Aluminium nitride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Analysis of metal ion-induced DNA damage, apoptosis, and necrosis in human (Jurkat) T-cells demonstrates Ni2+ and V3+are more toxic than other metals: Al3+, Be2+, Co2+, Cr2+, Cu2+, Fe3+, Mo5+, Nb5+, Zr2+

Author:

Caicedo, M.

Year:

2007

Bibliographic source:

J Biomed Mater Res 86A: 905–913, 2007

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The purpose of the comet assay is to identify substances that cause DNA damage. Under alkaline conditions (>pH 13), the comet assay can detect single and double stranded breaks, resulting, for example, from direct interactions with DNA, alkali labile sites or as a consequence of transient DNA strand breaks resulting from DNA excision repair.

GLP compliance:

not specified

Type of assay:

comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium chloride

EC Number:

231-208-1

EC Name:

Aluminium chloride

Cas Number:

7446-70-0

IUPAC Name:

aluminum trichloride

Test material form:

other: aluminium chloride solution

Details on test material:

Aluminium chloride solution purchased from Sigma. No further information given by the author

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

other: human Jurkat T-lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum

Metabolic activation:

without

Test concentrations with justification for top dose:

0.0 (control), 0.05, 0.1, 0.5, 1, and 5 mM

Vehicle / solvent:

N.A.

Controls

Untreated negative controls:

yes

Remarks:

medium

Negative solvent / vehicle controls:

no

True negative controls:

not specified

Positive controls:

no

Positive control substance:

other: Hydrogen peroxide

Details on test system and experimental conditions:

CELL CULTURE:
Jurkat T-lymphocytes were cultured in Dulbecco’s modified Eagle medium (DMEM; GIBCO, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT) at 37°C and 0.5% CO2. Jurkat T-lymphocytes were subcultured after they became confluent every 3–4 days, and all cells were used between passages 5 and 10. Cells were washed twice with PBS and resuspended in fresh new media at 1 x 10^6/well in 24-well plates

COMET ASSAY:
Metal-challenged and cultured Jurkat T-lymphocytes were cast in molten agarose (37°C) on glass slides (1x10^5 cells/mL at 1:10 ratio). Kit supplied (Comet assay, Trevigen, Gaithersburg, MD) lysis, and alkaline (pH > 13) solutions were used to lyse and unwind labile DNA at sites of damage, allowing the migration of unwound relaxed DNA strands out of the cell during electrophoresis (10 min at 22 mV). Stain (SYBR Green I nucleic acid gel stain) added to the cells was used to show fluorescent tails of electrophoresed/drifted DNA fragmentation from the cells after electrophoresis. The amount of DNA damage (measured by the comet tail length) was normalized to untreated controls, resulting in a DNA damage index (IDD). 34 measurements of 50 different cells from two separate slides at each concentration for each metal were performed with a public domain image analysis program for the single cell electrophoresis (comet) assay.

DETERMINATION OF APOPTOSIS:
Approximately 1x10^6 cells were incubated, with each metal in a 24-well culture plate in a final volume of 1 mL of media for 48 h. Ten microliters of a FITC-conjugated pan-caspase inhibitor (ApoStat) was added to the cell culture during the last 30 min of the duration of the experiment.
After intracellular staining, cells were harvested, washed twice with PBS, and resuspended in 400 µL of flow cytometry buffer. Flow cytometry was used to quantify intracellular caspase-9 activity with a FACScan flow cytometer (Becton Dickinson Co., San Diego, CA), according to fluorescence intensity in a linear histogram plot.
Scatter gates were set to exclude cellular debris. A threshold of 50% caspase-9 positive cells was used to determine the concentration at which a metal is toxic or not. All tests were conducted in triplicate for each metal at each concentration.

DETERMINATION OF CELL VIABILITY:
Cell viability was determined in a FACScan flow cytometer (Becton Dickinson Co.) using propidium iodide (PI) staining (Calbiochem, San Diego, CA). Flow cytometry viability assays assessed plasma membrane integrity using PI, a fluorescent vital dye that stains nuclear DNA. PI cannot cross intact plasma membranes of viable cells. The PI stock solution was composed of 50 µg PI/mL in PBS (at pH 7.4) for flow cytometric assays, where 10 µL of this stock solution was used per 1x10^5 cells after 48-h incubation, with each of the metal solutions at each of the eight concentrations.
General viability/toxicity was ranked using a LC50 index (half lethal concentration, i.e., the concentration at which 50% of the cells were viable) to compare metals. All viability tests were conducted in triplicate for each metal at each concentration.

DETERMINATION OF PROLIFERATION:
Proliferation assays were performed using 96-well cell culture plates (Sigma), at a density of 0.2 x 10^6 cells/well for 6 days in 150 µL/well of complete media at 37°C and 0.5% CO2, with or without metal treatments. Each metal concentration was tested in quadruplicate (four wells/metal concentration). [3H]-Thymidine (1 mCi/well) was added during the last 12 h of the 6-day culture period. A cell harvester was used to collect metal treated
and nontreated CD4+ T lymphocyte Jurkat cell membranes and a Beta plate counter was used to measure [3H]-thymidine incorporation. All tests wereconducted in quadruplicate for each metal at each concentration.

Statistics:

Student’s t test

Results and discussion

Test results

Species / strain:

other: Human Jurkat T-lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

TABLE 1: Concentrations of Metals Required to Induce a a Significant Harmful Effect (DNA Damage, Apoptosis, Viability, and Proliferation Inhibition) on T-Helper Jurkat Cells
Metal Concentrations (mM)	DNA Damage	Apoptosis	Viability	Proliferation Inhibition	Average Concentration of Four Parameters
V	0.05	0.05	1.0	0.05	0.29
Ni	0.05	0.1	5.0	0.5	1.41
Co	5.0	5.00	0.5	0.1	2.65
Cu	>5	0.5	5.0	0.1	>2.65
Nb	>5	0.5	0.5	>5	>2.75
Mo	>5	1.0	>5	0.5	>2.87
Zr	5.0	0.5	5.0	>5	>3.875
Be	>5	5.0	1.0	5.0	>4
Cr	>5	>5	>5	>5	>5
Al	>5	5.0	>5	>5	>5
Fe	>5	5.0	>5	>5	>5
Significant effect:
DNA damage: IDD>75.
Apoptosis:>50% caspase 9-positive cells.
Viability: >50% PI-positive cells.
Proliferation inhibition: p<0.05 significance in metal-treated cells CPMs reduction compared to untreated controls.

Applicant's summary and conclusion

Conclusions:

Aluminum chloride solution is considered to be non-mutagenic in a Comet assay.

Executive summary:

In an in vitro Comet assay, human Jurkat T-lymphocyte cells cultured in vitro were exposed to aluminum chloride solution at concentrations of 0.05, 0.1, 0.5, 1.0 and 5 mM in the absence of mammalian metabolic acivation.

For assessment of cytotoxicity, apoptosis, cell proliferation and cell viability were measured. Aluminum chloride induced >50% caspase-9 positive cells at 5 mM concentration. Aluminum chloride solution showed no effects on cell proliferation and cell viability at the concentrations tested. No significant increase of DNA damage, measured by using an index of DNA damage (IDD, average tail lenghth of 50 cells) were observed.

Therefore, Aluminum chloride solution is not genotoxic in the Comet assay.