Oxidative stress in carcinogenesis. Correlation between lipid peroxidation and induction of preneoplastic lesions in rat hepatocarcinogenesis
Introduction
There are clear examples of the participation of reactive oxygen species (ROS) in hepatocarcinogenesis in the rat. Nakae et al. [1] showed that initiation with low doses of N-diethylnitrosamine (DEN) induced liver DNA-8-hydroxydeoxy-guanosine adducts and suggested that oxidative stress participates in hepatocarcinogenesis. Therefore, one can assume that initiation with high doses of DEN produces oxidative stress, as in the case of hepatocarcinogenic models such as that by Solt and Farber, initiated with DEN and promoted with 2-acetylaminoflourene (2-AAF) and partial hepatectomy (PH) as proliferative stimulus [2], or modifications of this, as in the Semple-Robert's [3] model. During a choline-deficient diet, the representative marker of oxidative DNA damage 8-hydroxydeoxy-guanosin is induced [4] and the same is true after administration of cipofibrate, one of the more efficient peroxisome proliferators that induces liver cancer in the rat [5]. Trimethylarsine oxide, an organic metabolite of inorganic arsenics, produced liver tumors in male Fischer 344 rats and authors implicate a possible mechanistic role of oxidative DNA damage and enhanced cell proliferation [6]. Also, in male Fischer 344 rats, it was demonstrated that α,α-bis(p-chlorophenyl)-β,β,β trichloroethane (DDT) induces eosinophilic foci and hepatocellular carcinoma (HCC) as a result of oxidative DNA damage [7].
It is not clear yet if participation of oxidative stress depends on the DNA oxygen adducts or if there is a concomitant alteration of signalization by the abrupt induction of ROS during initiation, or if both, DNA damage and a new intracellular reduced steady state are necessary for carcinogenesis to take place. Evidence with U937 cells treated with two well-known nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and DEN showed that ROS are produced; these activate nuclear factor κB (NF-κB); subsequently, cyclooxygenase-1 (COX-1) activity is induced, and this pathway increases prostaglandin E2 (PGE2) synthesis [8]. These results, and evidence that acetylsalicylic acid, a non-steroidal anti-inflammatory drug, and NNK lung carcinogenesis inhibitors block activation of NF-κB, induction of COX-1 and PGE2 synthesis, together lend support to the proposition that ROS participate in carcinogenesis. When NNK or DEN were substituted by their respective O-acetate derivatives, which do not need to be metabolized, they did not activate NF-κB or induce PGE2 synthesis, even though it is known that these nitrosamines as well as their acetates produce DNA adducts [8], [9].
There is evidence that oxidative stress is an obligatory component of carcinogenesis. It was recently communicated that COX-1 or COX-2-deficient mice had altered epidermal differentiation and, when treated with 7,12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-12,13-acetate (TPA), they presented reduced skin tumorigenesis, although DMBA stable DNA adducts were increased twice [10]. This study clearly shows that there is no correlation between adduct levels and tumorigenesis. Even though the authors do not demonstrate that oxidative stress is produced, a direct relation between COX-1 and COX-2 deficiency is hypothetically associated to a lesser degree of oxidative stress [10].
We propose that, in the rat hepatocarcinogenesis model, both direct alkylating DNA damage and alterations produced by ROS induction during carcinogen metabolism are necessary processes for liver cancer induction. The dilemma is how to differentiate the participation of DNA alteration by ethyl adducts [9] from the participation of cell modifications induced by ROS or, even more, from the obliged hypothetical participation of both to induce initiation. To gain insight into this problem, experiments were carried out to compare (a) induction of gamma-glutamyl transpeptidase-positive (GGT+) preneoplastic lesions on the 25th day, as an early end point of hepatocarcinogenesis in a DEN-2AAF-PH model or (b) in the same model, substituting DEN by N-ethyl-N-nitrosourea (ENU), a direct carcinogen, that is only carcinogenic in the rat liver under very special circumstances [11], [12], [13] or (c) by administration of quercetin, an antioxidant, administered previous to the DEN, 2AAF treatment. And as a key comparative feature, in these three different groups, LPX was measured during the initiation period.
Section snippets
Reagents and animals
All reagents were purchased from SIGMA (St Louis, MO, USA). Male Fischer-344 rats (180–200 g) were obtained from the CINVESTAV animal house, they had access to food (PMI Feeds, Inc., Laboratories Diet) and water at all times; food cups were replenished three times weekly. All animals received humane care and the study protocols were in compliance with the institutional guidelines for use of laboratory animals.
Experimental protocols
Two groups of rats were initiated with a dose of 200 mg/kg of DEN and a third group with
Preneoplastic lesions
Liver preneoplastic nodules were induced with a modified Semple-Roberts model [3], under three different conditions as shown in Fig. 1. The GGT+ lesions were measured at day 25 and our reference group initiated by DEN clearly presented abundant and high-intensity GGT+ stained preneoplastic lesions while the other two groups of either ENU as initiator or quercetin plus DEN presented a few high-intensity GGT+ stained preneoplastic lesions. However, all groups showed diffuse low-intensity GGT+
Discussion
To gain insight into oxidative stress participation in the induction of preneoplastic lesions, we analyzed the effects during cancer initiation with ENU, a direct and seldom rat liver carcinogen [18] and with DEN an indirect and very efficient rat liver carcinogen, followed by 2AAF administration as a promoter and PH as proliferative stimulus. We chose to evaluate GGT+ nodule formation at day 25 after initiation and LPX during 24 h after initiation. We show that DEN, a ROS-generating carcinogen
Acknowledgements
We would like to thank Samia Fattel and Evelia Arce Popoca for technical assistance during this project. We also wish to acknowledge the excellent technical support of Jorge Fernandez, head of the Animal House and Manuel Flores, Rafael Leyva and Ricardo Gaxiola. We thank Isabel Pérez Montfort for revising the English version of the manuscript. This work was supported by grants 31665-N and 34547-M from CONACyT, México, DF, Mexico. Fellowship from CONACyT: YSP;144244, CECL;112857, JIPC;144549,
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