

*

*

Months

CC

HO-1

1.5 5 8 13

1.5 5 8 13

HCC

HCCDAB C DAB C DAB C DAB

[___________________________]

Figure 1 Effect of p-dimethylaminoazobenzene treatment on

heme oxygenase (HO)-1 protein expression. Western blotting was

performed by standard procedures with polyclonal antibody to

HO-1. b-actin was used as a loading control. Other experimental

conditions are as described under Materials and methods. One

typical gel is shown. These results are representative of two

independents experiments. The densitometric data are mean6 SE

of six animals. ‘*’ indicates data significantly different (P < 0.05)

from that of control.



(a)

(c) (d)

(f)

(h)

(e)

(g)

(b)

(i) (j)

Figure 2 Representative findings of

heme oxygenase (HO-1) immunoreac-

tivity. (a) HO-1 expression in non-

atypical hepatocytes and in hyperplastic

Kupffer cells (day 104) (original mag-

nification ·250). (b) Negative HO-1

expression in necrotic hepatocytes

(arrow) with intense HO-1 expression

in marginal regenerative hepatocytes

and hyperplastic Kupffer cells (original

magnification ·250). (c) Isolated

ductular epithelial cell proliferation and

poor defined duct profiles, HO-1

immunostaining negative (original

magnification ·250). (d) Decreased

HO-1 expression in vacuolated AHF

surrounded by intense positive HO-1

macrophages (original magnification

·250). (e) Marked decreased HO-1

expression in adenomatous cells with

intense positive macrophages (original

magnification ·400). (f) Intense diffuse

HO-1 expression in trabecular hepato-

cellular carcinoma (original magnifica-

tion ·250). (g) Negative HO-1

expression in atypical cells from an

infiltrating undifferentiated hepatocar-

cinoma with weak positive comprised

hepatocytes (original magnification

·400). (h) Increased HO-1 expression

in nontumour hepatic tissue (bottom

half) at edge of carcinoma (arrow)

(original magnification ·250). Com-

parative heme oxygenase (HO-1)

expression according to carcinoma dif-

ferentiation degree. (i) Heterogeneous,

weak to negative immunostaining in

poorly differentiated trabecular hepa-

tocarcinoma (original magnification

·250). (j) Homogeneous, intense

immunostaining in well-differentiated

trabecular hepatocarcinoma (original

magnification ·250).



Neoplasms: The neoplasms found, in agreement with the

proposed classification, were identified as hepatocellular

adenoma and hepatocellular carcinoma.

The adenomas, mainly eosinophilic and vacuolated, exam-

ined at day 104 and later, were small and existed as distinct

nodules with well-differentiated hepatocytes. A marked

decreased or negative HO-1 immunostaining in adenomatous

tissue as compared to surrounding nonadenomatous tissue

was observed (Figure 2e).

Hepatocellular carcinomas were not obvious until 10

months. They were usually considerably larger and more irregu-

lar nodules. Several histological varieties of the trabecular

hepatocellular carcinoma were found; some of them showed

a well-differentiated pattern with a glandular appearance, and

others were well demarcated but poorly differentiated

tumours. Hepatoblastomas or cholangiomas were not found.

In hepatocellular carcinoma, various patterns of HO-1

immunostaining were observed (Figure 2f,g). In all nodular

areas (AHF, adenomas and carcinomas), the HO-1-positive

immunostaining was always of lower intensity than in the

surrounding normal hepatocytes (Figure 2h).

A decreased or negative relationship was found in poorly

differentiated tumours between the immunohistochemical

expression of HO-1 and tumour differentiation degrees;

instead, more differentiated tumours exhibited varying and

increased positive immunostaining patterns (Figure 2i,j).

No pertinent changes occurred in any organ other than liver

and the spleen, which at late stages showed marked enlarge-

ment with hypertrophy, mainly of the white splenic pulp.

Nodular lesions were not identified in sections from livers of

control animals at any time up to 14 months after laboratory

standard diet feeding. A minimal fatty change but no liver

necrosis or DEC proliferation was seen. Proliferated and pigment

containing macrophages were not observed at any stage.

Discussion

The hepatocarcinogenic protocol used here was designed to

induce tumours as a consequence of prolonged inflammation

and cell damage after oral administration of a carcinogenic

azo dye to mice. The model was very effective in terms of

tumour incidence (100%) and in reproducing all the steps in

the carcinogenic process. The continuous feeding with DAB

was useful to study the morphological changes that take place

during HCC. It was possible to identify early preneoplastic

lesions, which were characterized by proliferation of pheno-

typically AHF and oval cells. The emergence of such cells

generally precedes the development of HCC (Dominguez-

Malagon & Gaytan-Graham 2001). Oval cells proliferate in

response to several carcinogens and toxic agents (Saeter &

Seglen 1990). Although oval cells may be facultative liver

stem cells, they have the potential of evolving into tumours,

histologically identified as hepatocellular carcinoma (Braun

et al. 1989); recently, it was shown that preneoplastic foci

can originate from mature hepatocytes; these findings are

consistent with the hypothesis that dedifferentiation of mature

hepatocytes may occur during the course of a carcinogenic

regimen (Gourny et al. 2002).

It is well known that three stages are identified in HCC:

initiation, promotion and progression (Pitot et al. 1996).

Initiation of cellular transformation involves an irreversible

genetic change, occurring either spontaneously or as a result

of exposure to chemical or physical agents. During the promo-

tion stage which depends on the continuous presence of a

promoter agent (Dominguez-Malagon & Gaytan-Graham 2001),

selected cells clonally expand into AHF which grow progres-

sively as compared with surrounding normal tissue (Seo et al.

1988). The progression stage may be characterized by the

development of malignant neoplasm (Pitot & Dragan 1991).

The present study indicates that chronic exposure to DAB

promotes AHF at day 54 and DEC proliferation in portal areas

at day 89, both of these events are characteristic features of

early preneoplastic hepatic lesions. A significant finding was

foci of liver necrosis after 67 days of DAB feeding. In this in

vivo bioassay, the alterations provoked by the carcinogen

would be fixed through cell proliferation, reflected by hepato-

megaly and in response to necrosis, as we have previously

reported (Caballero et al. 2001; Vazquez et al. 2002). Under

the experimental conditions described here, we demonstrated

the presence of adenomas at day 104 and hepatocellular car-

cinomas at 10 months of DAB feeding. In a recent study, we

employed an experimental design administrating tamoxifen

citrate to promote hepatocarcinogenesis initiated with DAB

in mice. Animals also developed trabecular hepatocellular

carcinoma (Caballero et al. 2001; Vazquez et al. 2002).

One important component observed in the histological char-

acterization of this model was the proliferation and enlarge-

ment of Kupffer cells from day 54 as well as the presence of

macrophages surrounding foci of necrosis and nodular lesions,

indicative of the inflammatory response occurring under the

current protocol.

Oxidative stresses such as oxidant stimuli, inflammation,

exposure to xenobiotics and ionizing irradiation elicit various

tissue injuries and provoke cellular responses, principally

involving transcriptional activation of genes encoding proteins

which participate in the defence reactions (Camhi et al. 1995).

Chronic inflammation is leading to cancer in animals, and it is

therefore a risk factor of human cancer (Dominguez-Malagon

& Gaytan-Graham 2001). The incidence in liver cancer is low



in humans (but not in some strains of mice) unless the liver is

chronically damaged (Ames & Gold 1990).

HO-1 plays a major protective role against oxidant stimuli

(Clark et al. 2000; Tanaka et al. 2003). The strong adaptative

response of HO-1 expression on the diverse array of stress

stimuli suggests an important role for HO other than heme

degradation (Maines 1997; Maines 2000). In fact, the over-

expression of HO-1 is associated with the resolution of inflam-

mation and may act as a feedback mechanism (Wagener et al.

2001). Enhancement of HO-1 expression by gene transfer

provides cellular resistance against haemoglobin-heme toxicity

(Abraham et al. 1995; Yang et al. 1999). Recent observations

suggest that the mechanism of HO-1-mediated control of

inflammation may originate from the modulation of adhesion

molecule expression (Willis et al. 1996; Wagener et al. 1997).

Our results reveal and underline the importance of HO-1

expression in normal tissue, and its up-regulation suggests a

cell-protective response to the stress provoked by the gen-

eration of ROS during the carcinogen metabolism (Caballero

et al. 2001; Caballero et al. 2002). Similarly, HO-1 overexpression

in Kupffer cells and macrophages surrounding necrotic and

nodular areas very likely reflects the protective role of this

protein to the stress reaction triggered by inflammation.

Our findings are consistent with recent data reported

about the induction of HO-1 in isolated hepatocytes and

macrophages during acetaminophen-induced hepatotoxicity

(Chiu et al. 2002). Similarly, transgenic mice lacking func-

tional HO-1 are more sensitive to endotoxin-induced tox-

icity and highly susceptible to the development of chronic

liver inflammation (Poss & Tonegawa 1997).

Western blot analysis of whole liver samples confirmed the

induction of HO-1 expression along the experimental trial

with DAB.

The in vivo bioassay used here turned out to be a valuable

model to follow the complex cascade of morphological events

and the associated cellular response of HO-1. Within this

context, an important observation was the decrease of HO-1

immunostaining in early preneoplastic lesions (AHF) and the

marked decrease of HO-1 immunoreactivity in nodular lesions

compared to surrounding nontumoural tissue. An attenuated

response of HO-1 in nodules was also documented by employ-

ing Western blot analysis.

Furthermore, the relationship between HO-1 immuno-

reactivity and tumour differentiation was a relevant finding.

To the best of our knowledge, this is the first report on the

association between diminution of HO-1 expression and the

neoplastic transformation, and these findings give further sup-

port to the assigned protective role of HO-1 to oxidative stress

(Yang et al. 1999; Motterlini et al. 2000; Otterbein & Choi

2000; Vazquez et al. 2002).

Previous reports have demonstrated that HO-1 overexpression

is cytoprotective by attenuating the oxidative stress-mediated

pro-inflammatory reaction cascade. Within this context, HO-1

overexpression constitutes an alternative therapeutic approach to

disrupt inflammatory tissue deterioration occurring in a wide

range of diseases (Wang et al. 1998).

More promising is the observation that down modulation of

HO-1 expression correlated with malignancy progression in

chemical experimental carcinogenesis. Our data emphasize the

protective role of HO-1 in this model of chronic liver damage

and point to activation of HO-1 as a potential therapeutic tool

in liver cancer.

Acknowledgements

We are very grateful to Dr Nora Navone, Department of

Genitourinary Oncology, MD Anderson Cancer Center,

University of Texas, Houston, Texas, USA for generously

providing some reagents. We also thank Mrs B. Corvalan and

M. Binaghi for valuable technical assistance. A. Batlle and

E. Vazquez are members of the Career of Scientific Researcher

at the Argentine National Research Council (CONICET).

F. Caballero holds the post of Research Assistant at the

CONICET. R. P. Meiss is Chief and A. M. Gimenez is staff

member of Department of Pathology at the National Academy

of Medicine, Argentina. This work has been supported by grants

from the CONICET; the University of Buenos Aires, Argentina;

AICR, UK; and the Science and Technology Argentine Agency.

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Immunohistochemical analysis of heme oxygenase-1 in preneoplastic and neoplastic lesions during chemical hepatocarcinogenesiss

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