Biotransformation of chlorpropham (CIPC) in isolated rat hepatocytes and xenoestrogenic activity of CIPC and its metabolites by in vitro assays.
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 17beta-oestradiol bound to the oestrogen receptor-alpha (ERalpha) 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 x 10(-5) and 5 x 10(-4) M, respectively. In contrast, neither CIPC nor 4OH-CIPC impaired the binding of 17beta-oestradiol to ERalpha at concentrations ranging from 10(-9) to 10(-4) M, whereas at concentrations of >5 x 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.
Nakagawa
Y
Y
Division of Pharmacology, Tokyo Metropolitan Research Laboratory of Public Health, 3-24-1, Hyakunin-cho, Shinjuku-ku, Tokyo 169-0073, Japan. yoshio_1_Nakagawa@member.metro.tokyo.jp
Nakajima
K
K
Suzuki
T
T
D000818:Animals; D001711:Biotransformation; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D002748:Chlorpropham / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000378:metabolism*; D004968:Estrogens, Non-Steroidal / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism* / Q000473:pathology; D006540:Herbicides / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D008297:Male; D011485:Protein Binding; D051381:Rats; D011916:Rats, Inbred F344; D011959:Receptors, Estradiol / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004968:Estrogens, Non-Steroidal; D006540:Herbicides; D011959:Receptors, Estradiol; D002748:Chlorpropham; D004958:Estradiol