Ricerc@Sapienza

ACC is an endocrine tumor associated with a poor prognosis and a high risk of recurrence (Else et al. 2014). Currently MTT, an isomer of the insecticide DDT with adrenolytic activity, is the main compound used in therapy for advanced ACC. It inhibits enzymatic activity in the mitochondria of steroidogenic cells, leading to a reduction in cortisol levels (Lehmann et al. 2013). Therapeutic MTT concentrations ranging from 14 and 20 mg/L are associated with anti-neoplastic activity (Kerkhofs et al. 2013), with an average response rate of up to 55% (Baudin et al. 2001). The recent ESMO guidelines on management of adrenal cancer recommend the use of MTT in an adjuvant setting in patients with stage III ACC with Ki-67>10% and incomplete (R1) or unrecognizable (Rx) surgical resection (Berruti et al. 2012). As demonstrated, MTT affects the enzymatic cascade in steroidogenic cells (Lehmann et al. 2013), potentially including testis tissue. Further thought must be given to the question of treatment with a drug interfering with steroidogenesis in a man regard to the endocrine and reproductive function. The lack of data in the literature on this topic probably results from the rarity of this tumor but particularly from the high incidence of mortality due to the aggressiveness of ACC. Currently, clinical endocrinologists are frequently faced with the need to make a decision about the treatment of male patients of reproductive age suffering from ACC. Open issues to be addressed are related to impaired cells and damage to the steroidogenesis machinery in the testis. Data on this topic are very scarce, except for a few date regarding the needs to personalized androgen replacement in patients with ACC treated with MTT (Chortis et al. 2013).
We intend to use in vivo model to study the effect of MTT on testicular functions.
MTT has always been considered an adrenolytic drug able to impair steroidogenesis, resulting in focal degeneration in the fasciculata and reticular zona of the adrenal cortex (Bergenstal et al. 1960). Recently, several studies have noted the effect of MTT on mitochondrial machinery. In a previous study, performed in the H295R cell line, we demonstrated in a total cell extract or in a mitochondria-enriched fraction that MTT treatment interfered with the expression of D-3-PGDH isoforms and adrenodoxin reductase (AdR), enzymes strongly involved in the mechanism of redox potential and electron transfer from NADPH to ferredoxin, which provides electrons to the mitochondrial P450 (CYP) cytochromes (Stigliano et al. 2008). Another study by Poli et al. demonstrated that the cytotoxic effect of MTT seems to be mainly mediated by a specific mitochondrial disruption, leading to the impairment of steroidogenesis (Poli et al. 2013). Mitochondrial machinery plays a crucial role in the initiation of steroidogenesis through StAR, which transports cholesterol to the inner mitochondrial membrane by presenting it to key enzymes, such as CYP11A, responsible for the biosynthesis of pregnenolone, the precursor for all steroid hormones. It is commonly observed that mitotane therapy prevents clinical hyperandrogenism in women with Cushing's disease (Baudry et al. 2012). Moreover, Salenave et al. demonstrated a reduction in testosterone and its immediate precursor ¿4-androstenedione in premenopausal women affected by ACC and Cushing¿s disease and treated chronically with mitotane (Salenave et al. 2015). In a previous study on the ovary, we demonstrated that treatment of CD1 mice with mitotane induced a decrease in early antral follicles with a subsequent increase in the secondary follicles, measured by the increased levels of Anti Mullerian Hormone and decreased levels of FSH receptor. Moreover, we observed a significant decrease in Cyp11a1 and Cyp17a1 mRNA level, in mitotane-treated animals. The ovulation, induced by PMSG/hCG stimulation, was also significantly impaired, with a reduction in the number of ovulated oocytes and fewer corpora lutea in treated animals. Likewise, the mating experiment demonstrated a delay in the time of conception as well as fewer pups per litter in mitotane-treated mice. Experiments performed on the COV-434 cell line, showed a significant inhibition of growth followed by apoptosis. In conclusion, our study highlights the key points of ovarian folliculogenesis affected by MTT and demonstrates impairment of the ovulation process with a negative impact on conception, which is nevertheless preserved (Innocenti et al 2017). In the present study we want to investigate the effect of mitotane on the male gonad in order to understand the precautions to be taken before the treatment of patients with ACC.