Elsevier

Nutrition

Volume 22, Issue 9, September 2006, Pages 940-946
Nutrition

Basic nutritional investigation
Modulation of xenobiotic-metabolizing enzymes and redox status during chemoprevention of hamster buccal carcinogenesis by bovine lactoferrin

https://doi.org/10.1016/j.nut.2006.05.017Get rights and content

Abstract

Objective

Chemoprevention by dietary constituents has emerged as a novel approach to control oral cancer incidence. We therefore evaluated the chemopreventive efficacy of bovine milk lactoferrin (bLF) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis.

Methods

Hamsters were divided into four groups. The right buccal pouches of animals in groups 1 and 2 were painted with 0.5% DMBA three times a week for 14 wk. Animals in group 2, received in addition, basal diet containing 0.2% bLF. Group 3 animals were given 0.2% bLF alone. Group 4 animals served as control. The status of carcinogen-metabolizing enzymes, the extent of lipid peroxidation and glutathione-dependent antioxidants in the buccal pouch and liver as well as bone marrow micronuclei incidence were used as biomarkers.

Results

All the hamsters painted with DMBA alone for 14 wk, developed HBP carcinomas that showed diminished lipid peroxidation and increased activities of carcinogen-metabolizing enzymes and antioxidants with enhanced bone marrow micronuclei. In the liver of tumor bearing animals, the increase in phase I enzymes and lipid peroxidation was accompanied by reduced activities of antioxidant and phase II detoxification enzymes. Administration of bLF decreased the incidence of DMBA-induced micronuclei and HBP carcinomas by decreasing phase I enzymes, modulating lipid peroxidation and enhancing antioxidant and phase II enzyme activities.

Conclusion

The chemopreventive effects of bLF is mediated by reducing DMBA-induced genotoxicity and modulating carcinogen-metabolizing enzymes and oxidant-antioxidant profile in the target organ as well as in the liver.

Introduction

Oral cancer, a common malignancy worldwide, is an important contributor to cancer morbidity and mortality and to overall international cancer burden. In India, oral cancer incidence constitutes 9.8% of estimated cancer cases, accounting for 75,000–80,000 new cases annually [1]. Chemoprevention has evolved as a promising approach to control the incidence of oral cancer. However, it is important to establish chemoprevention in an experimental tumor model that closely resembles human oral squamous cell carcinoma before embarking on clinical trials.

The hamster buccal pouch (HBP) carcinogenesis model is the best-known animal system for investigating the efficacy of chemopreventive agents. HBP carcinomas induced by the application of 7,12-dimethylbenz[a]anthracene (DMBA) to the cheek pouch of Syrian hamsters are morphologically and histologically similar to human oral squamous cell carcinomas. In addition, hamster tumors express many biochemical markers that are expressed in human oral cancer [2], [3]. The HBP model has been extensively used in this laboratory to demonstrate the chemopreventive efficacy of medicinal plants and dietary agents [4], [5], [6].

Chemoprevention by dietary agents has emerged as a cost-effective method for preventing oral cancer incidence. These agents may act by multiple pathways to block tumorigenesis [7]. Among the diverse pathways, modulation of carcinogen-induced genotoxicity, inhibition of carcinogen activation by altering the activities of phase I and II enzymes, and scavenging of reactive oxygen species (ROS) by antioxidant defense systems have assumed significance.

Over the past decade, bovine milk lactoferrin (bLF), an 80-kDa iron-binding glycoprotein present in whey protein fraction of milk, has attracted the focus of attention because of its wide range of beneficial effects. It is present in raw milk at a concentration of 0.02–0.2 mg/mL. Although bLF is denatured during pasteurization, the protein can be detected in natural cheese and cheese whey and intake of dairy foods is believed to result in significant ingestion of bLF. Bovine LF has been documented to exhibit a wide range of pharmacologic properties including antimicrobial, antioxidant, immunomodulatory, antimetastatic, and anticarcinogenic activities. The anticancer activity of bLF has been demonstrated in experimental lung, bladder, tongue, colon, and liver carcinogeneses [8]. However, the protective effect of bLF in oral cancer has not been sufficiently documented.

The present study was designed to evaluate the mechanism underlying the chemopreventive potential of bLF on DMBA-induced genotoxicity and buccal pouch carcinogenesis in Syrian hamsters. The incidence of bone marrow micronuclei, an indicator of DNA damage, the status of phase I (cytochrome P450) and phase II (glutathione S-transferase [GST] and DT-diaphorase [DTD]) enzymes, extent of ROS-induced lipid peroxidation, and scavenging by reduced glutathione (GSH)-dependent antioxidants were used as biomarkers of chemoprevention.

Section snippets

Chemicals

DMBA was purchased from Sigma Chemical Company (St. Louis, MO, USA), and bLF (lot no. 020119) of ≥96.2% purity was obtained from Morinaga Milk Industry Co., Ltd. (Tokyo, Japan). The iron content of bLF was 18 mg/100 g. All other reagents used were of analytical grade.

Animals and diets

The experiment was carried out with male Syrian hamsters that were 8–10 wk old, weighed 100–110 g, and were obtained from the Central Animal House, Annamalai University (Annamalai, India). The animals were housed five to a

Macroscopic and microscopic observations

Table 1 summarizes the mean body weights, food consumption, and the frequency of bone marrow micronuclei in the different groups. Hamsters in group 1 showed a tendency to be lower in body weight gain during the experiment and the mean final body weights were decreased compared with the control group (group 4). No significant differences in body weights were observed in groups 2–4. The amount of diet consumed in groups 1–4 was not significantly different.

A significant increase in the frequency

Discussion

In the present study, topical application of DMBA to the HBP for 14 wk resulted in well-developed squamous cell carcinoma with a very high mean tumor burden associated with severe hyperplasia, hyperkeratosis, and dysplasia. This was accompanied by an imbalance in phase I and II xenobiotic-metabolizing enzymes, cellular redox status, and increased frequency of bone marrow micronuclei. Being an indirect carcinogen, DMBA is metabolically activated by cytochrome P450 mono-oxygenases to form

Acknowledgments

The authors are thankful to Dr. S. K. Abraham, School of Life Sciences, Jawaharlal Nehru University, New Delhi, for help with analysis of micronuclei.

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Financial support from the Indian Council of Medical Research, New Delhi, India, in the form of a Senior Research Fellowship to K. V. P. Chandra Mohan is gratefully acknowledged.

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