Vitamin A and β-carotene inhibitory effect during 1,2-dimethylhydrazine induced hepatocarcinogenesis potentiated by 5-azacytidine

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Abstract

5-Azacytidine is being used for reactivation of tumor suppressor genes. However, its administration during DNA repair pontentiates hepatocarcinogenesis. We observed chemopreventive activities by vitamin A and β-carotene during early hepatocarcinogenesis. Thus, in the present study we evaluated vitamin A and β-carotene chemopreventive potential during early hepatocarcinogenesis potentiated by 5-azacytidine. Wistar rats received vitamin A (VAA group), β-carotene (βCA group) or corn oil (CO and COA groups). After three weeks of treatment, all animals were initiated with 1,2-dimethylhydrazine. Twelve hours later VAA, βCA and COA groups received a single dose of 5-Azc. Hepatocytes were selected/promoted by 2-acetylaminofluorene and 70% partial hepatectomy. All animals were sacrificed six weeks after initiation. Compared to CO group (without 5-azacytidine), COA group presented higher (p < 0.05) nodule multiplicity, larger (p < 0.05) γ-GT positive lesions that occupied a larger (p < 0.05) area of liver section. Compared to COA group, VAA group presented decreased (p < 0.05) nodule multiplicity while βCA group tended to present smaller γ-GT positive lesions and to decrease occupied liver section. These results reinforce vitamin A and β-carotene chemopreventive potential. Considering that 5-azacytidine potentiates hepatocarcinogenesis, more studies are needed to elucidate the efficacy and safety of this drug for cancer control.

Introduction

CpG island hypermethylation is an important epigenetic mechanism leading to tumor suppressor gene silencing during carcinogenesis (Jones and Baylin, 2002). In several cancers, p16, E-caderin and retinoic acid receptor β promoters are frequently hypermethylated (Lee et al., 2003, Yang et al., 2003). Thus, DNA hypomethylating agents, such as 5-Azc, have been considered for tumor suppressor gene upregulation during carcinogenesis (Esteller, 2003).

5-Azc covalent binding to DNA methyltransferase leads to a global DNA hypomethylation, including genes previously silenced by hypermethylation (Bender et al., 1998). However, 5-Azc could also induce chromosome instability, mutations (Karpf and Jones, 2002) and altered testicular histology (Doerksen et al., 2000). Moreover, it also modulates carcinogenesis (Carr et al., 1984, Prasanna et al., 1995). Specifically during hepatocarcinogenesis, 5-Azc acted as a promoter agent after initiation with DEN (Carr et al., 1984) and potentiated initiation with 1,2-DMH when administrated specifically during DNA repair (Denda et al., 1985).

Using the RH model of hepatocarcinogeneis with DEN as the initiation agent, we observed chemopreventive activities by vitamin A and β-carotene when administrated specifically during initiation (Moreno et al., 1991), early promotion (Rizzi et al., 1997), both initiation and promotion (Moreno et al., 1991, Moreno et al., 1995, Fonseca et al., 2005) and progression (Silveira et al., 2001, Moreno et al., 2002) phases of carcinogenesis.

There is no data on vitamin A and β-carotene chemopreventive activities in a hepatocarcinogenesis model potentiated by 5-Azc. Thus, we evaluated in the present study the effect of vitamin A and β-carotene treatment in Wistar rats during initiation and promotion phases of the RH model initiated with 1,2-DMH and potentiated by 5-Azc administration during DNA repair (Denda et al., 1985).

Section snippets

Animals and experimental design

Male Wistar rats (n = 36) from the colony of the Faculty of Pharmaceutical Sciences, initially weighing 77 g, maintained in cages of four animals, at constant temperature (22 °C), with 12-h light–dark cycle and receiving water and commercial diet ad libitum, were used.

Fig. 1 illustrates the experimental protocol design. At the end of 7-day acclimatization period, rats were randomly divided into four experimental groups. VAA and βCA groups were treated with vitamin A (1 mg/100 g body weight; retinyl

Results

During the experimental period, no differences were observed regarding growth and diet intake between the groups (data not shown).

Table 1 presents data on final body and absolute and relative liver weights, and nodule incidence and multiplicity of CO, COA, VAA and βCA groups. No differences (p > 0.05) were observed between the different groups regarding body and liver weights and nodule incidence. Compared to CO group (not treated with 5-Azc), COA group presented higher (p < 0.05) nodule

Discussion

The results of the present study show that 5-Azc administration potentiated hepatocarcinogenesis induced by 1,2-DMH, as previously described (Denda et al., 1985). 5-Azc is incorporated into DNA during S phase of the cell cycle round elicited by the carcinogen, when DNA repair synthesis occurs (Denda et al., 1985). However, in the absence of the carcinogen, 5-Azc administration after PH, when its incorporation should be maximum, does not result in any carcinogenic affect (Carr et al., 1984,

Conflict of interest statement

There are no conflicts of interest that could influence the outcome of the present study.

Acknowledgements

The authors thank Miss Silvania M.P. Neves for providing the care and maintenance of the animals. This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Técnico e Cientı´fico (CNPq).

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