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Basic toxicokinetics

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basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Metabolism and toxicity of benzophenone in isolated rat hepatocytes and estrogenic activity of its metabolites in MCF-7 cells
Nakagawa Y et al.
Bibliographic source:
Toxicology 156, 27-36

Materials and methods

Objective of study:
Test guideline
no guideline followed
Principles of method if other than guideline:
The metabolism and cytotoxicity of benzophenone and estrogenic activity of its metabolites was studied in freshly isolated rat hepatocytes and cultured MCF-7 human breast cancer cells, respectively.
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): Benzophenone
- Analytical purity: >97%
- Supplier: Tokyo Casei Co. (Tokyo, Japan)

Test animals

other: Hepatocytes isolated from male F344/DuCrj rats and cultured MCF-7 human breast cancer cells
Details on test animals or test system and environmental conditions:
Rat Hepatocytes
- Source: Charles River Japan Inc.

MCF-7 cells
- Source: American Type Culture Collection (Manassas, VA)

Administration / exposure

Route of administration:
other: incubation
Details on exposure:
Isolation and incubation of hepatocytes
Male F344/DuCrj were used. The hepatocytes were isolated by collagenase perfusion of the liver. Hepatocyte viability was assessed by Trypan blue exclusion, and initial cell viability in each experiment was more than 85%. Hepatocytes (10E+6 cells per ml) were suspended in Krebs–Henseleit buffer. All the incubations were performed in rotating, round-bottomed flasks at 37°C, under a constant flow of humidified carbogen. Reactions were started by the addition of benzophenone dissolved in DMSO; final concentration, <1%). Corresponding control groups received an equivalent volume of DMSO. In some experiments using DCNP as a inhibitor of sulfate conjugation, the compound (100 uM) dissolved in DMSO was added to the hepatocyte suspension 15 min before the addition of benzophenone. Aliquots of incubation mixture were taken at intervals to monitor cell death and the concentrations of intracellular adenine nucleotides, and benzophenone and its metabolites.

Biochemical assays
ATP, ADP, and AMP in hepatocytes were measured by HPLC. Cell death of hepatocytes was assessed by Trypan blue uptake under a light microscopy.

Determination of benzophenone and its metabolites by HPLC
An equal volume of chilled methanol was added to the cell suspension and then the mixture was filtered through a membrane cartridge. The eluate (20 ul) was injected onto an analytical Capcell Pak C18 or Capcell Pak CN column equipped with a photodiode-array detector (200–350 nm) or a UV absorbance detector (260 nm). The mobile phase was methanol/0.1 M ammonium dihydrogen phosphate and the flow rate was 1.0 ml/min. Benzophenone, p-hydroxybenzophenone and benzhydrol were identified by UV spectra profiles or by comparing HPLC retention times with those of authentic compounds in both column systems. The recoveries for benzophenone, p-hydroxybenzophenone and benzhydrol were checked by the addition of known amounts of authentic compounds to hepatocyte suspensions, and were more than 85%. Sulfate conjugate derived from benzophenone in the cell suspensions was enzymaticaly hydrolyzed with sulfatase to yield free p-hydroxybenzophenone and was determined as free p-hydroxybenzophenone by HPLC. Briefly, aliquots of cell suspensions were treated with a cell disrupter in ice-water for 20 s and then filtered through an ultrafiltration cartridge (limit of elution; < molecular weight 5000). The eluate adjusted to pH 4.5 with sodium acetate buffer was incubated with sulfatase for 3.5 h at 37°C and then the mixture (20 ul) was injected into the HPLC system.

MCF-7 cell proliferation assay
Cells were cultured in phenol red free-RPMI-1640 medium, supplemented with 5% FCS, 15 mM HEPES, 50 U/ml penicillin and 50 ul/ml streptomycin and 10 ng/ml insulin at 37°C with 5% CO2 in air at saturating humidity, and cells were routinely passed at approximately 70% confluence. Prior to initiating experiments, cells were seeded and attached in 96-well multiwell plates at 4 * 10E+3 cells per well in 0.3 ml RPMI-1640 medium, supplemented with 5% estrogenfree-FCS. After 24 h, the medium was replaced with the same volume of above the medium containing benzophenone, its metabolites, bisphenol A (1 uM) and 17b-estradiol (1 nM). The concentrations of benzophenone and its metabolites in this study range from 10 nM to 500 uM. Estrogen- free FCS was prepared using the dextran-charcoal procedure. Cells were cultured for 6 days and cell numbers in each well were determined using a cell proliferation assay kit, 30 ul of WST-8 solution, 5 mM 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt and 0.2 mM 1-methoxy phenazinium methylsulfate dissolved in 150 mM KCl, was added to wells containing 0.3ml of medium with cells. After incubation for 60–90 min at 37°C in a humidified 5% CO2 atmosphere, the absorbance of the well was read at 450 nm (reference wavelength at 650 nm) by a microculture plate reader. The assay, a modified tetrazolium colorimetric assay, is based on metabolic reduction of WST-8 to its corresponding formazan and the coloration obtained is directly proportional to cell number. The coloration was found to be linear for up 120 min after addition of the dye-containing medium, with cell numbers exceeding 3 * 10E+4.
Details on study design:
In this study, the action/mechanism and metabolism of benzophenone in freshly isolated rat hepatocytes was investigated, i.e. examination of the potential of estrogenic activity of benzophenone and its metabolites in MCF-7 cells (oestrogen-responsible human breast cancer cells).
One-way analysis of variance, followed by a Dunnett’s or Sheffe’s multiple comparison test. Statistical significance was assumed at p < 0.05.

Results and discussion

Metabolite characterisation studies

Metabolites identified:
Details on metabolites:
Although at high concentrations benzophenone itself was toxic, the compound at a low toxic level (0.25 mM) was converted in rat hepatocytes to at least three metabolites: p-hydroxybenzophenone, its sulfate conjugate and benzhydrol.

Applicant's summary and conclusion

The study showed that benzophenone (at a low toxic level) in rat hepatocytes is enzymatically converted to at least three metabolites: benzhydrol, p-hydroxybenzophenone and its sulfate conjugate. There was a weak estrogenic effect of p-hydroxybenzophenone on MCF-7 cell proliferation. The parent compound and benzhydrol were inactive in this respect.
Executive summary:

The metabolism and cytotoxicity of benzophenone and estrogenic activity of its metabolites have been studied in freshly isolated rat hepatocytes and cultured MCF-7 human breast cancer cells, respectively. The incubation of hepatocytes with benzophenone (0.25–1.0 mM) elicited a concentration- and time-dependent cell death, accompanied by loss of intracellular ATP and depletion of adenine nucleotide pools. Benzophenone at a low-toxic level (0.25 mM) in the hepatocyte suspensions was converted to benzhydrol, p-hydroxybenzophenone and its sulfate conjugate, without marked loss of cell viability.

In another experiment, MCF-7 cells (estrogen-responsible breast cancer cells) were cultured in estradiol free medium and then exposed to 10 nM–500 μM benzophenone or its metabolites for 6 days. Although at higher concentrations all the compounds were toxic, except for benzophenone and benzhydrol, 10–100μM p-hydroxybenzophenone significantly increased cell proliferation. These results indicate that benzophenone is enzymatically converted to benzhydrol, p-hydroxybenzophenone and its sulphate conjugate in rat hepatocytes. Even if there is less free p-hydroxybenzophenone than benzhydrol and sulfate conjugate in hepatocyte suspensions, p-hydroxybenzophenone itself acts as a weak xeno-estrogen on MCF-7 cells.