Use of Rat and Human Liver Slices for the Detection of Steroid Hormone‐Induced DNA‐Adducts in vitro by Means of the ³²P‐Postlabeling Technique

Abstract

Precision cut liver slices from humans and rats were used to investigate the covalent binding of xenobiotics to the DNA by means of the (32)P-postlabeling technique. Human liver slices were incubated with the structurally related steroid hormones chlormadinone acetate (5 mu g/ml), cyproterone acetate (0.01-5 mu g/ml), megestrol acetate (5 mu g/ml), and the positive control 2-aminofluorene (0.01-5 mu g/ml), which is known for its marked ability to form DNA-adducts in vivo. Rat liver slices were incubated with cyproterone acetate in concentrations of 0.1, 1, and 5 mu g/ml. The functional viability and metabolic activity of the slices were shown to be sufficiently maintained during the incubation time by measurement of the intracellular K(+)-content and the metabolic turnover of the model substrate 7-ethoxycoumarin, respectively. All three test substances and the control induced DNA-adducts in human liver slices, however, with a different adduct pattern. While the total DNA-adduct levels obtained with cyproterone acetate and megestrol acetate were in the same order of magnitude (on average 1000 DNA-adducts/10(9) nucleotides after incubation with 5 mu g /ml), the relative adduct labeling calculated for chlormadinone acetate was about 400. Following in vitro incubation of rat liver slices with cyproterone acetate, the relative adduct labeling values increased proportionally with increasing concentrations and added linearily to in vivo generated DNA-adducts. At the level of liver slices, different DNA-adduct patterns were induced by cyproterone acetate in rat and man. In contrast to the finding of others, using rat hepatocytes, the relative adduct labeling values of cyproterone acetate and megestrol acetate were in the same order of magnitude after incubation with human liver slices. The present study indicates that liver slices are a useful tool to investigate the in vitro DNA-adduct inducing potential of xenobiotics.

Long term phenobarbital administration does not promote the multiplication of hepatocytes replicating after single cyproterone acetate administration

“…Because this drug is widely used in clinic, many studies have adressed its carcinogenic potential in human and most of the studies have come to the agreement that the liver cancer risk associated with the use of CPA is low, if it exists (Hinkel et al, 1996;Kasper, 2001;Kattan et al, 1994;Laron and Kauli, 2000;Rabe et al, 1996). Nevertheless, progestogens are still classified as potentially carcinogenic in human by the International Agency for Research on Cancer (IARC) and it was reported that in human liver slices, CPA induced DNA adducts (Baumann et al, 1996).…”

Cyproterone Acetate: A Genotoxic Carcinogen?

“…Formation of cyproterone acetate-DNA adducts in human liver cells was also reported by Bauman et al (1996) who used human liver slices of male and female donors for their studies and found a dose-dependent formation of one main adduct in a concentration range of 0.1-5 mg cyproterone acetate/ml. In comparison to isolated hepatocytes the liver slice system has some advantages in that the structural heterogeneity of the liver is preserved and thus the slices more closely resemble the in vivo situation than hepatocytes (Baumann et al 1996). In general, the good concordance between genotoxic effects found in human and rat cells at the in vitro level in response to cyproterone acetate suggests that the female rat appears to be appropiate for modelling adverse effects related to genotoxicity in the human liver.…”

Workshop Overview: Scientific and Regulatory Challenges for the Reduction, Refinement, and Replacement of Animals in Toxicity Testing,

“…One approach is to compare the effect of a drug in cultured human and animal tissue, providing information about the potential species-specificity of drug-induced adverse effects, otherwise unavailable in the preclinical phase of safety testing. Precisioncut tissue slicing has been a very useful tool in this respect (Smith et al, 1985), and has been increasingly used for interspecies comparisons of toxicity (Fisher et al, 1991(Fisher et al, , 1995Price et al, 1996), metabolism (Steensmae/ al., 1994Connors et al, 1996), liver enzyme induction (Glockner et al, 1995;Heinonen et al, 1996;Lake et al, 1996a), and genotoxicity (Baumann et al, 1996;Beamand et al, 1996;Lake et al, 1996b). Using the same preparative technique in all species, slices are relatively simple to prepare from almost any organ, including important targets of toxicity, such as the liver (Smith et al, 1985), lung (Fisher et al, 1994;Price et al, 1995a,b), and kidney (Ruegg, 1994).…”