**1. Introduction**

Intracellular reactive oxygen species (ROS) [1–3] are crucial for normal cell metabolism [3–6] and ROS generation is highly regulated by either enzymatic (catalases, peroxidases and dismutases) or non-enzymatic (vitamin A, C or E) reductive molecules. Disturbances in cellular redox balance can lead to an over-accumulation of ROS [1]. Cells also produce ROS after exposure to radiation or chemotherapeutics [7], many of which induce ROS to toxic levels as part of their mechanism of action [8].

The combination of doxorubicin (Dox) and cyclophosphamide is often used to treat breast cancer [9–11]. Dox is an anthracycline agen<sup>t</sup> that causes apoptosis by intercalating into double-stranded DNA and inhibiting topoisomerase-II [12]. A second mechanism of action involving ROS has also been described [5,13,14].

Cyclophosphamide is an alkylating agen<sup>t</sup> that requires hepatic metabolic activation [15], which generates 4-hydroxycyclophosphamide and aldophosphamide [16]. Aldophosphamide is metabolized into phosphoramide mustard and acrolein, which increases ROS production in a variety of cell lines [17,18].

Dox and cyclophosphamide are associated with a variety of adverse e ffects in vivo, including T-cell suppression, chronic cardiotoxicity and premature ovarian failure [19–28]. It has been proposed that chemotherapeutics cause premature ovarian failure by targeting proliferating granulosa cells in growing follicles [22,23]. Since granulosa cells synthesise anti-Müllerian hormone (AMH), the loss of follicles due to chemotherapeutic-induced cytotoxicity causes a consequent depletion in circulating AMH, which results in the activation and recruitment of dormant primordial follicles into the growing pool [23]. It is thought that the in vivo administration of repeated cycles of Dox and cyclophosphamide diminishes the cohort of active growing follicles, reduces the reserve of primordial follicles, and results in premature ovarian failure [21,23–29].

Although the toxicity of cyclophosphamide and Dox (as single agents) on the ovary is well established [21,23–29] there are no reports that describe the e ffect of the combination of Dox and cyclophosphamide on proliferating granulosa cells.

The tocopherols (alpha, beta, gamma and delta) and tocotrienols (alpha, beta, gamma and delta) that together form Vitamin E [30] act as free radical scavengers in cell membranes [31]. α-tocopherol (αToc) is the most abundant form in nature, while γ-Tocopherol ( γToc) is the most common form in the human diet [31]. It has been proposed that Dox-induced cardiotoxicity is the result of ROS-induced membrane lipid peroxidation [32], and vitamin E deficiency results in histological features that are comparable to Dox-treated cardiac tissue [19,32]. Although the administration of αToc to breast cancer patients before chemotherapy elevated serum concentrations 8-fold, there were no other observable e ffects [19]. However, the e ffects of αToc on post-chemotherapeutic ovarian function were not examined. γToc delayed the formation of breast cancer tumours in rodent models [33], induced apoptosis in breast cancer cells in vitro [34,35] and may prevent breast cancer in vivo [36,37]. Additionally, a mixture of γ and delta tocopherol down-regulated the expression of estrogen receptor and inhibited estradiol-induced human MCF-7 breast cancer cell proliferation in vitro [36].

Both α and γ tocopherol are antioxidants with the potential to reduce chemotherapeutic-induced ROS damage, and consequently reduce premature ovarian failure. Reduced ROS, however, could also lead to decreased e fficacy against breast cancer cells. γToc has both reductive power and anticancer activity [33], and this led to our hypothesis that gamma tocopherol, but not alpha tocopherol, would augmen<sup>t</sup> the cytotoxic activity of the combination of Dox and cyclophosphamide against breast cancer cells in vitro, whilst simultaneously reducing ROS generation.
