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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: publication, detailed information of analogue was used for read-across.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1995

Materials and methods

Objective of study:
other: to investigate GSTc- d GSTm-catalyzed glutathione S-conjugate formation in rat and uman hepatocytes and in Hep G2 cells.
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
CTFG biosynthesis and the activities of GSTc and GSTm were examined in rat and human hepatocytes and in Hep G2 cells. Hepatocytes of >88% viability were obtained from rat or human liver slices by collagenase or collagenase + dispase digestion, respectively. HepG2 cells were grown in modified Earle’s medium supplemented with 10% (v/v) fetal calf serum. Cells and subcellular fractions were exposed to chlorotrifluoroethene, and CTFG formation was quantified by HPLC.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
no details given
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
Isolated rat hepatocytes were prepared from male, Sprague-Dawley rats (200-300 g; Charles River Laboratories, Wilmington,MA). Pathologically normal human liver tissues were obtained from donors for transplantation. Samples of liver tissues were excised and placed in ice-cold buffer (Viaspan; DuPont-Merck Pharmaceutical Co., Wilmington, DE) for transport to the laboratory.

Administration / exposure

Statistics:
Data were analyzed with a two-tailed Student’s t test. A level of P < 0.05 was chosen for acceptance or rejection of the null hypothesis.

Results and discussion

Any other information on results incl. tables

GSTc and GSTm Activities: GSTc and GSTm activities in human liver tissue and Hep G2 cells were compared with those of rat liver tissue. GSTc and GSTm activities in human liver tissue were compa- rable with those of rat, although interdonor differences were observed in GSTc activities. Hep G2 cells examined 8 days after subculture also showed GSTc and GSTm activities, which were,

however, lower than those of rat or human liver tissue. The ratios of GSTc to GSTm activities were 31.6 in rat, 23.4-36.7 in human, and 13.5 in Hep G2 cells. GST activities of Hep G2 cell 9,000g supernatant fractions prepared 2 and 8 days after subculture were similar (18.5 ± 1 .8 and 17.7 ± 1 .6 nmol/mg protein/min, respectively, N=3). The presence of GSTm in Hep G2 cells was also demonstrated by Western blotting with anti-GSTm antibodies. An immunore- active protein (M, 19,000) was detected in rat microsomes and in human and Hep G2 cells microsomes, although the bands were less intense than in rat microsomes. No immunoreactive proteins were observed in cytosol from human liver or Hep G2 cells.

Enzyme-Catalyzed CTFG Formation: The tissue: air partition coefficients determined for chlorotrifluoroethene were blood, 4. 1; liver, 3.4; muscle, 2.8; and fat, 90.3. The olive oil:air partition coefficient was 90.4. The concentration of chlorotrifluoroethene in incu- bation mixtures based on the partition coefficient was 49.3 µmol/ml. Rat and human liver and Hep G2 cell subcellular fractions catalyzed CTFG formation from chlorotrifluoroethene and glutathione. The rate of CTFG formation measured after 20 min of incubation was proportional to the protein concentration in the range of 0.1- 1.0 mg/ml for cytosol and microsomes of rat and human liver, and 0.05-0.2 mg/ml for cytosol and microsomes of Hep G2 cells. The rate of CTFG formation in the presence of cytosol (0.5 mg protein/ml for rat and human liver, and 0.1 mg proteinlml for Hep G2 cells) and microsomes (same protein concentrations as cytosol) was linear for 30 min. In all preparations, microsomal fractions exhibited higher specific activities with chlorotrifluoroethene than did cytosolic fractions: 3.4-fold in rat, 1.2- to 2.5-fold in human, and 2.0-fold in Hep G2 cells.

Cytotoxicity of Chiorotrifluoroethene: In preliminary studies, rat hepatocytes were incubated with 2, 5, or 10 ml of chlorotrifluoroethene in closed 25-ml flasks. Addition of 5 or 10 ml of chlorotrifluoroethene produced a time-dependent decrease in cell viability to 2 ml of chiorotrifluoroethene was comparable with that of control cells. Based on these results, 2 ml of chlorotnfluoroethene/25 ml flasks was used. Hepatocytes >88% viable were obtained from both rat and human liver tissue with the isolation procedure described herein. Rat and human hepatocytes incubated in the absence of chlorotrifluoroethene were >81% viable after 60 min of incubation. No cytotoxicity was observed in human hepatocytes or Hep G2 cells incubated with 2 ml of chlorotrifluoroethene/25 ml flasks up to 60 min of incubation.

Biosynthesis of CTFG in Cells: The concentration of chlorotrif- luoroethene in incubation mixtures based on the partition coefficient was 8.3 µmol/ml. Time-dependent formation of CTFG was observed in rat and human hepatocytes and in Hep G2 cells incubated with chlorotrifluoroethene in the presence of N-acetyl-L-cysteine. CTFG formation was observed in human hepatocytes, although hepa- tocytes from donor 4 showed low rates of CTFG biosynthesis. Hep G2 cells incubated 2 days after subculture also synthesized CTFG in a time-dependent manner, although the rate of formation was lower than in rat hepatocytes.

To investigate further the biosynthesis of CTFG in Hep G2 cells, cells were incubated with chlorotrifluoroethene 2, 4, or 8 days after subculture in the absence or presence of glutathione precursors. Time- dependent formation of CTFG was observed in all groups on all experimental days. Hep G2 cells examined 2 days after subculture synthesized the largest amount of CTFG in all groups, and the amounts declined gradually at 4 and 8 days after subculture. Among the precursors studied, only N-acetyl-L-cysteine stimulated the biosynthesis of CTFG in Hep G2 cells.

Effects of Chlorotrifluoroethene on Glutathione and Glutathine Disulfide Concentrations: Cellular glutathione concentrations in control cells decreased from 11.6 to 8.6 nmol/106 cells during e first 40 min of incubation. Cellular glutathione concentrations in ontrol cells examined at 4 and 8 days after subculture were 9.8 and 6.0 nmol/l06 cells, respectively. Glutathione concentrations in cells posed to chlorotrifluoroethene in the absence of N-acetyl-L-cysteine decreased to 4.8 nmol/106 cells after 60 min of incubation, whereas the glutathione concentrations in cells incubated with N-acetyl-L- cysteine were comparable with those of control cells. Incubation of Hep G2 cells with chlorotrifluoroethene caused no significant change in the glutathione disulfide concentrations. Chlorotrifluoroethene also had no effect on glutathione disulfide concen- trations in rat or human hepatocytes.

Synthesis of Glutathione in Hep G2 CelLs: Total concentrations of CTFG, glutathione, and glutathione disulfide in Hep G2 cells exposed to chlorotrifluoroethene in the absence of glutathione precursors were measured after 20 min of incubation on 2, 4, and 8 days after subculture. The concentrations were similar to cellular glutathione contents at the beginning of incubation on 2, 4, and 8 days after subculture ( 11.6, 9.8, and 6.0 nmol, respectively). Total concentrations of CTFG, glutathione, and glutathione disulfide were also measured in Hep G2 cells incubated with N-acetyl-L- cysteine, a L-cysteine precursor. On all experimental days, the total concentrations were larger than those in cells incubated in the absence of N-acetyl-L-cysteine, indicating that Hep G2 cells utilized N-acetyl- L-cysteine as a L-cysteine source. The difference in total concentrations of CTFG, glutathione, and glutathione disulfide between cells incubated with and without N-acetyl-L-cysteine was greatest at 2 days after subculture and declined gradually at 4 and 8 days after subculture. L-Cysteine concentrations in cells incubated in the presence of N-acetyl-L-cysteine were, however, constant over 8 days after subculture. L-Cysteine concentrations in Hep G2 cells incubated in the absence of N-acetyl-L-cysteine were below the limit of detection (7 nmol/l06 cells).

Applicant's summary and conclusion

Conclusions:
These results demonstrate that human hepatocytes and Hep G2 cells are competent to synthesize CTFG and that Hep G2 cells may provide a useful model for studying human liver-catalyzed glutathione S-conjugate formation.
Executive summary:

CTFG biosynthesis and the activities of cytosolic and microsomal glutathione S-transferases were measured in rat and human hepatocytes and in human hepatoma-derived Hep G2 cells.Hepatocytes of >88% viability were obtained from rat or human liver slices by collagenase or collagenase + dispase digestion, respectively. HepG2 cells were grown in modified Earle’s medium supplemented with 10% (v/v) fetal calf serum. Cells and subcellular fractions were exposed to chlorotrifluoroethene, and CTFG formation was quantified by HPLC.Both human liver and Hep G2 cell subcellular fractions catalyzed CTFG formation, and human and rat microsomal fractions exhibited higher specific activities than cytosolic fractions with chlorotrifluoroethene as the substrate. Time-dependent formation of CTFG was observed in all cell preparations. The presence of microsomal glutathione S-transferase was demonstrated by Western blotting with antimicrosomal glutathione S-transferase antibodies in rat and human liver tissue and in Hep G2 cells. Cytosolic and microsomal glutathione S-transferase activities were lower in Hep G2 cells than in rat and human liver tissues. These results demonstrate that human hepatocytes and Hep G2 cells are competent to synthesize CTFG and that Hep G2 cells may provide a useful model for studying human liver-catalyzed glutathione S-conjugate formation.