Hepatitis B virus-induced liver injury and altered expression of carcinogen metabolising enzymes: the role of the HBx protein

Toxicology Letters 102]103 Ž1998. 595]601

Hepatitis B virus-induced liver injury and altered expression of carcinogen metabolising enzymes: the role of the HBx protein Pascale Chomarat a , Jerry M. Rice b , Betty L. Slagle c , Christopher P. Wildd,U a

Unit of En¨ ironmental Carcinogenesis, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon Cedex 08, France b Unit of Carcinogen Identification and E¨ aluation, International Agency for Research on Cancer, Lyon, France c Di¨ ision of Molecular Virology, Baylor College of Medicine, Houston, TX 77030, USA d Molecular Epidemiology Unit, School of Medicine, Algernon Firth Building, Uni¨ ersity of Leeds, Leeds LS2 9JT, UK Accepted 11 September 1998

Abstract Hepatitis B virus ŽHBV. and aflatoxins are major risk factors for hepatocellular carcinoma ŽHCC. exhibiting a synergistic interaction in the development of this disease. The molecular mechanisms of this interaction remain to be elucidated but an altered carcinogen metabolism in the presence of hepatitis-induced liver injury is one hypothesis. The availability of biomarkers of aflatoxin exposure and metabolism permits this hypothesis to be examined in human populations whilst animal models, such as HBV transgenic mice permit parallel studies in an experimental setting. The hepatitis B virus X protein ŽHBx. is suspected to play a role in the hepatocarcinogenic process by virtue of its capacity to transactivate oncogenes and several other cellular genes via cis-acting elements. In previous studies in HBV transgenic mice expressing the HB surface antigen and X genes we observed a marked induction of specific cytochrome P450s ŽCYP. ŽKirby et al., 1994a.. In the current study we investigated the status of CYP, glutathione S-transferases ŽGST. and antioxidant enzymes in mice carrying only the X gene under the control of the a-1 antitrypsin regulatory elements ŽATX mice.. Livers of ATX mice showed no major pathological alterations compared to age-matched non-transgenic control mice. Immunohistochemical staining for CYP1A, 2A5 and GST expression and determination of related enzymatic activities Ž7-ethoxyresorufin O-deethylation, 7-methoxyresorufin O-deethylation, coumarin 7-hydroxylation and GST activities. revealed no differences between control and ATX mice. In addition, no differences in antioxidant enzymes were observed. Overall, these results support the conclusion that HBx expression alone is insufficient to induce transactivation of CYP and GST genes or to alter the antioxidant

U

Corresponding author. Tel.: q44 0113 2336602; fax: q44 0113 2336603; e-mail: [email protected] Abbre¨ iations: CYP, cytochrome P450; HBV, hepatitis B virus; HBx, hepatitis B virus X protein; HCC, hepatocellular carcinoma; NFk B, nuclear factor kappa B; 7-OH-C, coumarin 7-hydroxylation; GST, glutathione S-transferase; GSH, glutathione 0378-4274r98r$ - see front matter Q 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274Ž98.00254-9

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system and that the induction in other HBV models is a result of inflammatory injury in the liver, a feature absent in ATX mice. These data are compared to biomarker studies of enzyme activities in aflatoxin-exposed human populations with and without HBV infection. Q 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cytochrome P450; GST; X protein; Liver cancer

1. Introduction Biomarkers can improve exposure assessment, disease diagnosis and identify individuals susceptible to a given exposure but in addition are valuable tools with which to investigate causal mechanisms in disease development. One advantage is that the same biomarkers can be used in both experimental and field studies providing a link between these approaches. An example where this has been fruitful is in studying the role of aflatoxins and hepatitis B virus ŽHBV. in the development of hepatocellular carcinoma ŽHCC. ŽMontesano et al., 1997.. These major risk factors for HCC exhibit a synergistic interaction in the development of this disease. The molecular mechanisms of this interaction remain to be elucidated but an altered carcinogen metabolism in the presence of hepatitis-induced liver injury is one hypothesis ŽWild et al., 1993a. and this hypothesis can be tested in experimental models, e.g. HBV transgenic mice, or in human populations using phenotyping and genotyping for aflatoxin metabolising enzymes ŽEaton and Gallagher, 1994.. The X protein ŽHBx. encoded by HBV has been suggested to play a role in hepatocarcinogenesis ŽFeitelson and Duan, 1997.. HBx does not bind directly to DNA, but modulates the expression of proto-oncogenes and other cellular genes, by physical interaction with nuclear transcriptional factors ŽFeitelson and Duan, 1997.. Transgenic mice transfected with the X gene Žnt 7071856. containing viral regulatory elements develop HCC ŽKim et al., 1991. whereas those carrying the X gene Žnt 1376]1840. under the control of the human a-1 antitrypsin regulatory elements ŽATX mice. do not develop liver pathology ŽLee et al., 1990., but show an increased incidence of HCC following treatment with diethylnitrosamine ŽSlagle et al., 1996.. HBV transgenic mice that

overexpress HBsAg and carry the X gene, show characteristic features of liver cell injury and chronic hepatitis leading to HCC development ŽChisari et al., 1989.. In these mice cellular enzymes involved in carcinogen metabolism are induced in association with liver injury ŽChemin et al., 1996; Kirby et al., 1994a. although it is possible that HBx contributes to the effects on carcinogen metabolising enzymes via gene transactivation. We therefore examined this question in ATX mice where no inflammation or regenerative hyperplasia occurs. The data are discussed in relation to biomarker studies designed to address the same question of altered aflatoxin metabolism in people chronically infected with HBV. 2. Materials and methods 2.1. Chemicals Chemicals were from Boehringer Mannheim ŽMannheim, Germany., Sigma ŽSt Louis, MO., Molecular Probes Inc. ŽEugene, OR. or Fluka Chemie ŽBuchs, Switzerland.. 2.2. Animals and histopathology HBx transgenic mice ŽATX mice. were generated by breeding ŽICR= B6C3. male mice carrying the ATX transgene with non-transgenic ICR female mice ŽLee et al., 1990.. Transgenic and non-transgenic F1 littermate males, 12 days and 3 months of age, were used. Sections of the major liver lobes were fixed in formalin for histopathological examination Žhematoxylin and eosin staining.. Liver portions of a separate series were also frozen in liquid nitrogen and maintained at y808C for biochemical analyses. HBx protein was detected in the livers of ATX mice as described using a rabbit anti-X antiserum ŽSlagle et al., 1996..

P. Chomarat et al. r Toxicology Letters 102]103 (1998) 595]601

2.3. Immunohistochemistry

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Immunohistochemistry was performed essentially as described previously ŽKirby et al., 1994a; Chemin et al., 1996. with the following antibody dilutions: anti-CYP1A, 1:1000, anti-CYP2A5, 1:750, anti-GSTp , 1:2500 ŽDr Tsuda, Tokyo, Japan., and anti-GSTYaYa Žclass a ., 1:600. Negative controls comprised sections processed in the absence of primary antibody.

GSH-peroxidase activity were measured as previously described ŽKirby et al., 1994a; Chemin et al., 1996; Chomarat et al., 1997.. Each sample was tested on at least two occasions in duplicate or triplicate. Comparison of enzymatic activities in the different groups was made by Student’s t-test or the alternative t-test depending on similarity or otherwise of standard deviations. All tests were two-sided; P- 0.05 was considered the level of significance.

2.4. Biochemical assays

3. Results

Microsomes were prepared as described previously ŽKirby et al., 1994a; Chemin et al., 1996.. Total CYP content, O-dealkylations of 7ethoxyresorufin ŽEROD. and 7-methoxyresorufin ŽMROD., coumarin 7-hydroxylase Ž7-OH-C., GST activities toward CDNB Žtotal GST. or DCNB ŽGSTm ., catalase activity, reduced GSH and

3.1. Hepatic histopathology As previously ŽLee et al., 1990; Slagle et al., 1996., livers of ATX mice aged 12 days and 3 months showed minimal liver alterations, compared to control mice. For 12-day-old mice, some foci of extramedullary hematopoiesis could be

Fig. 1. Immunohistochemical staining of CYP1A in control and ATX livers. Three-month control ŽA. and ATX ŽC. liver sections are stained with hematoxylinreosin Ž=40.. CYP1A staining was performed in parallel on the same control ŽB. and ATX ŽD. liver sections Žmagnification =40., showing symmetrical centrilobular staining. Žsee Materials and Methods for details..

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apparent decrease in HBx in older animals is consistent with previous reports ŽLee et al., 1990; Slagle et al., 1996.. 3.2. Hepatic cytochrome P-450 status

Fig. 2. Detection of HBx in the livers of ATX mice. Detergent extracts of control Žnontransgenic . and ATX mice were prepared from 12 day- and 3 month-old mice Žsee bottom of figure.. Aliquots of extracts were analysed as described in Materials and Methods. The antibody used for immunoprecipitation is designated at the top of each lane as either ‘y’ Žnegative control antibody. or ‘q’ Žanti HBx antibody.. Migration of a 14.1-kD molecular weight marker is shown at the left, and the arrow labelled ‘X’ denotes migration of the HBx protein. The HBx protein is detected only in liver tissue of ATX mice; the apparent decrease in HBx levels in the livers of 3 month-old mice is consistent with previous reports ŽLee et al., 1990..

observed among mostly normal hepatocytes. At 3 months, no marked variation in liver histology was noted between control and ATX mice with no evidence of widespread inflammation and no massive hepatocellular necrosis ŽFig. 1A,C.. HBx was detected in ATX livers from 12-day and 3month-old mice ŽFig. 2, lanes four and eight. but was not present in age-matched, non-transgenic control animals ŽFig. 2, lanes two and six.. The

There was a significant increase Ž) 10-fold. in total CYP in 3-month-old compared to 12-day-old mice ŽTable 1. but total CYP contents were similar in control and ATX mice at both ages studied. As CYP regulation is specific to different isoforms, CYP1A and 2A5 isoforms were studied as these have previously been shown to be modified in HBV transgenic mice with associated liver injury ŽKirby et al., 1994a; Chemin et al., 1996.. Hepatic expression of CYP1A was compared in control and ATX mice aged 12 days and 3 months by immunohistochemical ŽIHC. analysis. In 12 day-old control mice, CYP1A staining was constitutively expressed in centrilobular areas with more intense staining around central veins Žnot shown.. At the same age, livers of ATX mice demonstrated a similar staining pattern of CYP1A, although two of five showed in addition some foci of positive hepatocytes in more periportal regions Žnot shown.. In 3-month-old mice, no significant variation in the centrilobular distribution was noted between control and ATX mice ŽFig. 1B,D.. As shown in Table 1, no significant variation in these MROD or EROD activities was observed between age-matched control or ATX mice confirming the lack of altered CYP1A expression seen at the single cell level by IHC. IHC staining for CYP2A5 in livers of both control and ATX

Table 1 Hepatic enzymes in microsomes of control and ATX mice Time

Groupa

Total CYPb

12 days

Control ATX

0.24" 0.09 0.25" 0.42

3 months

Control ATX

3.48" 0.17 3.09" 0.06

a

ERODc

MRODc

7-OH-Cc

66 " 40 51 " 48

113 " 78 72 " 50

22 " 6 16 " 0

225 " 73 156 " 42

210 " 46 218 " 53

50 " 17 62 " 31

Pathology was evaluated for individual livers and scored on the basis of examination of liver sections and severity of necroinflammatory infiltrates, bile duct hyperplasia, hepatocellular alterations and neoplasia. No pathology was seen in any of the mice. b Expressed in nmolrmg of protein as mean "S.D. of five mice. c Expressed in pmolrminrmg of protein as mean " S.D. of five mice.

P. Chomarat et al. r Toxicology Letters 102]103 (1998) 595]601

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mice showed faint staining of cells around the central veins Žnot shown., with no difference in mice in the two groups at either 12 days or 3 months of age. Consistent with the IHC data, no significant change in microsomal metabolism of coumarin, a CYP2A5 substrate, was observed between control and ATX age-matched mice ŽTable 1.. As with the EROD and MROD data, enzymatic activities were higher in the older group of mice. 3.3. Glutathione S-transferase status IHC staining revealed that GSTYaYa and GSTp were expressed predominantly in centrilobular and periportal hepatocytes, respectively with no difference between control and ATX mice of either 12 days or 3 months of age ŽFig. 3.. Similarly there were no differences in CDNB Žtotal GST. or DCNB ŽGSTm activity. metabolism in ATX mice compared to control mice ŽTable 2.. 3.4. Antioxidant capacity As shown in Table 2, a slight increase Žapprox. 30%. in catalase activity was observed in ATX mice at 12 days and 3 months of age compared to control mice, but this was not statistically significant. There were no significant differences in GSH content or GSH peroxidase activity between age-matched control and ATX mice.

Fig. 3. Immunohistochemical staining of GSTp in control and ATX livers. Three-month control ŽA. and ATX ŽB. liver sections were stained with an antibody to GSTp Žas described in Materials and Methods ŽMagnification =40..

4. Discussion Studies from several animal models of liver cancer, including HBV transgenic mice ŽKirby et

Table 2 Hepatic enzymes in cytosols of control and ATX mice Time

Groupa

12 days

Control ATX

358 " 111 450 " 25

3 months

Control ATX

1352 " 450 1412 " 492

a

CDNB activityb

DCNB activityb

Catalase activityc

GSH amountd

GSH peroxidaseb

8.4" 4.7 9.8" 0.9

218 " 77 286 " 23

6.1" 2.2 8.6" 1.4

346 " 91 366 " 124

26.2" 8.5 44.4" 16.0

277 " 68 352 " 92

3.6" 0.8 4.2" 1.0

360 " 133 403 " 244

Pathology was evaluated for individual livers and scored on the basis of examination of liver sections and severity of necroinflammatory infiltrates, bile duct hyperplasia, hepatocellular alterations and neoplasia. No pathology was seen in any of the mice. b Expressed in nmolrminrmg of protein as mean "S.D. of 5 mice. c Expressed in m molrminrmg of protein as mean "S.D. of 5 mice. d Expressed in m grmg of protein as mean "S.D. of 5 mice.

600

P. Chomarat et al. r Toxicology Letters 102]103 (1998) 595]601

al., 1994a; Chemin et al., 1996., Helicobacter hepaticus infected mice ŽChomarat et al., 1997. and Opisthorchis ¨ i¨ errini infected hamsters ŽKirby et al., 1994b., have revealed alterations in the expression of CYP 1A, 2A5 and some GST isoenzymes. In contrast, although HBx is overexpressed in livers of ATX mice, no significant alteration in these enzymes or antioxidant levels was observed between control and ATX mice. In the ATX mouse model, the X protein was localized to both the cytoplasm and nucleus of hepatocytes surrounding the central vein ŽLee et al., 1990; Slagle et al., 1996. and was still expressed at the latest timepoint used in our study Ž3 months. ŽSlagle et al., 1996.. Thus, the lack of effect on CYP, GST or antioxidant levels was not linked to a limited localization of HBx to the nuclear compartment, something which might prevent some of its biological functions ŽDoria et al., 1995.. The younger ATX mice of 12 days of age were used because a synergy in HCC induction observed with diethylnitrosamine Žmetabolised by mouse CYP 2A5. ŽSlagle et al., 1996. occurred when the carcinogen was administered at this age. It therefore appears that expression of the transactivator HBx is not sufficient to induce the CYP and GST alterations observed in other models. This suggests that the induction is a result of liver inflammatory injury, a feature lacking in ATX mice but shared by the animal models in which CYP, GST andror antioxidant enzyme expression is altered ŽKirby et al., 1994a; Chemin et al., 1996; Chomarat et al., 1997.. Consistent with this idea, numerous in vitro studies have also demonstrated the regulatory role of many cytokines ŽIL-1, IL-4, IL-6., on CYP and GST expression ŽFukuda et al., 1992; Langouet et al., 1995.. Many studies have reported a role of reactive oxygen species in the initiation and progression of carcinogenesis. In this regard, free radical scavengers ŽGSH. and antioxidant enzymes are frequently found downregulated in tumour cells. In the ATX mouse model, no alteration in antioxidant enzymes was noted, indicating a lack of induction of oxidative stress even when HBx is overexpressed. Again, these results contrast with the decreased hepatic antioxidant capacity in

HBV transgenic mice that overexpress HBsAg ŽHagen et al., 1994. and in early stages of infection with H. hepaticus ŽChomarat et al., 1997.. Aflatoxins are metabolised in the human liver by cytochrome P450s ŽCYPs., glutathione Stransferases ŽGST. and possibly epoxide hydrolase ŽEaton and Gallagher, 1994.. Given the possibility of phenotyping individuals for CYP activities some attempts have been made to assess the impact of HBV infection on these activities in small numbers of subjects ŽHorsmans et al., 1995.. A less direct approach to assessing the impact of HBV infection on aflatoxin metabolism using biomarkers has been to examine the level of binding of aflatoxin to DNA or proteins with respect to viral status assuming that this will reflect interindividual differences in metabolism ŽAllen et al., 1992; Wild et al., 1993b.. Although further work is required to draw conclusions from these studies they illustrate that the availability of such biomarkers permits this type of question to be addressed in the field. The interchange between data obtained in experimental models and field studies is based on the common endpoints used in both approaches and provides a foundation for understanding causal mechanisms in the disease process. Acknowledgements Pascale Chomarat was supported by the French Foundation ‘Ligue Contre le Cancer’ and by a Special Training Award from the International Agency for Research on Cancer. This work was supported by grants from the National Institute of Environmental Health Sciences, grant ES06052 Žawarded to CPW. and the National Cancer Institute, grant CA54557 Žawarded to BLS.. The authors thank M. Laval and N. Lyandrat for excellent technical assistance and G. Mollon for help with photography. The provision of antibodies to CYP2A5 by Dr M. Lang and to GST YaYa by Dr M. Hayes is gratefully acknowledged. References Allen, S.J., Wild, C.P., Wheeler, J.G., et al., 1992. Aflatoxin exposure malaria and hepatitis B infection in rural Gambian children. Trans. R. Soc. Trop. Med. Hyg. 86, 426]430.

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