Use of the γH2AX assay for assessing the genotoxicity of polycyclic aromatic hydrocarbons in human cell lines.

The development of in vitro genotoxic assays as an alternative method to animal experimentation is of growing interest in the context of the implementation of new regulations on chemicals. However, extrapolation of toxicity data from in vitro systems to in vivo models is hampered by the fact that in vitro systems vary in their capability to metabolize chemicals, and that biotransformation can greatly influence the experimental results. Therefore, much attention has to be paid to the cellular models used and experimental conditions. Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic ubiquitous pollutants. Human exposure to PAHs is mainly from food origin. In this study, a detailed analysis of the biotransformation capabilities of three human cell lines commonly used for in vitro testing (HepG2, ACHN and Caco-2) was undertaken using 3 model PAHs (benzo(a)pyrene [B(a)P], fluoranthene [FLA] and 3-methylcholanthrene [3-MC]). Concomitantly the genotoxicity of these PAHs was investigated in different cell lines, using a new genotoxic assay (H2AX) in 96-well plates. The metabolic rates of B(a)P, FLA and 3-MC were similar in HepG2 and Caco-2 cell lines, respectively, though with the production of different metabolites. The ACHN cell line was shown to express very limited metabolic capabilities. We demonstrated that the PAHs having a high metabolic rate (B(a)P and 3-MC) were genotoxic from 10(-7) molar in both HepG2 and Caco-2 cells. The present study shows that H2AX measurement in human cell lines competent for the metabolism, is an efficient and sensitive genotoxic assay requiring less cells and time than other currently available tests.

Urinary and biliary metabolic patterns of chlorothalonil in germ-free and conventional rats.

The metabolic fate of chlorothalonil, a broad spectrum fungicide that is known to be metabolized via glutathione conjugation, was examined through the analysis of urine and bile metabolites. The role of digestive microflora in the metabolism of chlorothalonil was assessed by comparing the metabolic patterns in germ-free and conventional rats. Low urinary and biliary excretion of radioactivity was observed in both conventional and germ-free rats. However, the urinary excretion of radioactivity was higher in conventional than in germ-free rats. Radio-HPLC analysis of urine and bile showed a complex metabolic profile in both conventional and germ-free rats. Methylthio metabolites of chlorothalonil were determined in ethyl acetate extracts of urine and bile of conventional and germ-free rats. These metabolites were excreted in a higher amount in the urine of conventional rats than in the urine of germ-free rats. This study shows the complexity of chlorothalonil metabolism and the role of the digestive microflora in chlorothalonil metabolism.

Biotransformation of pentachlorophenol, aniline and biphenyl in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes: comparison with in vivo metabolism.

1. The biotransformation of pentachlorophenol (PCP), aniline and biphenyl in rainbow trout (Oncorhynchus mykiss) isolated liver cells was investigated to examine if fish hepatocytes represent a suitable alternative to the in vivo approach for studying the biotransformation of chemicals. Each compound was incubated at two concentrations (10 and 60 microM) for 2 h. For comparison, the metabolic profile of these xenobiotics was also studied in urine and bile of trout orally exposed to 1.8-4.0 mg/kg wet wt of each compound. 2. In vitro as in vivo, PCP glucuronide and to a lesser extent PCP sulphate were the metabolites formed by trout from PCP. 3. Aniline was mainly metabolized to acetanilide and to a lesser extent to 2-aminophenol by isolated hepatocytes, but neither hydroxylated acetanilide nor conjugates were found in vitro whereas they were present in bile and urine of trout treated with this chemical. 4. Trout hepatocytes metabolized biphenyl to hydroxylated and dihydroxylated products and the corresponding glucuronides. These results correlated well with the metabolic profile obtained from the bile of trout exposed to this pesticide. 5. It is concluded that although hepatocytes are well suited for several types of biotransformation studies, the fact that this system may in some cases produce a different metabolic pattern than in vivo should be considered when attempting to extrapolate in vitro to in vivo data.

Ex vivo gastrointestinal biotransformation of chlorothalonil in the germ-free and conventional rat.

1. The metabolism and absorption of chlorothalonil and corresponding diglutathione and dicysteine conjugates was studied using isolated everted gastrointestinal sacs of the conventional and germ-free rat. An HPLC method was used to analyse mucosal and serosal fluids. Thiol metabolites of chlorothalonil were determined by GC/MS. 2. Low absorption of the substrates was observed, with < 4% of the radioactivity being recovered from the serosal buffers and the digestive tissues. A major part of the radioactivity was recovered from the mucosal fluids and it corresponded to unchanged chlorothalonil. Traces of unchanged chlorothalonil and mono-, di- and trimethylthio metabolites were present in serosal fluids as well as unidentified polar peaks. An important transformation (> 75%) of the chlorothalonil conjugates was observed. The di- and trimethylthio metabolites of chlorothalonil were detected from both sides of the everted gut sac of rat incubated with the diglutathione and dicysteine conjugates. 3. Few differences were observed between the conventional and germ-free rat: absorption was higher in the duodenum of germ-free rat, but tissue retention was more significant in the duodenum of the conventional rat.