Biotransformation of chlorpropham (CIPC) in isolated rat hepatocytes and xenoestrogenic activity of CIPC and its metabolites by in vitro assays

Abstract

1. The metabolism and action of chlorpropham (isopropyl N-(3-chlorophenyl)carbamate; CIPC, a post-harvest agent) were studied in freshly isolated rat hepatocytes, and the oestrogen-like activity of CIPC and its metabolites was assessed by in vitro assays. The exposure of hepatocyte suspensions to CIPC caused concentration- (0.25–1.0 mM) and time- (0–3 h) dependent cell death, which was assessed by Trypan blue exclusion, accompanied by losses of cellular adenosine triphosphate and adenine nucleotide pools, and formation of cell bleb.

2. CIPC at a weakly toxic level (0.25 or 0.5 mM) was metabolized to isopropyl N-(3-chloro-4-hydroxyphenyl)carbamate (4OH-CIPC) and subsequently to its glucuronide and sulfate conjugates (major metabolites) or alternatively to the minor metabolites 3-chloroaniline (3CA) and 3-chloroacetanilide. CIPC (0.25 mM) added to hepatocyte suspensions was distributed equally between hepatocytes and the extracellular medium during the incubation. The glucuronide rather than the sulfate conjugate of 4OH-CIPC predominantly increased in the medium with time, while the amount of unconjugated free 4OH-CIPC in the extracellular medium increased by approximately threefold compared with the amount in the cell fraction after 0.5 h and then decreased rapidly accompanied by increases in the conjugates. This indicates that unconjugated free 4OH-CIPC produced in hepatocytes was temporarily excreted in the extracellular medium and subsequently converted to the conjugates via re-influx into hepatocytes.

3. Diethylstilbestrol (DES), bisphenol A (BPA) and 4-hydroxybenzoic acid butyl ester (butylparaben), which are known xenoestrogenic compounds, competitively displaced 17β-oestradiol bound to the oestrogen receptor-α (ERα) in a concentration-dependent manner; IC₅₀ values of DES, BPA, butylparaben and its derivative 3-chloro-4-hydroxybenzoic acid butyl ester (3-chloro-butylparaben) were approximately 10⁻⁸, 10⁻⁵, 5 × 10⁻⁵ and 5 × 10⁻⁴ M, respectively. In contrast, neither CIPC nor 4OH-CIPC impaired the binding of 17β-oestradiol to ERα at concentrations ranging from 10⁻⁹ to 10⁻⁴ M, whereas at concentrations of >5 × 10⁻⁴ M, the binding affinity of 4OH-CIPC was greater than that of CIPC. In a proliferation assay of MCF-7 cells, CIPC, 4OH-CIPC and 3CA did not increase cell numbers at concentrations ranging from 10⁻⁹ to 10⁻⁵ M, but these compounds at a concentration of 10⁻⁴ M induced a considerable decrease in cell numbers relative to the control. The results suggest that even if CIPC is metabolized to 4OH-CIPC by hepatocytes, the chlorine adjacent to the 4-hydroxy group added to the intermediate as well as 3-chloro-butylparaben obstructs the appearance of oestrogen-like effects via an interaction between the intermediate and the ER.