Registration Dossier

Administrative data

Endpoint:
neurotoxicity
Remarks:
other:
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Neurotrophic and neurotoxic effects of zinc on neonatal cortical neurons
Author:
Chen C & Liao S
Year:
2003
Bibliographic source:
Neurochem. Int. 42: 471–479

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
A study was conducted to determine the cellular responses and neuronal viability following exposure to different concentrations of zinc chloride in primary cultures of neonatal rat cortical neurons.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Zinc

Test animals

Species:
other: Mixed cortical cultures and cortical neurons prepared from cerebral cortices of 1-day-old rats
Strain:
other: Mixed cortical cultures and cortical neurons prepared from cerebral cortices of 1-day-old Sprague–Dawley rats
Sex:
not specified
Details on test animals and environmental conditions:
No data

Administration / exposure

Route of administration:
other: Not applicable
Vehicle:
other: Not applicable
Details on exposure:
Not applicable
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
24 h
Frequency of treatment:
Not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:
0–0.2 mM
Basis:
nominal conc.
No. of animals per sex per dose:
Not applicable
Control animals:
other: Not applicable
Details on study design:
No data

Examinations

Observations and clinical examinations performed and frequency:
Not applicable
Specific biochemical examinations:
OTHER:
Immunohistochemical staining: After the medium was aspirated, the cells (24-well) were washed twice with phosphate-buffered saline (PBS) [0.1M
Na2HPO4, 0.1M NaH2PO4, and 0.9% NaCl], fixed with 4% paraformaldehyde in phosphate buffer (PB) [0.1M Na2HPO4 and 0.1M NaH2PO4] for 10 min, permeabilized with 10% methanol for 15 min, and washed with PBS. The cells were blocked with 5% milk in PBS for 30 min. The cells were then incubated with primary antibody (500×) overnight at 4 oC, followed by washing with PBS. After washing, the cells were incubated with alkaline phosphatase
conjugated secondary antibody (500×) for 1 h at room temperature. After washing, the color was developed using NBT/BCIP and observed by light microscope. A visible cell body and length of neurite process more than two cell bodies defined the MAP-2 positive neuron. The number of cells was the average of five areas (500 µm2) in each well.
Viability assay: Cell viability was assessed by MTS reduction assay.
Immunoblot: Protein extracts (100 µg) were resolved by SDS-polyacrylamide gel electrophoresis and immunoblotting with primary antibody (Chen et al., 2001).
Measurement of intracellular zinc content: The amount of zinc was estimated in 1 mL homogenization buffer containing 0.001% (w/v) N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (Marin et al., 2000).
Measurement of intracellular glutathione (GSH) content: GSH was measured as described by Papadopoulos et al., 1998.
Assessment of intracellular free radicals: Levels of intracellular free radical were analyzed by the fluorescent signal after the oxidation of a non-fluorescent 2,7-dichlorofluorescein (Molecular Probe) by reactive oxygen species (Zhu et al., 1994).
Measurement of ATP content: Intracellular ATP levels were assayed using a luciferin/luciferase-based method (DeLuca and McElroy, 1984), based on the oxidation of luciferin by luciferase in the presence of ATP.
Determination of neuronal death: Cell death was detected by TUNEL (TdT-mediated dUTP nick-end labeling) using the Cell Death Kit (Roche Molecular Biochemicals).
DNA agarose gel electrophoresis: DNA gel electrophoresis was performed as described in Ahn et al., 1998.
Neurobehavioural examinations performed and frequency:
Not applicable
Sacrifice and (histo)pathology:
Not applicable
Other examinations:
None
Positive control:
None
Statistics:
The data was expressed as mean values ± S.E.M.). Each experiment was repeated at least twice using different batches of culture preparations. Statistical analysis was carried out using one-way analysis of variance (ANOVA), followed by Dunnett’s test to assess the statistical significance between treated and untreated groups through all experiments. A level of P <0.05 was considered statistically significant.

Results and discussion

Results of examinations

Clinical signs:
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Other effects:
not examined
Description (incidence and severity):
Migrated information from 'Further observations for developmental neurotoxicity study'



Details on results (for developmental neurotoxicity):Not applicable (migrated information)
Details on results:
OTHER FINDINGS: Neural viability: Treatment of mixed cortical cultures with concentrations of zinc chloride within a serum-free DMEM for 24 h produced dual effects on cell viability. Lower concentrationsof ZnCl2 (0.05 mM) protected neurons from washing-, and serum-deprivation-induced decreases in the number of visible neurons (71% of the control), cell viability (81% of the control), and MAP-2 content (87% of the control). However, higher concentrations of ZnCl2 (0.2 mM) caused marked decreases in the number of visible neurons (7% of the control), cell viability (19%), and MAP-2 (32% of the control) and GFAP contents (39% of the control). Taken together, treatment of mixed cortical cultures with ZnCl2 showed both neurotrophic and neurotoxic features.
Cellular responses to zinc: Higher concentration of exogenous ZnCl2 (0.2 mM) triggers marked influx of zinc into cortical neurons, depletes intracellular ATP and GSH content, and decreases oxidative stress. In contrast, a lower concentration of ZnCl2 (0.05 mM) is unable to cause these changes. The antiapoptotic action of low amounts of zinc was found both in mixed cultures and neuron-enriched cultures indicating the independence of glial mediator. Neurotrophic action was not accompanied by significant alteration in those cellular responses but required chelatable zinc.

Effect levels

Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the test conditions, higher concentrations of zinc (0.15 and 0.2 mM) triggered excessive zinc influx, glutathione depletion and ATP loss leading to necrotic neuronal death. In contrast, lower concentrations of zinc (0.05 and 0.1 mM) attenuated serum-deprivation induced apoptotic neuronal death.
Executive summary:

A study was conducted to determine the cellular responses and neuronal viability following exposure to different concentrations of zinc chloride in primary cultures of neonatal rat cortical neurons.

Mixed cortical cultures and cortical neurons were prepared from cerebral cortices of 1-day-old Sprague–Dawley rats. Mixed cortical cultures were exposed to various concentrations of ZnCl2 (0–0.2 mM) for 24 h in a serum-free DMEM. Neuronal viability was detected by the immunoreactivity of MAP-2 (B and C) and MTS reduction.

Under the test conditions, higher concentrations of zinc (0.15 and 0.2 mM) triggered excessive zinc influx, glutathione depletion and ATP loss leading to necrotic neuronal death. In contrast, lower concentrations of zinc (0.05 and 0.1 mM) attenuated serum-deprivation induced apoptotic neuronal death.