Inhibitory effects of combined administration of antibiotics and anti-inflammatory drugs on lung tumor development initiated by N-nitrosobis(2-hydroxypropyl)amine in rats
Masahiro Tsutsumi1,
Hiromichi Kitada,
Kazumi Shiraiwa,
Makoto Takahama,
Toshifumi Tsujiuchi,
Hiroyuki Sakitani,
Yasutaka Sasaki,
Koichi Murakawa,
Masatoshi Yoshimoto and
Yoichi Konishi
Department of Oncological Pathology, Cancer Center, Nara Medical University, 840 Shijo-cho Kashihara, Nara, 634 Japan
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Abstract
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The effects of antibiotics and anti-inflammatory drugs on the promotion stage of lung carcinogenesis initiated with N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats were investigated in two experiments with a similar protocol. In experiment 1, rats received tap water containing 2000 p.p.m. BHP for 12 weeks followed by basal diet or basal diet containing 0.02% erythromycin (EM), 0.04% ampicillin (ABPC), 1.5% sho-saiko-to, 0.02% EM plus 1.5% sho-saiko-to or 0.04% ABPC plus 1.5% sho-saiko-to for 8 weeks after BHP administration. The development of adenocarcinomas (AC), squamous cell carcinomas (SqC) and adenosquamous carcinomas (ASqC) was completely inhibited in rats given ABPC plus sho-saiko-to and the numbers of lung lesions including alveolar hyperplasias, adenomas and carcinomas were decreased in rats given EM plus sho-saiko-to or ABPC plus sho-saiko-to. Neutrophil and macrophage infiltration into alveolar spaces of the lung were also markedly suppressed. In experiment 2, rats received BHP in the same manner as in experiment 1 and basal diet or basal diet containing 0.04% ABPC, 0.006% piroxicam, 0.04% ABPC plus 0.006% piroxicam and 0.04% ABPC plus 0.75% ougon for 8 weeks. The incidence and number of carcinomas, including ACs, SqCs and ASqCs were decreased in rats given ABPC plus piroxicam or ABPC plus ougon. Bacteria, mainly Escherichia coli, were detected in broncho-alveolar lavage of rats receiving BHP. The results suggest that chronic inflammation might be involved in the progression of lung carcinogenesis by BHP in rats and its suppression may therefore be useful as a chemopreventive strategy in lung cancer clinics.
Abbreviations: AC, adenocarcinoma; ABPC, ampicillin; Ad, adenoma; AH, alveolar hyperplasia; ASqC, adenosquamous carcinoma; BAL, broncho-alveolar lavage; BHP, N-nitrosobis (2-hydroxypropyl)amine; EM, erythromycin; NSAID, non-steroidal anti-inflammatory drug; PCNA, proliferating cell nuclear antigen; SM, squamous metaplasia; SqC, squamous cell carcinoma.
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Introduction
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Prolonged inflammation, due to infection or exposure to a toxin, causes continued production of molecules that are protective but may also act as carcinogens or promoters. It is generally accepted that infectious agents such as Helicobacter pylori for the stomach (1), hepatitis B and C viruses (2,3) and liver flukes for the liver (4), papilloma viruses for the uterine cervix (5) and EpsteinBarr virus for lymphoid tissue (6) play important roles in the development of malignant neoplasms in these organs, at least partly through chronic inflammation. Further, prostaglandin synthesis inhibitors are effective at inhibiting carcinogenesis by decreasing inflammation (7), especially cyclooxygenese-2 inhibitors in the colon (810) and urinary bladder (11). It is also reported that inflammatory changes induce pro-inflammatory cytokines and non-protein factors that may be involved in carcinogenesis (12).
Previously, we established a lung cancer model with a single i.p. or oral administration of N-nitrosobis(2-hydroxypropyl)amine (BHP) inducing high incidences of adenocarcinomas (ACs), squamous cell carcinomas (SqCs) and adenosquamous carcinomas (ASqC) in rats (13,14). BHP can also be synthesized in vivo and in vitro from environmental precusor amines [bis(2-hydroxypropyl)amine and tris(2-hydroxypropyl)amine] under acidic conditions in the presence of sodium nitrite and endogenously synthesized BHP has an organotropism similar to that of the exogenously administered carcinogen (15). Genetic changes detected so far in this model are frequent mutations of Ki-ras but not Ha-ras and p53 (16), mutation and deletion of p16 and overexpression of midkine (17) and vascular endothelial growth factor (18). Careful histopathological assessment of lung carcinogenesis by BHP suggested a contribution of chronic inflammation exemplified by infiltrating neutrophils and macrophages. Therefore, in the present study we examined the influence of suppression of chronic inflammation by antibiotics and/or anti-inflammatory drugs [anti-inflammatory herbal medicines and a non-steroidal anti-inflammatory drug (NSAID)] on lung carcinogenesis in rats.
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Materials and methods
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Male SPF Wistar rats (Nihon SLC, Shizuoka, Japan), 6 weeks old, were housed five to a plastic cage in an air-conditioned room maintained at 24°C and 60% humidity with a 12 h light/dark cycle. The basal diet was a commercial stock diet, CE-2 (Nihon Clea Co. Ltd, Tokyo, Japan). BHP was purchased from Nacalai Tesque (Kyoto, Japan) and drinking water containing 2000 p.p.m. of this carcinogen was prepared once a week. The basal diet and diets containing test compounds and drinking water or water containing BHP were supplied ad libitum and their intakes and body weights were measured once a week. As the anti-inflammatory drugs, sho-saiko-to, ougon and piroxicam were used in this study. Sho-saiko-to is a herbal medicine consisting of seven herbs with anti-inflammatory activity used for chronic hepatitis patients. Ougon is a herbal medicine included in sho-saiko-to with stronger anti-inflammatory effects than the other herbs. Piroxicam is a long half-life NSAID with relative low toxicity. This study consisted of two experiments based on similar protocols to those shown in Figure 1
. In experiment 1, groups 16 received tap water without BHP throughout the experimental period and basal diet or diet mixed with 0.02% erythromycin (EM) (Dainaboto Co. Ltd, Osaka, Japan), 0.04% ampicillin (ABPC) (Meiji Co. Ltd, Osaka, Japan), 1.5% sho-saiko-to (Tsumura Co. Ltd, Tokyo, Japan), 0.02% EM plus 1.5% sho-saiko-to or 0.04% ABPC plus 1.5% sho-saiko-to, respectively, for 8 weeks starting from week 12; groups 712 received tap water containing 2000 p.p.m. BHP for 12 weeks followed by the same doses of test compounds over the same period as in groups 16, respectively. In experiment 2, groups 15 received tap water without BHP throughout the experimental period and basal diet, 0.04% ABPC, 0.006% piroxicam (Sigma, St Louis, MO), 0.04% ABPC plus 0.006% piroxicam or 0.04% ABPC plus 0.75% ougon (Scutellariae radix; Tsumura Co. Ltd, Tokyo, Japan), respectively, for 8 weeks starting from week 12; groups 610 received tap water containing 2000 p.p.m. BHP for 12 weeks followed by the same doses of test compounds as in groups 15. All rats were killed under ether anesthesia at week 20 of the experiment. The lungs were macroscopically examined, removed and immediately fixed in ice-cold 100% ethanol. Two millimeter thick slices from all lobes were cut and processed routinely for paraffin embedding and staining of sections with hematoxylin and eosin. Lung lesions were diagnosed according to the criteria described previously (13,14).

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Fig. 1. Experimental protocol. (A) Groups 16 in experiment 1 and groups 15 in experiment 2; (B) groups 712 in experiment 1 and groups 610 in experiment 2.
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For evaluation of cell proliferation activity, immunohistochemical staining of proliferating cell nuclear antigen (PCNA) was performed with EPOS anti-PCNA antibody (DAKO Japan, Kyoto, Japan) followed by counterstaining wtih methyl green (18). PCNA labeling indices were generated from counts of >100 cells in 15 alveolar hyperplasia (AH) lesions selected randomly from groups 7 and 12 in experiment 1 and groups 6 and 9 in experiment 2.
For bacterial examination, broncho-alveolar lavage (BAL) was perfomed before killing of three rats in each group in experiment 2. Neck skin was cut and saline was injected into the lungs aseptically through the trachea. BAL fluid was spread on trypt-soya agar (Nissui, Tokyo, Japan) containing 5% defibrinated equine blood and incubated at 37°C for 24 h. After colony isolation, Gram stain, catalase and oxidase staining and the bacteria lysis test with potassium hydroxide were performed and api 20E and api Staph kits (Bio Mérieux S.A., Etoile, France) were used to identify bacterial species (19).
Statistical analyses were carried out using the
2 and Student's t-tests.
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Results
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Inhibition of BHP-initiated lung tumor development by combined administration of antibiotics and anti-inflammatory drugs
Final body weights demonstrated growth retardation in carcinogen-treated groups 712 in experiment 1 and groups 610 in experiment 2, as compared with groups 16 and 15, respectively. However, growth was not inhibited by any of the test compounds in either experiment 1 or 2. There were no statistically significant differences in food intake during the BHP and test compound administration periods, values ranging from 10.5 to 15.8 and 10.0 to 14.0 g/day per rat in the two experiments. No lung lesions developed in rats without BHP administration. Incidences and numbers of lung lesions for groups 712 in experiment 1 are shown in Table I
. The development of carcinomas, including AC, SqC and ASqC was totally blocked in rats of group 12. The numbers of AH were also decreased in groups 8 (P < 0.05), 9 (P < 0.05), 11 (P < 0.01) and 12 (P < 0.01) and of total lung lesions in groups 11 and 12 (P < 0.05). Incidence and numbers of lung lesions in experiment 2 are shown in Table II
. Incidences of AC were significantly reduced in groups 9 and 10 and the numbers of adenomas (Ad) were decreased in groups 8 (P < 0.01), 9 (P < 0.05) and 10 (P < 0.05). The numbers of AC were also decreased in groups 9 (P < 0.01) and 10 (P < 0.01). In groups 9 and 10, SqC and ASqC were not observed.
Inhibition of neutrophil and macrophage infiltration in the lungs by antibiotics and anti-inflammatory drugs
Histologically, neutrophil infiltration was seen in the alveolar walls and alveolar spaces, along with macrophage infiltration and some lymphocyte infiltration and edema in areas of AH in rats of group 7 in experiment 1 (Figure 2A
) and group 6 in experiment 2. Such histological changes were also consistently observed in Ad and AC, but not in non-lesional areas of the lung. In experiment 1, neutrophil and macrophage infiltration and inflammatory reactions were reduced in the lungs of groups 812, as shown in Table III
and Figure 2B
, compared with group 7. Inflammation was also suppressed in the lungs of rats from groups 710 in experiment 2. PCNA labeling indices for AH were also significantly reduced in rats from group 12 as compared with group 7 (P < 0.01), to 38.8 ± 4.1 and 44.3 ± 3.0 in experiment 1 (Table III
). The PCNA labeling indices for AH were also significantly reduced in rats from group 9 compared with group 6 (P < 0.01) in experiment 2, to 36.1 ± 2.3 and 43.7 ± 4.1, respectively. Representative sections immunohistochemically stained for PCNA are shown in Figure 3A and B
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Fig. 2. Representative histological appearance of inflammation in preneoplastic lesions (experiment 1). (A) Alveolar hyperplasia in a group 7 rat; (B) alveolar hyperplasia in a group 12 rat.
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Table III. Histological findings of inflammatory reactions and PCNA labeling indices in the lungs of rats in experiment 1
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Fig. 3. Representative findings for PCNA staining of preneoplastic lesions (experiment 1). (A) Alveolar hyperplasia in a group 7 rat; (B) alveolar hyperplasia in a group 12 rat.
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Identification of bacterial species in BAL fluid
The results of bacterial examination of BAL fluid in experiment 2 are shown in Table IV
. Bacterial species Aerococcus sp., Staphylococcus haemolytics and Bacillus sp. were identified in broncho-alveolar fluid of four out of 15 rats in groups 1, 2, 4 and 5, respectively. Escherichia coli, Aerococus sp., Staphylococcus epidermidis, Proteus mirabilis and Klebsiella pneumoniae were identified in rats of groups 610.
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Discussion
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It has been proposed that suppression of inflammation might be an important anti-promotion/anti-progression strategy. The promotion stage is characterized by growth of initiated cells into populations of preneoplastic cells, due to alterations in gene expression and cell proliferation, and the progression stage is characterized by transformation of the preneoplastic into neoplastic cells as a result of additional alterations (12). In the experimental protocol used in the present studies, AH were observed in the lungs of rats at the end of BHP administration. K-ras mutations are detectable in AH (16), while midkine expression increases from AH to Ad to AC (17) and VEGF and its receptors may play important roles in the acquisition of malignant potential by preneoplastic lung lesions induced by BHP (18).
In the present experiment, progression of lung carcinogenesis was inhibited by suppressing chronic inflammation in the alveolar walls and spaces in lesions by combined administration of antibiotics and a herbal medicine or NSAID. EM and ABPC are commonly used wide spectrum antibiotics and sho-saiko-to consists of seven herbs which activate natural killer cells and macrophages (20,21). Chemopreventive effects against hepatocellular carcinoma development in cases of viral infection have already been reported (22). Ougon is a herbal medicine included in sho-saiko-to with stronger anti-inflammatory effects than the other herbs. Piroxicam, a long half-life NSAID, is known to exhibit relatively strong chemopreventive potential against colon (23), tongue (24), urinary bladder (25) and liver (26) neoplasms. In the present study, higher rates of cell proliferation were found in AH, putative neoplastic lesions for AC, with inflammatory cell infiltration than in those without inflammatory cell infiltration. It is therefore hypothesized that oxygen radicals produced in inflammatory cells may cause oxidative DNA damage and gene alterations which accelerate the progression from preneoplastic to neoplastic lesions. In fact, it has been reported that activated phagocytes associated with chronic inflammation produce nitric oxide and other superoxides, and these oxygen radicals may stimulate an increased rate of cell division and decreased repair of DNA damage (27). Moreover, chronic inflammation induces alterations in gene expression, for example with regard to some cytokines and arachidonic acid cascades (12). In fact, increased levels of prostaglandins in various tumor cell lines (28) have been reported and the effectiveness of piroxicam in the present study provides support for the involvement of chronic inflammation in our lung carcinogenesis model. Under our experimental conditions, E.coli and other enterobacterial species were identified in rats treated with BHP. The doses of antibiotics used in these experiments were relative low and the antibiotics could not kill the bacteria completely, therefore, some bacteria were detected in BAL fluid, but the doses were effective in reducing any inflammatory reaction. It was difficult to demonstrate infection by other pathogenic bacteria, such as Gram-positive cocci, because the rats were housed in a clean air room. Rats bearing lung lesions may be susceptible to bacterial infection and prolonged inflammation. In the present study, the numbers of bacteria in the rat lungs could not be estimated, but the combination of antibiotics and anti-inflammatory drugs clearly reduced inflammatory cell infiltration while inhibiting the development of lung non-small cell carcinomas. Therefore, chronic inflammation may promote lung carcinogenesis initiated by BHP, so the present results may point to novel approaches to the chemoprevention of lung non-small cell carcinoma.
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Acknowledgments
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This study was supported by a Grant for Scientific Research Expenses for Health and Welfare Programs, and the 2nd term Comprehensive 10-year Strategy for Cancer Control and Cancer Prevention, from the Ministry of Health and Welfare.
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Notes
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1 To whom correspondence should be addressedEmail: ykonishi{at}nmu.gw.cc.naramed-u.ac.jp

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Received June 29, 1999;
revised October 19, 1999;
accepted October 29, 1999.