Reaction mass of cobalt sulphide and nickel sulphide and trinickel disulphide

Immunotoxicity

Administrative data

Endpoint:

immunotoxicity

Remarks:

other: in vitro human lymphocyte immunophenotype & NK function

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally acceptable scientific standards, well documented and acceptable for assessment.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

The effect of nickel compounds on immunophenotype and natural killer cell function of normal human lymphocytes

Author:

Zeromski J, Jezewska E, Sikora J, Kasprzak KS

Year:

1995

Bibliographic source:

Toxicology. 97: 39-48

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Peripheral blood mononuclear cells (PBMC) isolated from human blood (obtained from a blood bank) were exposed to Ni3S2 for 24 hrs. Immunophenotype and natural killer cell cytotoxicity were evaluated.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trinickel disulphide

EC Number:

234-829-6

EC Name:

Trinickel disulphide

Cas Number:

12035-72-2

Molecular formula:

Ni3S2

IUPAC Name:

trinickel disulfide

Details on test material:

- Name of test material (as cited in study report): nickel subsulfide (Ni3S2)
- Physical state: particles of median size >= 30 um

Test animals

Species:

other: human

Strain:

other: peripheral blood mononuclear cells (PBMCs) isolated from blood obtained from unidentified blood bank

Sex:

not specified

Details on test animals or test system and environmental conditions:

PBMC fractions (2 X 10^6 cells per 2 ml) were placed in 24-well tissue culture plates in RPMI-1640 medium for an 18-h pre-exposure incubation at 37°C in a 5% CO2 atmosphere.

Administration / exposure

Route of administration:

other: in vitro

Vehicle:

not specified

Details on exposure:

The PBMCs were cultured with three different concentrations, 0.01, 0.02, or 0.04 mM, of Ni3S2 alone, NiSO4 alone, and Mg(CH3COO)2 alone, or with three equimolar mixtures of a nickel and magnesium salt at the above concentrations, and the cells were incubated for an additional 24-h period. Control lymphocytes were cultured under the same conditions in the RPMI-1640 medium alone. After the incubation (all in triplicate), the cells were examined for immunophenotype expression and cytotoxic NK function.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

24 hrs

Frequency of treatment:

Once

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.01, 0.02, and 0.04 mM Ni3S2
Basis:
other: Preliminary dose-finding study indicated that this dose range was nontoxic to cells

No. of animals per sex per dose:

Not applicable

Details on study design:

Dose Finding Study: Prior to the experiments, the toxicity of the individual metal salts for lymphocytes was tested in order to establish an optimal concentration which did not cause toxic effects on lymphocytes in short-term culture. The tests were carried out in plastic 96-well microplates kept for 24 h in a CO2 incubator. The cells (2 x 10^5 cells per 200 ul) were cultured in RPMI-1640 medium in the absence or presence of 0.01-1.0 mM concentrations of Ni3S2 and other metal salts. After a 24-h incubation, the percent of dead cells was determined by means of trypan blue exclusion using the cell culture without added metal salt as control.

Evaluation of immunophenotype: Indirect immunofluorescence was conducted to determine immunophenotype of control and treated PBMC cells. A cell suspension was overlayed on poly-L-lysine-coated slides. Cell sediments were fixed for 5 min in 1% buffered formalin and subsequently treated with mouse monoclonal antibodies (mAb) to lymphocyte or macrophage differentiation antigens (listed in Table 1 of publication). Following a 20-min incubation with the mAb and subsequent washing in phosphate buffered saline (pH 7.6), rabbit anti-mouse Ig(Fab)2 fluorescein-labelled reagent was used as a secondary antibody for another 20 min. Slides were then washed, mounted in buffered glycerin and evaluated using an Orthoplan fluorescence microscope with incident light illumination. Positive cells were counted in relation to total cells. At least 200 cells per sample were assessed.

Evaluation of NK cell cytotoxicity: PBMC were tested as effector cells versus K562 target cells labelled with 51Cr. Target cells (4 X 10^6 cells per sample) were incubated with 100 uCi of Na2·51CrO4 for 90 min at 37°C with gentle shaking. After labelling, the cells were washed five times with, and finally suspended in, RPMI-1640 medium. The test was carried out in plastic round-bottom microplates in triplicate. 100 ul aliquots of the effector cells were mixed with the same volume of target cells at effector-to-target cell ratios (E:T) of 12:1, 25:1, and 50:1. After 4-h incubation (37°C; 5% CO2), a 100 ul sample of the supernatant was collected from each well and radioactivity counted in a gamma counter. The result was calculated from the formula:

% Cytotoxicity = [(E - S)/(T - S)] x 100

Where E = activity of 51Cr released from target cells in the presence of effector cells. S = activity of 51Cr released spontaneously under identical conditions from target cells alone; this value did not exceed 10-15% of the maximal release (T). T = maximum activity of 51Cr released when all target cells are destroyed. T was determined by the addition of 10% Triton X-100 to the suspension of target cells.

Examinations

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: No data


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

Sacrifice and pathology:

Not applicable

Cell viabilities:

SPLEEN: No data

THYMUS: No data

BONE MARROW: No data

Humoral immunity examinations:

ANTIBODY PLAQUE FORMING CELLS (PFC) ASSAY: No data

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): No data

Specific cell-mediated immunity:

ONE-WAY MIXED LYMPHOCYTE CULTURE (MLC) ASSAY: No data

DELAYED-TYPE HYPERSENSITIVITY (DTH) REACTION: No data

CYTOTOXIC T-LYMPHOCYTE (CTL) ASSAY: No data

Non-specific cell-mediated immunity:

NATURAL KILLER (NK) CELL ACTIVITY: Yes
- Method: PBMC were tested as effector cells versus K562 target cells labelled with 51Cr. Target cells (4 X 10^6 cells per sample) were incubated with 100 uCi of Na2·51CrO4 for 90 min at 37°C with gentle shaking. After labelling, the cells were washed five times with, and finally suspended in, RPMI-1640 medium. The test was carried out in plastic round-bottom microplates in triplicate. 100 ul aliquots of the effector cells were mixed with the same volume of target cells at effector-to-target cell ratios (E:T) of 12:1, 25:1, and 50:1. After 4-h incubation (37°C; 5% CO2), a 100 ul sample of the supernatant was collected from each well and radioactivity counted in a gamma counter. The result was calculated from the formula:

% Cytotoxicity = [(E - S)/(T - S)] x 100

Where E = activity of 51Cr released from target cells in the presence of effector cells. S = activity of 51Cr released spontaneously under identical conditions from target cells alone; this value did not exceed 10-15% of the maximal release (T). T = maximum activity of 51Cr released when all target cells are destroyed. T was determined by the addition of 10% Triton X-100 to the suspension of target cells.

- Dose groups: each dose (0.01, 0.02, or 0.04 mM) or exposure condition (i.e., single metal salt alone, or equimolar mixture with magnesium salt) was run in triplicate.

- No. of animals: not applicable

Other functional activity assays:

SPLEEN CELL PROLIFERATION ASSAY (ANTI-CD3 MEDIATED T CELL PROLIFERATION): No data

ENUMERATION TOTAL B CELLS, TOTAL T CELLS AND T CELL SUBPOPULATIONS: No data

Other examinations:

Evaluation of immunophenotype: Indirect immunofluorescence was conducted to determine immunophenotype of control and treated PBMC cells. A cell suspension was overlayed on poly-L-lysine-coated slides. Cell sediments were fixed for 5 min in 1% buffered formalin and subsequently treated with mouse monoclonal antibodies (mAb) to lymphocyte or macrophage differentiation antigens (listed in Table 1 of publication). Following a 20-min incubation with the mAb and subsequent washing in phosphate buffered saline (pH 7.6), rabbit anti-mouse Ig(Fab)2 fluorescein-labelled reagent was used as a secondary antibody for another 20 min. Slides were then washed, mounted in buffered glycerin and evaluated using an Orthoplan fluorescence microscope with incident light illumination. Positive cells were counted in relation to total cells. At least 200 cells per sample were assessed.

Positive control:

Not reported

Statistics:

Significance levels of differences among the results were determined with the use of Student's t test.

Results and discussion

Results of examinations

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Gross pathological findings:

not specified

Details on results:

NON-SPECIFIC CELL-MEDIATED IMMUNITY: Ni3S2 distinctly suppressed the NK cytotoxicity of cultured PBMC at all concentrations and E:T ratios tested. In contrast, Mg(CH3COO)2 alone tended to enhance NK cytotoxicity and to reverse the suppressive action of the nickel salts (see figures 4 & 5 in paper).

OTHER FINDINGS - Immunophenotyping: Ni3S2 had a marked inhibitory effect on the PBMCs. It consisted of a significant decrease in the number of CD4-positive cells at concentrations of 0.02 and 0.04 mM Ni3S2 and a decrease in NK (CD56-positive) cell number at all Ni3S2 concentrations tested. However, the percentages of CD3, CD8, CD20, and CD11a cells did not show major alterations. Mg(CH3COO)2 prevented the inhibitory effects of Ni3S2 on CD56 and CD4 cells.

Specific immunotoxic examinations

Cell viabilities:

not specified

Humoral immunity examinations:

not specified

Specific cell-mediated immunity:

not specified

Non-specific cell-mediated immunity:

effects observed, treatment-related

Other functional activity assays:

not specified

Other findings:

effects observed, treatment-related

Effect levels

1

Dose descriptor:

dose level:

Effect level:

>= 0.05 other: mM

Sex:

not specified

Basis for effect level:

other: cell viability (from dose finding study): effect level = dose at which % cells alive below 95%.

2

Dose descriptor:

dose level:

Effect level:

> 0.04 other: mM

Sex:

not specified

Basis for effect level:

other: overall effects other: decrease in the number of CD3-positive cells (relative to control)

3

Dose descriptor:

dose level:

Effect level:

>= 0.02 other: mM

Sex:

not specified

Basis for effect level:

other: overall effects other: decrease in the number of CD4-positive cells (relative to control)

4

Dose descriptor:

dose level:

Effect level:

> 0.04 other: mM

Sex:

not specified

Basis for effect level:

other: overall effects other: decrease in the number of CD8-positive cells (relative to control)

5

Dose descriptor:

dose level:

Effect level:

< 0.01 other: mM

Sex:

not specified

Basis for effect level:

other: overall effects other: decrease in the number of CD56-positive cells (relative to control)

6

Dose descriptor:

dose level:

Effect level:

> 0.04 other: mM

Sex:

not specified

Basis for effect level:

other: overall effects other: decrease in the number of CD20-positive cells (relative to control)

7

Dose descriptor:

dose level:

Effect level:

> 0.04 other: mM

Sex:

not specified

Basis for effect level:

other: overall effects other: decrease in the number of CD11a-positive cells (relative to control)

8

Dose descriptor:

dose level:

Effect level:

< 0.01 other: mM

Sex:

not specified

Basis for effect level:

other: overall effects cell-mediated immunity (NK cell activity): suppression of the NK cytotoxicity of PBMC cells

Applicant's summary and conclusion

Conclusions:

The primary effects of Ni3S2 on human peripheral blood mononuclear cells in vitro consist of depletion of the CD4 and NK cell subsets and reduction of the NK cytotoxic function. The magnesium salt Mg(CH3COO)2 prevented these Ni3S2 effects.

Executive summary:

Zeromski et al. (1995) examined a variety of immunotoxicity endpoints associated with Ni3S2 exposures in human lymphocytes (i.e., peripheral blood mononuclear cells – PBMCs) derived from samples of blood donors as obtained from an unidentified blood bank. Initially, a dose-finding study was conducted to identify doses of Ni3S2 that did not affect short-term cell viability in culture. Second, a series of assays was conducted to assess the natural killer activity of the PBMCs associated with Ni3S2 exposure in vitro.

The cell viability test was carried out in plastic 96-well microplates kept for 24 hr in a CO2 incubator. The cells (2 x 105 cells per 200 ul) were cultured in RPMI-1640 medium in the absence or presence of 0.01-1.0 mM concentrations of Ni3S2 and other metal salts. Trypan blue exclusion illustrated that 0.01, 0.02, and 0.04 mM Ni3S2 concentrations left greater than 95% of the cell populations alive and were thus considered “nontoxic” for the purposes of examining lymphocyte immunophenotype and function. PBMCs (2 X 106 cells per 2 ml) were cultured in 24-well tissue culture plates in RPMI-1640 medium for an 18-h pre-exposure incubation at 37°C in a 5% CO2 atmosphere, and then exposed to the three non-toxic concentrations of Ni3S2 alone or in an equimolar mixture with magnesium acetate for 24 hr. The immunophenotype of the cells was determined by indirect immunofluorescence, using monoclonal antibodies to major differentiation antigens of peripheral blood mononuclear cells. Exposure of cells to Ni3S2 resulted in the decline of CD4 (at 0.02 and 0.04 mM) and natural killer cell populations (i.e., CD56; at all concentrations). However, Ni3S2 did not affect CD3, CD8, CD20, or CD11a cell populations.

The natural killer activity of the PBMCs was studied by measuring the cytotoxic effect of Ni3S2 -exposed and control PBMCs toward K562 target cells. Target cells (4 X 106 cells per sample) were radiolabelled with 51Cr during a 90 min incubation at 37°C. The cells then were washed and suspended in, RPMI-1640 medium. 100 ul aliquots of the effector cells (i.e., PBMCs) were mixed with the same volume of target cells at effector-to-target cell ratios (E:T) of 12:1, 25:1, and 50:1. After 4-h incubation (37°C; 5% CO2), a 100 ul sample of the supernatant was collected from each well and radioactivity counted in a gamma counter. At all exposure concentrations, Ni3S2 suppressed the natural cytotoxic activity of PBMCs toward the target cells. The metal salt magnesium acetate, however, prevented all Ni3S2 -mediated immunotoxic effects studied. The authors broadly concluded that nickel subsulfide had deleterious effects on human peripheral blood mononuclear cells in short-term in vitro culture, but the presence and magnitude of these effects was dependent on the cell subsets. In this in vitro study, Ni3S2 selectively depleted helper/inducer T lymphocytes (i.e., CD4-presenting PBMCs) and natural killer cells (i.e., CD56-presenting PBMCs), while also reducing natural killer cell function. And since magnesium inhibited these effects, the authors further suggest that magnesium supplementation might be a useful prophylactic against immunotoxic effects of nickel exposure. STUDY RATED BY AN INDEPENDENT REVIEWER