Trans-4-hydroxy-2-nonenal, an aldehydic lipid peroxidation product, lacks genotoxicity in lacI transgenic mice

Cancer Letters 148 (2000) 81±86 www.elsevier.com/locate/canlet

Trans-4-hydroxy-2-nonenal, an aldehydic lipid peroxidation product, lacks genotoxicity in lacI transgenic mice Akiyoshi Nishikawa a,*, Fumio Furukawa a, Ken-ichiro Kasahara a, Shinichiro Ikezaki a, Toshiaki Itoh b, Takayoshi Suzuki b, Koji Uchida c, Masaaki Kurihara d, Makoto Hayashi b, Naoki Miyata d, Masao Hirose a a

b

Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan c Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, Japan d Division of Organic Chemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Received 6 July 1999; received in revised form 12 August 1999; accepted 16 August 1999

Abstract In order to cast light on the signi®cance of lipid peroxidation products for carcinogenesis, the lacI mutant frequency (MF), micronucleus induction and cell proliferation were analyzed in lacI transgenic mice treated with trans-4-hydroxy-2-nonenal (HNE), a typical example. Male mice were ip injected with HNE at doses of 0, 5 or 50 mg/kg bw and 48 h thereafter, peripheral blood was collected for analyzing micronucleus induction. After 14 days, the mice were sacri®ced to allow tissue sampling for examination of lacI MF and cell proliferative activity. Sixty percent of the mice given 50 mg/kg HNE died within 5 days after the treatment, but no other mortalities were observed. Histopathologically, marked pulmonary hemorrhage was found in the 50 mg/kg HNE group mice that survived until day 14. Immunohistochemically, HNE-modi®ed proteins were detected in their alveolar macrophages. The HNE treatment did not increase lacI MF in the liver, kidney and lung and no signi®cant increase in micronucleus induction or cell proliferation in major organs was found in either treatment. Moreover, no tumors developed in the 5 mg/kg HNE-treated mice which survived until week 78. Our results thus indicate that HNE lacks in vivo genotoxicity in lacI transgenic mice even when lethal doses are applied. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Trans-4-hydroxy-2-nonenal; lacI mutation; Micronucleus; Cell proliferation; BigBlue

1. Introduction Trans-4-hydroxy-2-nonenal (HNE), an aldehydic lipid peroxidation product, is highly reactive with biomolecules, forming DNA and protein adducts [1,2]. It has tested positive in in vitro chromosomal aberration tests [3,4] but proved consistently negative in bacterial mutagenicity tests [5]. However, HNE * Corresponding author. Tel.: 181-3-3700-9819; fax: 181-33700-1425.

could be endogenously epoxidized to form 2,3epoxy-hydroxynonanal (EH) which has been tested positive for bacterial mutagenicity [5] and causes development of renal preneoplastic tubule lesions in new-born mice, in contrast to HNE itself [5]. It has hitherto not been elucidated whether exogenous HNE may exert genotoxicity in vivo. Accumulated experimental data have revealed the reliability of commercially available lacZ- or lacItransgenic mice for detecting mutagenicity in target organs for carcinogenicity [6±10]. Recently, we have

0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00318-3

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2.2. Experimental design The mice were divided into 3 groups, each consisting of 10 animals. Groups 1, 2 and 3, respectively, received a single intraperitoneal injection of HNE dissolved in 0.5 ml of olive oil at a dose of 0, 5 or 50 mg/kg body weight. The animals were observed daily for toxicological signs and ®ve per group were sacri®ced to allow sampling of tissues for the lacI mutation assay after 14 days [9]. Thereafter, the remaining surviving animals in each group were kept for a 78-week carcinogenicity bioassay. 2.3. Assay for lacI mutant frequency

Fig. 1. Photomicrographs of lung histology (a) and immusohistochemistry for HNE-modi®ed proteins (b) in a rat given 50 mg/kg of HNE.

provided supportive evidence from studies of mutagens such as 2-amino-3,4-dimethylimidazo[4,5±f]quinoline (MeIQ) and dimethylnitrosamine (DMN) [6±8]. In the present study, the mutagenicity, clastogenicity and ability of HNE to induce cell proliferation were investigated in lacI-transgenic mice.

2. Materials and methods 2.1. Chemical and animals HNE was synthesized according to the method reported by Esterbauer et al. [11] and its purity con®rmed to be over 95% by gas chromatography. Male 7-week-old lacI-transgenic C57BL/6 BigBlue w mice were purchased from Stratagene (La Jolla, CA) and maintained under standard laboratory conditions with free access to Oriental MF basal diet (Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water in accordance with institutional guidelines. The experiment was started after an acclimation period of 1 week.

Total genomic DNA was extracted from the liver, kidney and lung according to the method of Rogers et al. [12]. As an alternative method for DNA isolation, a nuclear isolation step [7] was included without phenol or chloroform. Homogenates in 3 ml of Dounce buffer containing 20 mg/ml RNase were overlayed onto 3 ml of 0.5 M sucrose solution. After centrifugation at 2000 g for 10 min at 48C, the supernatant was removed by pipetting and the nuclear precipitate was resuspended in 3 ml of Dounce buffer. Then 3 ml of proteinase K solution consisting of 2 mg/ml proteinase K, 2% SDS and 0.1 M EDTA at pH 7.5 was added and incubation performed for 1±3 h at 508C until the mixture became clear. After the digestion, an equal volume of 5 M ammonium acetate was mixed gently to precipitate SDS and other contaminants. The mixture was kept on ice for 30 min and centrifuged at 10 000 £ g for 10 min at 48C. Then DNA was precipitated by adding 0.6 volumes of 2-propanol. The isolated DNA was dissolved in a small amount of TE buffer and subjected to in vitro packaging and mutant detection, with a modi®ed protocol from the Stratagene BigBlue w Instruction Manual (08/1992). In brief, a lambda packaging reaction was carried out at 378C and the resultant phages were then adsorbed to the E. coli SCS-8 strain for 20 min at room temperature. The lacI mutant frequency (MF) was determined from the incidence of blue plaques on 25 £ 25 cm NZY agar plates (35 ml top agarose containing 0.7 mg/ml of X-gal on 40 ml bottom agar). 2.4. Micronucleus assay Forty-eight hours after the HNE treatment, a 5 ml

A. Nishikawa et al. / Cancer Letters 148 (2000) 81±86

aliquot of peripheral blood was collected without anticoagulant from the tail vein, placed on an acridine orange-coated glass slide, covered with a coverslip, and supravitally stained as described previously [13]. One thousand reticulocytes per animal were analyzed by ¯uorescence microscopy within a few days of slide preparation and the numbers of cells with micronucleus were recorded. 2.5. Histopathology and histochemistry Tissues for histopathology and immunohistochemistry were the same as for the lacI MF analysis. The tissues were immediately ®xed in methanol Carnoy's ®xative (methanol: chloroform: acetic acid 6:3:1) at autopsy, processed for embedding in paraf®n, sectioned and subjected to histological examination and immunohistochemistry with anti-proliferating cell nuclear antigen (PCNA) antibody (Dako, Kyoto, Japan) and anti-HNE-modi®ed proteins [2,14] followed by demonstration of binding with the streptoavidin-biotin-peroxidase complex (ABC) method. PCNA-labeled cells visualized by diaminobenzidine were counted under the microscope and PCNA-labeling indices were generated as the percentages of positive liver, renal tubule and lung alveolar cells. 2.6. Statistics The statistical signi®cance of differences in lacI MFs and micronucleus induction were evaluated by the Fisher exact probability test and that for cell proliferation was determined using the Student t-test.

83

rhage in their lungs, suggesting that the main target organ for HNE toxicity could be the lung (Fig. 1a). Immunohistochemically, HNE-modi®ed proteins were positive in alveolar macrophages of group 3 mice (Fig. 1b). Mice in groups 1 and 2 demonstrated no and little pulmonary hemorrhage. Thus the remaining 5 each animals in the low dose and vehicle alone groups were maintained until week 78. The data for lacI MFs are summarized in Table 1 values for lung DNA being 26.4 (£10 26), 14.3 and 24.5 in the groups treated with 50, 5 and 0 mg/kg of HNE, respectively. The lacI MFs in liver DNA were 23.3 (£10 26) and 11.2 in the 50 and 0 mg/kg groups, respectively. Those for kidney DNA were 15.2 and 13.5 (£10 26), respectively. No statistically signi®cant increase was evident in any of these organs. The results of micronucleus assay are shown in Table 2. The mean incidence of micronucleated reticulocytes was 6.0 ( £ 10 23), 6.8 and 7.0 in the groups treated with 50, 5 and 0 mg/kg of HNE, respectively, with no signi®cant intergroup differences. Likewise, spontaneous induction ratio was a little high in this study as noticed in our previous report [7]. Fig. 2 demonstrates PCNA-labeling indices per 100 cells for each tissue. Among the cells of renal tubules, pancreatic acini, ducts and islets, liver cell trabeculae, intrahepatic biliary ducts, and pulmonary bronchioles and alveoli, only bronchial and alveolar cells showed a trend for increase in PCNA-labeling index, although this was also not statistically signi®cant. No neoplasms were found in the remaining 5 mg/kg HNE-treated mice which were maintained for 78 weeks after the treatment although lung adenoma

3. Results Six of 10 mice in group 3 were found dead by 5 days after the 50 mg/kg HNE treatment but no other mortalities occurred until day 14. Thus the LD60 value of HNE in lacI transgenic mice was estimated to be 50 mg/kg when given intraperitoneally, essentially comparable to the LD50 value found in a previous study [15]. Including surviving 4 mice in the high dose group, 5 each animals in the low dose and vehicle alone groups were sacri®ced for investigating histopathology, immunohistochemistry and lacI MF analysis. Histopathologically, surviving mice in group 3 showed remarkable intra-alveolar hemor-

Fig. 2. PCNA-labeling indices in different tissues of lacI transgenic mice. Data represent mean values ^ SE.

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Table 1 Lac I mutant frequency (MF) Treatment

HNE (50 mg/kg) HNE (5 mg/kg) Control a

Lung

Liver

Kidney

Plaques

Mutants

MF £ 10 26

Plaques

Mutants

MF £ 10 26

Plaques

Mutants

MF £ 10 26

1325194 420903 1388772

35 6 34

26.4 14.3 24.5

1329948 NE a 890559

31

23.3

25

15.2

10

11.2

1640381 NE 1403460

19

13.5

NE, not examined.

was noted in a control mouse which survived for the same duration after the vehicle administration. 4. Discussion The results of the present study thus indicate that HNE, an aldehydic lipid peroxidation product, lacks mutagenicity and clastogenicity in lacI-transgenic mice even when given at a lethal dose. Lipid peroxidation has been suggested as an endogenous event to yield possible DNA-damaging substances such as malondialdehyde (MDA) and HNE, with mechanistic signi®cance for non-genotoxic cytotoxic carcinogenesis like carbon tetrachloride-induced hepatocarcinogenesis [16]. Among a variety of lipid peroxidation products, HNE has been shown to form a speci®c exocyclic guanine adduct, 1,N 2-propanodeoxyguanosine [1,17]. In fact, similar exocyclic DNA adducts have been detected in human tissue DNAs [18]. An epoxide form of HNE, EH, can also covalently bind to guanine yielding 1,N 2-ethenodeoxyguanosine, which may be a responsible DNA lesion for tumorigenicity [5]. Endogenous formation of EH remains to be investigated although probable pathways such as metabolic activation in the liver, catalytic reactions of hydrogen peroxide and fatty acid hydroperoxides during lipid peroxidation and autoxidation have been hypothesized [19]. Because in vivo accumulation of proteins modi®ed by such aldehydes is believed to be involved in ageand disease-related impairment of cellular functions [20], the primary importance of HNE-depending pathways in hepatocytes seems to be protection of proteins from modi®cation by aldehydic lipid peroxidation products. It can be speculated that even if HNE has an ability to damage DNA, defense systems such as

glutathione (GSH) conjugation as well as the DNA repair systems would effectively preclude persistent effects [21]. In fact, we have shown that GSH is a favorite substrate of HNE as for simple alkylating agents [22], consistent with the hypothesis of Ames et al. [21]. Judging from the results of the present study, genotoxicity of exogenous HNE appears negligible. However, a possible indirect association of HNE and related EH with carcinogenesis still remains to be elucidated because it has been shown that HNE exerts a co-initiating effect on murine skin when given with B(a)P (Chung et al. unpublished data). This might partly involve depletion of defense or DNA repair potential. HNE has also been shown to exert a physiological in¯uence by modifying enzymes like multicatalytic proteinase [20] and phospholipase [23], and to affect cell kinetic-related cell growth [24], cell differentiation [25] and cell death [26], as well as activation of oncogenes like c-myc and c-myb [27±29]. Therefore, these biological effects of HNE must be taken into account when considering its cacinogenicity. In conclusion, our results indicate that HNE, one of the most potent in vitro clastogens yielded during lipid peroxidation, has no genotoxicity in vivo in lacI transgenic mice when given intraperitoneally. The in¯uTable 2 Micronucleus induction in peripheral blood Treatment

Number of mice examined

MNRETs/1000 RETs (mean ^ SD)

HNE (50 mg/kg) HNE (5 mg/kg) Control

5 10 9a

6.0 ^ 1.7 6.8 ^ 2.5 7.0 ^ 2.6

a

One mouse died before the collection of blood.

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ence of endogenously produced HNE and related aldehydes remain to be investigated. [12]

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