Inter-laboratory ring trial of in vitro DNA repair tests using rat hepatocytes: further testing and conclusive remarks

Abstract

The results of the extension of a collaborative study for the detection of chemical-induced DNA damage in rat hepatocytes in vitro are presented in this report. Three coded compounds, i.e. 1,4-butanediol dimethanesulphonate, hydrazine sulphate and sodium dichromate, were tested for DNA repair synthesis by seven different laboratories, either using autoradiographic procedures or the liquid scintillation counting technique. Inter-laboratory standardization was intentionally not requested in order to investigate the validity of each study design under routine conditions. 1,4-Butanediol dimethanesulphonate was clearly positive in most laboratories; sodium dichromate was generally positive, while the results on hydrazine sulphate were contradictory.

Busulfan induces steatosis in HepaRG cells but not in primary human hepatocytes: Possible explanations and implication for the prediction of drug‐induced liver injury

“…In keeping with these in vitro results, investigations in mice showed that repeated administration of 33‐mg/kg busulfan induced a significant decrease in hepatic GSH levels [13]. Finally, earlier investigations in cultured rat hepatocytes reported that busulfan induced DNA damage [14].…”

“…William's E medium, Dulbecco's phosphatebuffered saline (PBS) Gibco™, glutamine, penicillin, streptomycin, formaldehyde, Nile red, and Hoechst 33342 dyes were obtained from Thermo Fischer Scientific (Waltham, MA). Radiolabeled [U- 14 C]palmitic acid and [2-14 C]acetic acid, sodium salt were purchased from PerkinElmer (Waltham, MA). Hydrocortisone hemisuccinate was obtained from SERB Laboratories (Paris, France).…”

“…T A B L E 1 Information on the six donors whose liver pieces were used for the preparation of primary human hepatocytes (PHH). Mitochondrial fatty acid oxidation (FAO) was assessed by measuring the acid-soluble radiolabeled metabolites resulting from the mitochondrial oxidation of [U- 14 C] palmitic acid, as recently described [15]. In brief, culture medium was removed and cells were washed with warm PBS before addition of phenol red-free William's E medium containing 1% fatty acid-free BSA, 100-μM cold palmitic acid, [U- 14 C]palmitic acid (185 Bq per well, corresponding to 0.5 pM), 1-mM L-carnitine, and 1% DMSO.…”

“…Mitochondrial fatty acid oxidation (FAO) was assessed by measuring the acid-soluble radiolabeled metabolites resulting from the mitochondrial oxidation of [U- 14 C] palmitic acid, as recently described [15]. In brief, culture medium was removed and cells were washed with warm PBS before addition of phenol red-free William's E medium containing 1% fatty acid-free BSA, 100-μM cold palmitic acid, [U- 14 C]palmitic acid (185 Bq per well, corresponding to 0.5 pM), 1-mM L-carnitine, and 1% DMSO. After 3 h of incubation at 37 C, perchloric acid (final concentration, 6%) was added and plates were centrifuged at 2000g for 10 min, and supernatant was counted for [ 14 C]-labeled acid-soluble β-oxidation products using a Tri-Carb 4910TR liquid scintillation counter (PerkinElmer).…”