Nrf2 transactivator-independent GSTP1-1 expression in `GSTP1-1 positive' single cells inducible in female mouse liver by DEN: a preneoplastic character of possible initiated cells

Kimihiko Satoh1,6, Ken Itoh2, Masayuki Yamamoto2, Masanori Tanaka3, Makoto Hayakari4, Keizou Ookawa4, Takehiko Yamazaki4, Tsuyoshi Sato1, Shigeki Tsuchida4 and Ichiro Hatayama5

1 Department of Medical Technology, Hirosaki University, School of Health Science, Hon-Cho 66-1, Hirosaki 036-8564,
2 Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tennoudai, Tsukuba 305,
3 Second Department of Pathology, Hirosaki University, School of Medicine, Hirosaki 036-8562,
4 Second Department of Biochemistry, Hirosaki University, School of Medicine, Hirosaki 036-8562 and
5 Aomori Prefectural Institute of Health and Environment, Higashi-Tsukurimichi 1-1-1, Aomori 030-0913, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Whether single cells immunohistochemically positive for glutathione S-transferase P1-1 (GSTP1-1) induced in the female mouse liver by DEN (Hatayama et al., Carcinogenesis, 14, 537–538, 1993) are precursor initiated cells of preneoplastic foci, is of importance in chemical hepatocarcinogenesis. Nrf2 transactivates a wide variety of ARE (anti-oxidant response element)-mediated enzymes including GSTP1-1. Quantitative examination revealed that the basal expression of hepatic GSTP1-1 was 60% lower in Nrf2 gene knock-out female mice–/– than in wild type females, and that treatment with butyrated hydroxyanisole (BHA) increased by 10-fold GSTP1-1 expression in the liver of wild type female mice but not in knockout female mice–/–. Despite the lack of Nrf2, GSTP1-1-positive single cells were detected in livers of DEN-treated female–/– 3 months after treatment. Subsequent BHA feeding to the positive cell-bearing females for one more week clearly showed that the single cells were detectable with females–/– but not with females+/+,+/– due to the strong induction of GSTP1-1 in the surrounding hepatocytes. The sensitivity to DEN hepatocarcinogenesis was not significantly different among genotypes. These results demonstrate that Nrf2 is regulatory in normal hepatocytes but not in the single cells positive for GSTP1-1 inducible in the female mouse liver by DEN. The transcriptional distinction observed for the DEN-transformants is suggestive of a preneoplastic character of precursor initiated cells.

Abbreviations: BHA, 3(2)-t-butyl-4-hydroxyanisole; DEN, N,N-diethynitrosa-mine; GSTP1-1, glutathione S-transferase (GST) P1-1 species; single cells, GSTP1-1 positive single cells.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Whether single cells immunohistochemically positive for glutathione S-transferase P1-1 (GSTP1-1) induced in the rat and mouse livers by DEN (13) are precursor initiated cells of preneoplastic foci (4,5), remains to be elucidated. The Pi class GST is one of the most up-regulated enzymes in preneoplastic and neoplastic cells in various mammalian species (69). We were the first to demonstrate that injection of a single dose of diethylnitrosamine (DEN) results in the appearance of single cells and mini-foci heavily positive for GST-P (GSTP1-1) in the rat liver within 48 h of injection (1,2). Similar cells have also been detected in the liver of female mouse by us (3). Based on the phenotypic identity and other features, the DEN-transformant cells are considered to be precursors of preneoplastic foci (13,10). In order to characterize the possible initiated cells further, the mouse expression system is advantageous for several reasons. For example, the single cell populations are stably and reproducibly detectable in female livers over a period of >3 months. As a matter of course, gene knockout technology is applicable to the experimental animal system.

The GSTP1-1 expression can be tentatively classified into two types; a specific induction in preneoplastic and neoplastic cells by chemical carcinogens, and non-specific induction by non-carcinogenic agents such as anti-oxidants (7,8). In fact, GSTP1-1 is overexpressed in the normal male liver, and its concentration is ~10-fold higher than in the female liver. The Pi class species is inducible in the female up to the level seen in males following 1–2 weeks treatment with the antioxidant BHA (1214). The enzyme level also changes markedly upon castration (1517).

The 5'-upstream regions of the genes of the Pi class species of rat, mouse, and human, are fairly different to each other indicating that these marker species are similar but differently regulated under physiological conditions (1921). The transactivating factor Nrf2 (NFE-2 related factor 2), which belongs to the leucine zipper (bZip) transcription factor family, has been implicated as a key molecule involved in ARE (anti-oxidant response element)-mediated gene expression of enzymes and proteins, especially phase II drug-metabolizing enzymes including glutathione S-transferases (1517).

In an attempt to determine the characteristics of GSTP1-1-positive single cells, we used quantitative assays to examine the regulatory roles of Nrf2 with the gene-disrupted animals. Specifically, we examined the basal and inductive expression of GSTA3-3, GSTM1-1 and GSTP1-1, induction of GSTP1-1-positive single cells, and the response to the carcinogenic agent DEN in Nrf2 knockout mice.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
DEN, BHA, 2,4-dinitro-1-chlorobenzene (CDNB), and 1,2-dichloro-4-nitrobenzene (DCNB), were purchased from Wako Chemical Co. (Tokyo). All other chemicals were obtained from commercial sources and were of high reagent grade.

Animals and diets
Nrf2-disrupted male and female mice were prepared as reported previously (20) and wild type CD-1 mice, aged 5 weeks, were obtained from CLEA (Tokyo). The animals were maintained at the Institute for Animal Experiments of Hirosaki University and all animal experiments followed the Guidelines for Animal Experimentation of Hirosaki University.

Assays for glutathione-S-transferases
GSTs were assayed using CDNB, DCNB and ethacrynic acid as substrates in 100 mM Na2HPO4, pH 6.5 at 25°C, using the method described by Habig et al. (22). Qumene hydroperoxidase was assayed as described by Wendel (23).

Quantitation of GST isozymes
Antibodies against the three major forms, GSTA3-3, GSTM1-1, and GSTP1-1, were raised in rabbits and the protein content of each isozyme was quantitated by the single radial immunodiffusion method (SRID) as described previously (7,18).

DEN hepatocarcinogenesis
Nrf2-disrupted homozygous males–/– were mated with the heterozygous females+/– to obtain a number of hetero- and homozygotes (20). Newborn mice received a single intraperitoneal injection of DEN (10 mg/kg body wt) when 15 days old. The animals were fed basal diet ad libitum and killed at 3 and 6 months after treatment. The livers were quickly dissected and removed, and then fixed in ice-cold acetone.

Immunohistochemical staining
Immunohistochemical staining was carried out by the avidin–biotin–peroxidase complex (ABC) method (24).

Statistical analysis
All data were expressed as mean ± SEM. Differences between groups were examined for statistical significance using the Student's t-test.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Hepatic GST isozyme expression in Nrf2-gene disrupted mice
Basal expression of hepatic GSTs. Hepatic GST levels in knockout+/–, –/– and wild type+/+ mice were examined quantitatively by measuring enzymatic activity and protein content. GSTA3-3, GSTM1-1 and GSTP1-1 activities were assayed with specific substrates, cumene hydroperoxide, DCNB, and ethacrynic acid, respectively. In wild type animals, total CDNB activity in males was 3.9-fold greater than in females (Table IGo). This was mostly due to differences in GSTP1-1 activity. As shown in Figure 1Go, GSTP1-1 protein content in the male liver (5.1 ± 0.8 mg/g) was significantly higher than the other two forms. In contrast, the amount of GSTP1-1 (0.5 ± 0.1 mg/g) in females was significantly lower than those of the other two forms. The amounts of the two GSTs were relatively independent of sex.


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Table I. Hepatic GST activities in male and females of Nrf2-gene disrupted mice
 


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Fig. 1. Isozyme composition of three major GSTs in the liver of wild type male and female mice. {square}, GSTA3-3; , GSTM1-1; {blacksquare}, GSTP1-1.

 
Basal levels of the three GSTs in the Nrf2 gene-disrupted animal livers were generally lower than those in the wild type. Total GST activities in knockout males–/– and females–/– were 34.7 and 48.8% those of the males+/+ and females+/+, respectively. Furthermore, GSTA3-3 activities in knockout animals were 64.5 and 67.2% of those in the wild type males and females, respectively. Similarly, GSTP1-1 activity in the livers of knockout mice–/– was ~60% of that in the wild type for either sex. In contrast, GSTM1-1 activity and protein content in males–/– were as low as 9.2% and 7.9% of those in wild type males+/+, respectively. Further comparison showed that the enzymatic activities in heterozygote animals+/– were intermediate between those of homozygotes+/+ and homozygotes–/–, where GSTA3-3 and GSTP1-1 activities in males+/– were similar to those in wild types+/+ (Figure 2Go). Thus, the effects of Nrf2 gene disruption on the GST isozymes were in the order of GSTM1-1 >> GSTP1-1 > GSTA3-3 in males, and GSTM1-1 > GSTP1-1 > GSTA3-3 in females.



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Fig. 2. Isozyme composition of three major GSTs in the liver of wild type and Nrf2-gene disrupted mice. (A) male mice; (B) female mice. {square}, GSTA3-3; , GSTM1-1; {blacksquare}, GSTP1-1. *9.2%.

 
Effect of gene disruption on induction of hepatic GSTs in female mice by BHA. We next determined quantitatively the effects of Nrf2 gene disruption on the induction of hepatic GSTs in female knockout+/+, +/–, –/– and wild type mice treated with BHA for 12 days (1214). As a measure of induction, we determined the percent increment in hepatic GSTs in BHA-treated animals relative to that in control mice (Figure 3A–EGo). Induction of total CDNB activity in knockout homozygotes–/– (250%) was significantly lower than in control wild+/+ (850%) and heterozygotes+/– (950%) (Figure 3AGo). Furthermore, the expression levels of GSTM1-1 (Figure 3CGo) and GSTP1-1 (Figure 3D and EGo) in BHA-treated knockout homozygotes–/–, but not GSTA3-3 expression (Figure 3BGo), were less than those in control wild+/+ and knockout heterozygotes+/–. Similarly, while the levels of GSTP1-1 activity and protein increased by 340% and 530%, respectively, in BHA-treated wild type females+/+ and as high as 810% and 1240%, respectively, in knockout heterozygote females, those levels were as low as 230% and 280%, respectively, in knockout animals–/– (Figure 3D and EGo). These results indicate that the expression levels of GSTs in the livers of the three genotypes were in order of (+/–) >= (+/+) >> (–/–).



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Fig. 3. Induction of three major GSTs in the liver of wild-type and Nrf2 gene knockout+/–,–/– female mice following administration of BHA. Open and shaded bars represent the amounts of hepatic GSTs in animals fed basal diet and 0.75% BHA-containing diet for 12 days, respectively. GST activities were assayed as described in Table IGo. GSTP1-1 protein was quantitated by the SRID method. Values in parentheses denote induction (%).

 
DEN hepatocarcinogenesis in Nrf2-gene disrupted mice
Sensitivity to DEN in knockout females. The carcinogenic sensitivities of the three genotypic females (+/+, +/–, –/–) were examined according to the DEN hepatocarcinogenesis protocol (3). Multiple GSTP1-1 positive single cells were noted in the livers 3 months after injection of DEN, irrespective of genotype. However, mini-foci were not detected in these animals at that stage (Table IIGo). There was no statistical correlation among the single cell populations induced in the liver of the three genotypes. After a longer period of 6 months, single cells were also abundantly induced in the livers of all animals, together with some mini-foci. Although the number of single cells in the liver of knockout mice–/– was apparently greater than that in wild type mice, the difference was weak (P < 0.05), and the difference with that in knockout heterozygotes was not statistically significant. Thus, the sensitivities of homozygote and heterozygote knockout female mice to the hepatocarcinogenic effect of DEN were not different from that of wild type animals.


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Table II. Single cells and mini-foci positive for GSTP1-1 induced in the liver of wild type and knockout female mice by DEN
 
Induction of GSTP1-1-positive single cells in knockout female–/–, +/– livers and effect of subsequent BHA treatment. GSTP1-1-positive single cells were noted in the livers of Nrf2 gene disrupted female mice as well as in wild type females. In order to confirm the induction of minor cells, 0.75% BHA containing diet was subsequently fed to the single cell-bearing mice for one more week. As seen in the immunohistochemical staining patterns of control animal livers, hepatocytes strongly positive for GSTP1-1 were noted throughout the whole liver in control female mice+/+ untreated and treated with BHA (Figure 4d and eGo), but not in the livers of knockout females–/– (Figure 4fGo). In DEN-treated females, GSTP1-1-positive single cells were detected in the livers of wild type+/+ as well as in the knockout animals–/– (Figure 4g and hGo). However, while single cells were not detected in the wild type after treatment with BHA (Figure 4j and kGo), minor cell populations were detected in knockout mice–/– even after treatment with the anti-oxidant (Figure 4lGo). Immunohistochemical patterns of heterozygotes+/– were very similar to or identical with those of wild types as summarized in Figure 5Go. These results clearly indicate that Nrf2 plays a major role in the basal and induced expression of GST in normal hepatocytes but not in the expression of the marker species in `GSTP1-1 positive' single cells.



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Fig. 4. Immunohistochemical GSTP1-1 staining patterns of livers of wild type and knockout female mice. BHA, 0.75%BHA feeding for 1 week; DEN, i.p. administration of DEN followed by feeding basal diet for 3 months; DEN/BHA, DEN treatment followed by BHA feeding. See also Figure 5Go.

 


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Fig. 5. Schematic diagram for the induction of GSTP1-1-positive single cells in the livers of wild type and Nrf2 gene knockout–/– female mice. Arrowheads denote time of examination. Open and solid circles represent GSTP1-1 negative and positive liver patterns, respectively, and small dots represent GSTP1-1-positive single cells.

 

    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
To further characterize the potentially preneoplastic cells, i.e. DEN-induced GSTP1-1-positive single cells in the livers of female mice (3), we quantitatively examined in the present study two aspects of the regulatory roles of Nrf2 transactivator in the expression of Pi class GST using Nrf2 gene disrupted mice. The first was the effect of gene disruption on the basal and induced expression of three hepatic GST isozymes. The second was the induction of GSTP1-1-positive single cells in the liver of knockout females–/– as well as the hepatocarcinogenic effect of DEN in knockout mice.

Effect of Nrf2 gene disruption on non-carcinogenic induction of hepatic GSTP1-1
Quantitative analysis showed that the effect of Nrf2 gene disruption on the basal expression of GSTs was most prominent on the basal expression of GSTM1-1 in the liver of male mice; the basal activity and protein content were <10% of the wild type males. Since Mu class GSTs are not tumor specific species and distinct ARE sequences are not detectable in the 5'-upstream regions of the GSTM1-1 genes (29), the results indicate that Nrf2 regulates tumor-specific isozyme species as well as non-tumor specific (or tissue-specific) isozymes. Apart from the significant effect on GSTM1-1 expression in males, Nrf2 transactivator accounted for ~40% of the basal expression of GSTP1-1 in the liver of either sex. The effect of gene disruption was much more marked on the induction of expression of hepatic GSTP1-1 than on the basal expression, as the expression of the isozyme was significantly low in knockout females–/–, in agreement with the findings of Hayes et al. (25) and Ramos-Gomez et al. (26). Combined together, these results indicate that the role of Nrf2 in the induction of expression of GSTP1-1 is more important than the basal/constitutive expression of GSTP1-1 species in normal hepatocytes.

Effect of Nrf2 on carcinogenic induction of GSTP1-1
That Nrf2 transcription factor is a major positive regulator of GSTP1-1 expression implies that homozygous and heterozygous knockout mice might be more sensitive to carcinogens than wild type+/+, and that GSTP1-1-positive single cells and mini-foci might be undetectable in the female–/– livers in the early stages of DEN-induced hepatocarcinogenesis. However, no significant differences were observed in DEN carcinogenicity between homozygous and heterozygous female knockout mice, compared with those of wild type females. In addition, distinct GSTP1-1 positive single cell populations were induced in livers of homozygous and heterozygous female knockout mice as in those of wild type. The Nrf2-independent expression of the GSTP1-1-positive single cells was further confirmed by the subsequent treatment with BHA to single cell-bearing animals. As shown in the schematic diagram in Figure 5Go, no single cells were found in anti-oxidant treated heterozygous female knockout mice and wild type mice due to the strong induction of GSTP1-1 in the surrounding hepatocytes. In contrast, minor cell populations were detected in the livers of BHA-treated homozygous female knockout mice, in whom GSTP1-1 was hardly induced in normal hepatocytes. These results indicate that Nrf2 was involved in the regulation of the basal and induced expression of GSTP1-1 in normal hepatocytes but was not involved in the induction of `GSTP1-1 positive' single cells, foci and nodules (adenomas) in mice treated with DEN. Single cells could, therefore, be differentiated from normal hepatocytes based on the genetic regulation of GSTP1-1 expression by the Nrf2 transcription factor. The transcriptional distinction could be another characteristic feature of the single cell populations, which does not contradict the biochemical and pathological data so far obtained (15), suggesting that these cells are precursors of preneoplastic foci and nodules (adenomas).

Previous studies showed that the Nrf2 transcription factor synergistically heterodimerizes with Small maf (MafG and MafK) or c-Jun on binding to ARE, as positive and negative regulators of the expression of various phase II detoxicating enzymes as well as other proteins (2628). The present findings suggest that the process of induction of GSTP1-1 in the single cells (possible initiated cells) is not, however, related to Nrf2/Small maf/ARE-mediated ones, and is rather different from that in normal hepatocytes.

The above results suggest that Nrf2 protein could be one of the major transcription factors for GSTP1-1 expression, in addition to c-JUN and c-FOS (1517,30). However, it is rather strange that these transactivators do not play major roles in the carcinogenic induction of GSTP1-1 (31). No significant oncogenic changes have so far been detected in preneoplastic cell populations in experimental animals except for some occasional activation of c-myc or H-ras (3234). In this regard, Gijssel et al. (35) have also noted the lack of p53 protein expression in GSTP1-1 (GST-P)-positive preneoplastic foci and nodules in rats.

The present results suggest that GSTP1-1-positive single cells are formed to be preneoplastic cells, being transcriptionally distinct from normal hepatocytes. Are the single cell populations initiated cells? What is the initial oncogenic change(s), if any, in these cells? These are important questions that need to be resolved to clarify the molecular and cellular events involved in the initiation stage of chemical hepatocarcinogenesis in experimental animals as pointed out earlier by Farber and Pitot, as well as by Prehn (4,5,36,37).


    Notes
 
6 To whom correspondence should be addressed Email: kisatoh{at}cc.hirosaki-u.ac.jp Back


    Acknowledgments
 
This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science, Sports and Culture of Japan, Grants-in-Aid from the Intelligent Cosmos Academic Foundation (Sendai), and funds from NOF Corporation (Tokyo) and CHUGAI Pharmaceuticals (Tokyo).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 31, 2001; revised November 12, 2001; accepted November 14, 2001.