DNA fragmentation, DNA repair and apoptosis induced in primary rat hepatocytes by dienogest, dydrogesterone and 1,4,6-androstatriene-17β-ol-3-one acetate

Abstract

Four steroids that share the 17-hydroxy-3-oxopregna-4,6-diene structure – cyproterone acetate, chlormadinone acetate, megestrol acetate, and potassium canrenoate – have been shown previously to behave with different potency as liver-specific genotoxic agents, the response being markedly higher in female than in male rats, but similar in humans of both genders. In this study, performed to better define the relationship between chemical structure and genotoxicity, dydrogesterone (DGT) with double bonds C4C5 and C6C7, dienogest (DNG) with double bonds C4C5 and C9C10, and 1,4,6-androstatriene-17β-ol-3-one acetate (ADT) with double bonds C1C2, C4C5 and C6C7, were compared with cyproterone acetate (CPA) for their ability to induce DNA fragmentation and DNA repair synthesis in primary cultures of hepatocytes from three rats of each sex. At subtoxic concentrations, ranging from 10 to 90 μM, all four steroids consistently induced a dose-dependent increase of DNA fragmentation, which in all cases was higher in females than in males; their DNA damaging potency decreased in the order CPA > DNG > ADT > DGT. Under the same experimental conditions, the responses provided by the DNA repair-synthesis assay were positive or inconclusive in hepatocytes from female rats and consistently negative in hepatocytes from male rats. In the induction of apoptotic cells, examined in primary hepatocytes from female rats, CPA was more active than ADT and DGT, and DNG was inactive. Considered as a whole these findings suggest that a liver-specific genotoxic effect more marked in female than in male rats might be a common property of steroids with two or three double bonds.

Introduction

In the last 10 years evidence has been published that the three progestins, cyproterone acetate, chlormadinone acetate and megestrol acetate, and a competitive aldosterone antagonist, potassium canrenoate, which all share the 17-hydroxy-3-oxopregna-4,6-diene structure, give negative responses in standard genotoxicity assays, but are transformed by hepatocytes into DNA-reactive species and behave as liver-specific genotoxic carcinogens [1]. Therefore, these compounds should be considered not only as promoters but also as potentially capable of acting as initiators of liver cancer [1]. The three progestins were found to induce genotoxic effects in both rat and human hepatocytes, but the response was markedly higher in female than in male rats, and in contrast similar in humans of both genders. In this respect, it is worth noting that a clear-cut species specificity is displayed by drospirenone, another progestin with only one double bond (between C4 and C5), which was found to be markedly genotoxic to hepatocytes from rats of both genders, but completely inactive in those from both male and female humans [2]. In contrast, neither species nor sex specificity was observed with potassium canrenoate.

The aim of the present study was to verify the possible genotoxic activity of other progestins and to provide additional information on the relationship between chemical structure and ability to induce DNA lesions in liver cells. Three progestins that differ in the position and number of double bonds were examined for their capacity to induce DNA fragmentation and DNA repair synthesis in primary cultures of hepatocytes from rats of both genders: dydrogesterone (DGT), dienogest (DNG), and 1,4,6-androstatriene-17β-ol-3-one acetate (ADT) (Fig. 1). In addition, the three progestins were tested for their capacity to cause apoptosis, but this activity was examined only in primary hepatocytes from female rats, due the previously assessed more marked genotoxicity in this sex.