Potential effect of sodium nitrite on the expression of nuclear proto-oncogenes during 2-acetyl aminofluorene-induced hepatocarcinogenesis in rats

Chemico-Biological Interactions 108 (1997) 1 – 18

Potential effect of sodium nitrite on the expression of nuclear proto-oncogenes during 2-acetyl aminofluorene-induced hepatocarcinogenesis in rats Jeng-Dong Hsu a, Ching-Lin Hsu b, Fen-Pi Chou b, Pao-Hsiang Wen b, Chau-Jong Wang b,* a

Department of Pathology, Chung Shan Medical and Dental College, Hospital, No. 110, Section 1, Chien Kauo N. Road, Taichung 402, Taiwan, People’s Republic of China b Institute of Biochemistry, Chung Shan Medical and Dental College, No. 110, Section 1, Chien Kauo N. Road, Taichung 402, Taiwan, People’s Republic of China

Received 6 May 1997; received in revised form 27 September 1997; accepted 27 September 1997

Abstract 2-acetyl aminofluorene (AAF) reacts in acidic conditions with nitrous fume yielding N-nitroso-AAF (N-NO-AAF), as previously described, that exerts more toxic and mutagenic effects than its parental compound. In this study, the effect of sodium nitrite (NaNO2) on the tumorigenicity of AAF in rats fed with AAF and NaNO2 was observed. Wistar rats were divided into five groups: group I served as control; group II were treated with NaNO2 (0.3%); group III was given 0.02% AAF alone; groups IV and V received both AAF and NaNO2 (0.2 and 0.3% respectively) in their diet for 12 weeks. At the end of the experiment, all rats in groups III, IV and V developed early stage phenomena of hepatocellular carcinoma, including hepatomegaly with variable-sized foci and neoplastic nodules. Severe damage was observed in the rats treated with AAF and NaNO2. Feeding of AAF (0.02%) for 3 months elevated the levels of c-Fos, c-Jun and c-Myc proteins in the rat livers. The AAF-induced c-Jun, c-Fos and c-Myc expressions were significantly magnified (PB 0.001) by NaNO2. These data confirmed that the strengthening of AAF-induced hepatocarcinogenesis

Abbre6iations: AAF, 2-acetyl aminofluorene; DAB, diaminobenizidine; N-NO-AAF, N-nitroso-2acetyl aminofluorene. * Corresponding author. Fax: + 886 4 3890964. 0009-2797/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 0 9 - 2 7 9 7 ( 9 7 ) 0 0 0 8 9 - 6

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by NaNO2 should be associated with its enhancing effect on the AAF-induced increases in the expressions of c-Jun, c-Fos and c-Myc. © 1997 Elsevier Science Ireland Ltd. Keywords: 2-Acetyl aminofluorene; Sodium nitrite; Hepatocarcinogenesis; c-Fos; c-Jun; c-Myc

1. Introduction There are many known carcinogenic N-nitroso compounds that have been detected in a number of environmental sources including food [1,2]. It has been proposed that such carcinogens may be formed endogenously in digestive fluids from the reactions between nitrite in food and water that may be a risky factor for certain cancers, notably stomach and esophageal cancers [3,4]. In fact, the in vivo formation of nitrosamine has been demonstrated [5,6] in animal models and the combination of dietary nitrite administration with many carcinogens appeared to enhance the risk of neoplasia formation [7–10]. On the other hand, several N-nitroso-compounds have been considered to play a causative role in carcinogenesis in various species [11,12]. 2-Acetyl aminofluorene (AAF) is one of the most intensively used model compounds in studying the metabolism and carcinogenesis of arylamides and amines [13,14]. The metabolic activation of this compound includes various biochemical reactions such as N-hydroxylation, sulfate transfer, N-O-acetyl transfer, deacetylation, or 1-electron oxidation step to form the ultimate reactive intermediate [15– 19]. The relatively unstable N-nitrosoamides are powerful alkylating agents in in vivo and in vitro systems and they generate the presumed carbonium ion by a spontaneous pH-dependent hydrolysis, which in the case of alkylnitrosourethanes and alkylnitrosoguindine may be thiol catalyzed in vivo [20,21]. As expected, N-NO-AAF can be synthesized in good yield and reacts readily with various nucleophiles including amino acids, nucleosides and DNA. Therefore, N-NO-AAF is a strong mutagen and tetratogen [22] and in all likelihood, it could be produced in normal physiological conditions. Several lines of evidence indicate that some of the known oncogenes existing in rat liver tissue express under a variety of physiological conditions, including preand postnatal development [23,24], regeneration [25,26] and neoplasia. Among them, c-myc, c-fos and c-jun oncogenes had been detected in the chemically induced rodent hepatocarcinomas [27 – 33]. It appears that the expression of some oncogenes may be related to the neoplastic transformation process and their expression products may be used as tumor markers. To further explore the effect of NaNO2 on AAF hepatocarcinogenicity, we treated rats with AAF by feeding and examining the effect of NaNO2 on AAF-induced hepatic neoplasm and the increases in c-Jun, c-Fos and c-Myc expressions in rat liver.

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2. Material and methods

2.1. Animal treatment Male Wistar rats (120 – 150 g body weight) were purchased from National Taiwan University Hospital Animal Center. The animals were housed, three per cage in an environmentally controlled animal room. Experimental rats were divided into five feeding regimens, with six rats in each group. Rats in group I were fed only the basal diet (Purina Lab Chow), while groups II and III received 0.3% NaNO2 and 0.02% AAF (Sigma, St. Louis, MO), respectively and groups IV and V received NaNO2 plus AAF in diet. Each rat received about 20 – 25 g of diet per day for 12 weeks; food and water were provided ad libitum.

2.2. Autopsy and histology Immediately after death, a complete necropsy was performed and liver was examined for hepatic neoplasia formation. The liver was washed with physiologic saline and inspected for gross lesions. Tissues were fixed in 10% buffered formalin, processed for histological examination according to the conventional methods. Step sections (five section per block of tissue) were prepared from the liver tissue and stained with hematoxylin and eosin. The morphology, number and location of any lesion observation were registered.

2.3. Immunocytochemical assessment of proto-oncogenes expressions Determination of potential effect of NaNO2 on the AAF-induced proto-oncogenes expressions was performed following the procedures described by Lu et al. [34] with some modification. After excised from the animals, the livers were fixed in 10% buffered-formalin solution for 18–24 h, dehydrated, embedded in paraffin and cut into sections of 5 mm thickness. To perform the immunocytochemistry, the sections were deparaffinized in xylene, rehydrated in 0.05 M Tris buffer, pH 7.6, for 10 min and boiled in 0.01 M citrate buffer, pH 6.0, for 5 min. The sections were then removed and allowed to cool at room temperature for 20 min and rinsed two times with TBS for total of 30 min. Endogenous peroxidase activity was blocked by a 15 min incubation in 3% hydrogen peroxide. To increase antigenic exposure, tissue sections were incubated in 0.1% Triton X-100 for 45 min at room temperature. Following this, the samples were incubated with diluted primary antibodies, rabbit anti-c-Fos and c-Jun polyclonal antibodies (Oncogene Science) or monoclonal anti-c-Myc antibody (Santa Cruz Biotechnology), for 45 min at room temperature. After rinsing two times with TBS for a total of 20 min, the bound primary antibodies were detected by sequential incubation with biotinylated secondary antibody (Biotinylated anti-rabbit or anti-mouse immunoglobins, LSAB kit from DAKO) for 30 min, streptavidin peroxidase (LSAB kit from DAKO) for 15 min and DAB for 5 – 10 min, at room temperature with two rinses of TBS in

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between. The sections were then washed with distilled water and counterstained with Mayer’s hematoxylin. After dehydration and mounting, the expression of c-Jun, c-Fos and c-Myc in intact mouse liver was assessed by microscopic examination of the immunoperoxidase staining. The negative control for each experiment was a DAKO Antibody Diluent with background reductional compounds, which is intended for the use as a diluent in the preparation of primary antibodies and negative control reagents. The positively staining area, brown color, was determined by Leica Q500 MC image processing and analysis system against the negatively staining region shown relatively as a blue color.

3. Results The effect of NaNO2 on the hepatocarcinogenesis of AAF administration was studied. The results showed that during the 3 months study, the relative liver weight (liver/body weight) of the rats who received AAF (0.02%) by feeding in their diet was greater than that of the control group (Fig. 1). Groups IV and V with combined NaNO2 (0.2 and 0.3%) and AAF in their diet showed significant increases (P B0.001) in the AAF-induced hepatomegaly (Fig. 1).

Fig. 1. Effect of NaNO2 on the relative liver weight of AAF-treated rats. Rats received AAF with or without NaNO2 in diet for 12 weeks. Relative liver weight = (liver weight/body weight)× 100; *PB 0.001, compared with control; **PB 0.001 compared with AAF-treated groups.

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Fig. 2. Histopathological examination of the effect of NaNO2 on the liver of AAF-treated rat. (A) Liver section obtained from the control animal showed no noticeable change; (B) liver section obtained from the NaNO2-treated animal showed also no noticeable change; (C) liver section obtained from the AAF-treated animal showed a focus; (D) and (E) rat received both AAF and 0.2% NaNO2 and 0.3% NaNO2, respectively showed variable-sized foci and neoplastic nodules in the periphery of the portal areas. H and E, × 100.

The histopathological analysis showed that the livers, obtained from both the control and NaNO2-treated rats, had no remarkable change in gross under histopathological examination (Fig. 2A,B). On the contrary, numerous foci and neoplastic nodules with variable sizes, mildly forming in the periphery of the portal areas,

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Fig. 2. (Continued)

were observed in all AAF-treated rats (Fig. 2C showed one of the representatives). Livers of groups III, IV and V also manifested architectural distortion and extensive compression to surrounding parenchyma. Hepatocytes within the nodules consisted primarily of eosinophilic cells in a trabecular pattern and were separated by thin connective tissue strands (Fig. 2D). Neoplastic nodules are proliferative lesions and known to be induced by chemical carcinogens and at least they indicate an increased probability for the development of hepatocellular carcinoma. All rats in groups IV and V that received AAF together with NaNO2, 0.2% and 0.3%, respectively for 3 months, revealed moderate development of this kind of lesion

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Fig. 2. (Continued)

(Fig. 2D,E). Several hepatic damages for possible development of hepatocellular carcinoma were observed. To localize the expressions of c-Jun, c-Fos and c-Myc, immuno-cytochemical method was applied to the tissue sections obtained from the rats treated with AAF (0.02%) alone, AAF plus NaNO2 (0.2 or 0.3%) and NaNO2 alone using the biotin-avidin peroxidase detection system. As presented in Fig. 3, the expression of c-Jun, showing as brown staining in the rat liver, was increased predominantly (1.65-fold of control) by AAF treatment. The addition of NaNO2 (0.2 or 0.3%) to the diet of AAF-treated rats enhanced the AAF-induced c-Jun expression significantly (P B 0.001, to an extent of 1.97- or 2.32-fold of control, respectively) as analyzed by Leica image processing and analysis system (Table 1:Fig. 3D,E). When the same detection system was used to localized c-Fos antigen in rat liver, the results showed that AAF-treated group had a 1.49-fold increase in the expression than that of control rats (Fig. 4C). This augmentation in c-Fos expression was also magnified significantly (P B 0.001) by NaNO2 (0.2 or 0.3%) addition, with fold increase of 1.77 or 2.05 of control, respectively, when compared with the AAF alone-treated rats (Table 2:Fig. 4D,E). Similar results were also observed in the study of c-Myc. There was a 2.29-fold expression of c-Myc in the AAF-treated rats compared to that of controls (Table 3:Fig. 5C) and this induction was potentiated by the addition NaNO2 to an increase of 2.79- or 3.42-fold for 0.2 and 0.3% doses, respectively (Fig. 5D,E:Table 3).

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Fig. 3.

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Fig. 3. (continued) Fig. 3. Immunocytochemical examination of the effect of NaNO2 on the c-Jun expression in AAFtreated rat. Liver sections obtained from the animals of control (A), treated with NaNO2 (0.3%) (B), treated with 0.02% AAF alone (C), received both AAF and NaNO2 (0.2%) (D), and received both AAF and NaNO2 (0.3%) (E), were subjected to immunocytochemical examination for c-Jun expression using rabbit anti-c-Jun polyclonal antibody as primary antibody and biotin-avidin peroxidase detecting system. × 200. Arrows indicate representative stain areas.

4. Discussion The enhancing effect of NaNO2 on the hepatocarcinogenesis of AAF administration was investigated in the present study. In a 3 month study, the rats who received the combination of AAF and NaNO2 in their diet, had larger variable-sized foci and neoplastic nodules that have the potential for the development of hepatocarcinoma, compared with AAF-treated group. Since NaNO2 was nontoxic itself (low

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Table 1 Effect of NaNO2 on c-Jun protein level in AAF-treated liver Treatmenta

c-Jun foci areab (mm2)

Mean 9S.D.c (mm2)

Fold of control

Control

0.32, 0.39, 0.46, 0.50, 0.61, 0.59, 0.76, 0.75, 0.86, 0.91,

0.37 9 0.05

—

0.47 9 0.05

1.27

0.61 90.04*

1.65

NaNO2 (0.3%) AAF (0.02%) AAF+NaNO2 (0.2%) AAF+NaNO2 (0.3%)

0.35, 0.42, 0.41, 0.48, 0.55, 0.63, 0.75, 0.70, 0.83, 0.85,

0.40 0.31 0.41 0.43 0.60 0.65 0.71 0.72 0.84 0.88

0.73 9 0.03*

c

1.97

0.86 90.03*

c

2.32

a

Control rats were on a synthetic basal diet, while experimental rats received AAF with or without NaNO2 in the diet for 3 months. b The foci area was determined in five randomly selected fields from six liver tissues of every group, by Leica image processing and analysis system. c Mean9 S.D. of six animals. * PB0.001 compared with control group; c PB0.001 compared with AAF group.

dose) and not related to the metabolic process of AAF, it is plausible that the enhancing effect of NaNO2 on the carcinogenic activity of AAF is the result of the endogenous nitrosation of AAF that strengthens its tumorigenicity rather than modulation on the metabolic pathway. This is in agreement with previous studies, which had shown that the formation of nitroso-compounds through endogenous nitrosation was potentially more carcinogenic than the administration of NaNO2 and many various carcinogens [5,6]. The formation of N-NO-AAF from the reaction of AAF with nitrous fume in acetic acid has been demonstrated [22]. It is also possible that it also takes place in normal physiological conduction like other compounds, as previously described [5,6]. The mutagenicity of N-NO-AAF in TA 98 is stronger than those of N-methyl-N%-nitro-N-nitrosoguanidine (MNNG) and N-acetoxy-N-2-fluorenylacetamide (N-AcO-AAF). The mechanism of N-NO-AAF-induced carcinogenesis is thought to be via the interaction of its fluorenyl-2-diazonium ion with cellular macromoleculars, such as DNA, RNA and protein. Recent studies revealed that N-NO-AAF is a strong electrophile and reacts readily with histidine, lysine, cysteine, glutathione, tryptophan, adenosine, cytidine at neutral pH. Without metabolic activation (i.e. rat liver S9 fraction), N-NO-AAF exhibits more direct and strong damaging effect on DNA than its parental compounds at equal concentration in C3H10T1/2 mouse fibroblast and Chinese hamster ovary (CHO) cells [35]. Basing on the observation that N-NO-AAF induced the ouabain-resistance mutation and cycle-dependent transformation in C3H10T1/2 cells Lin and Kuo (1990) also demonstrated that N-NO-AAF is a new direct-acting mutagen and is more toxic than its parental compounds [36]. Taken all together, we suggested that the showing of stronger tumorigenicity, when AAF administration was supple-

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Fig. 4.

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Fig. 4. (continued) Fig. 4. Immunocytochemical examination of the effect of NaNO2 on the c-Fos expression in AAFtreated rat. Liver sections obtained from the animals of control (A), treated with NaNO2 (0.3%) (B), treated with 0.02% AAF alone (C), received both AAF and NaNO2 (0.2%) (D), and received both AAF and NaNO2 (0.3%) (E), were subjected to immunocytochemical examination for c-Fos expression using rabbit anti-c-Fos polyclonal antibody as primary antibody and biotin-avidin peroxidase detecting system. × 200. Arrows indicate representative stain areas.

mented with NaNO2, may be generated from the toxicity of AAF along with the endogenous nitrosation of AAF to N-NO-AAF (a more potential carcinogen). Nuclear oncogenes, such as c-jun, c-fos, c-myc and also c-Ha-ras, have been reported to be activated during rat hepatocarcinogenesis; and a strong association between an increased level of c-myc expression and cellular proliferation was observed in both regenerating liver and in numerous experimental systems [37–40]. One of the best characterized experimental tumor model systems is the chemically

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Table 2 Effect of NaNO2 on c-Fos protein level in AAF-treated liver Treatmenta

c-Fos foci areab (mm2)

Mean9 S.D.c (mm2)

Fold of control

Control

0.36, 0.43, 0.39, 0.48, 0.63, 0.64, 0.76, 0.73, 0.86, 0.90,

0.43 9 0.08

—

0.52 90.08

1.21

0.64 90.01*

1.49

NaNO2 (0.3%) AAF (0.02%) AAF+NaNO2 (0.2%) AAF+NaNO2 (0.3%)

0.37, 0.48, 0.47, 0.58, 0.64, 0.64, 0.75, 0.77, 0.85, 0.92,

0.40 0.57 0.47 0.62 0.62 0.65 0.74 0.80 0.87 0.88

0.76 9 0.03*

c

1.77

0.88 9 0.03*

c

2.05

a

Control rats were on a synthetic basal diet, while experimental rats received AAF with or without NaNO2 in the diet for 3 months. b The foci area was determined in five randomly selected fields from six liver tissues of every group, by Leica image processing and analysis system. c Mean9 S.D. of six animals. * PB0.001 compared with control group; c PB0.001 compared with AAF group.

induced hepatocarcinogenesis in rats [41,42]. The expressions of these ‘primary response genes’ that are involved in cell growth and differentiation are induced by tumor promoters like phorbol esters [43]. These genes have received particular attention, since they are also overexpressed in many human tumors, among other hepatocarcinoma [44]. However, few reports had been focused on the activation of proto-oncogenes like c-fos during rat liver carcinogenesis [45,46]. In the present Table 3 Effect of NaNO2 on c-Myc protein level in AAF-treated liver Treatmenta

c-Myc foci areab (mm2)

Mean9 S.D.c (mm2)

Fold of control

Control

0.14, 0.31, 0.32, 0.31, 0.41, 0.59, 0.68, 0.67, 0.85, 0.79,

0.24 9 0.06

—

0.36 9 0.05

1.50

0.55 90.08*

2.29

NaNO2 (0.3%) AAF (0.02%) AAF+NaNO2 (0.2%) AAF+NaNO2 (0.3%)

a

0.21, 0.27, 0.34, 0.41, 0.56, 0.58, 0.69, 0.69, 0.81, 0.80,

0.23 0.26 0.44 0.32 0.50 0.63 0.64 0.67 0.82 0.82

0.67 90.02*

c

2.79

0.82 90.02*

c

3.42

Control rats were on a synthetic basal diet, while experimental rats received AAF with or without NaNO2 in their diet for 3 months. b The foci area was determined in five randomly selected fields from six liver tissues of every group, by Leica image processing and analysis system. c Mean9 S.D. of six animals * PB0.001 compared with control group; c PB0.005 compared with AAF group.

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Fig. 5.

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Fig. 5. (continued) Fig. 5. Immunocytochemical examination of the effect of NaNO2 on the c-Myc expression in AAFtreated rat. Liver sections obtained from the animals of control (A), treated with NaNO2 (0.3%) (B), treated with 0.02% AAF alone (C), received both AAF and NaNO2 (0.2%) (D), and received both AAF and NaNO2 (0.3%) (E), were subjected to immunocytochemical examination for c-Myc expression using rabbit anti-c-Myc polyclonal antibody as primary antibody and biotin-avidin peroxidase detecting system. × 200. Arrows indicate representative stain areas.

study, c-Myc, c-Fos and c-Jun protein levels were increased in all AAF-treated animals at early stages. These results are in a good agreement with other findings [44,47]. The expressions of c-Myc, c-Fos and c-Jun in the livers of rats fed with a combination of AAF and NaNO2 were significantly higher than that of AAF alone-treated rats. The potential effect of NaNO2 on the expressions of nuclear proto-oncogenes induced by AAF may be the result of endogenous nitrosation of AAF to N-NO-AAF. In summary, the administration of NaNO2, along with AAF,

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enhanced the AAF-induced increases in c-Jun, c-Fos and c-Myc levels in the livers of rats, suggesting that NaNO2 may strengthen the hepatocarcinogenesis of AAF by, at least in part, increasing AAF-activated expression of nuclear proto-oncogenes.

Acknowledgements These studies were partially supported by the Chung-Shan Medical and Dental College Research Fund, CSMC 83-NS-B-005, and National Science Counial Grant, NSC 86-2621-B040-002Z, Republic of China.

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