Zinc oxide

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

Remarks:

Neurodevelopmental

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not specified

Reliability:

other: not rated acc. to Klimisch et al.

Rationale for reliability incl. deficiencies:

other: please refer to the 'Remarks' field

Remarks:

This mechanistic developmental neuro-toxicity study in rats supports the evidence of an impairment of maternal zinc deficiency [10 µg Zn/g diet (marginally zinc deficient), or 7 µg Zn/g diet (zinc deficient)] on the development of offspring brain in comparison to a control diet containing 25 µg Zn/g in the diet. Although a relevant route of exposure was chosen and two deficient diets were used, and the methods and results are described appropriately, the results can only be regarded as supportive, because this study represents a mechanistic study to address a very specific endpoint of neuro-toxicity and no regulatory relevant endpoints were addressed and is thus an unsuitable system in a regulatory context.

Data source

Reference

Reference Type:

publication

Title:

Maternal zinc deficiency reduces NMDA receptor expression in neonatal rat brain, which persists into early adulthood

Author:

Chowanadisai, W., Kelleher, S.L. and Lönnerdal, B.

Year:

2005

Bibliographic source:

Journal of Neurochemistry 94, 510-519

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

In this non-guideline mechanistic developmental neuro-toxicity study (non-GLP) in rats, the effects of maternal and early zinc deficiency on NMDA receptor expression and neurotrophins was evaluated. Female Sprague-Dawley rats were dosed with control diet (25 µg Zn/g diet), marginally zinc deficient diet (10 µg Zn/g diet), or zinc deficient diet (7 µg Zn/g diet) from 70 days prior to conception through weaning at PN20. On PN20, pups were weaned onto either their maternal diet (control, marginal zinc deficient, or zinc deficient) or control diet (marginal zinc deficient to control, zinc deficient to control) until PN65. Pups were sacrificed and examined at PNs 2, 11, and 65 in order to investigate on effects of prenatal zinc deficiency, neonatal zinc deficiency, and long-term consequences of early zinc deficiency, respectively. The brains of the pups were examined for NMDA receptor subunit NR1, NR2A, and NR2B mRNA and protein levels, polysialic acid-neural cell adhesion molecule (PSA-NCAM) protein levels, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NFG) protein levels, and zinc levels in brain and plasma. Moreover, PSA-NCAM expression in brain tissue was examined via immunohistochemistry.

GLP compliance:

not specified

Remarks:

publication

Type of method:

in vivo

Test material

Test material information

Constituent 1

Reference substance name:

Zinc

EC Number:

231-175-3

EC Name:

Zinc

Cas Number:

7440-66-6

Molecular formula:

Zn

IUPAC Name:

zinc

Test material form:

not specified

Specific details on test material used for the study:

not specified

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Wilmington, MA, USA)
- Age at study initiation: not specified
- Weight at study initiation: (P) Females: about 250 g
- Housing: stainless steel hanging cages
- Diet: egg white protein-based semi-purified experimental diet; ad libitum
- Water: deionized water; ad libitum
- Acclimation period: 7 days (during acclimation period fed with standard non-purified diet)

ENVIRONMENTAL CONDITIONS
- Temperature: temperature-controlled facility (not further specified)
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION:
Rats were fed an egg white protein-based semi-purified experimental diet based on the AIN-93 recommendations (Reeves et al. 1993)*. The experimental diets differed only in zinc content, containing 25 µg Zn/g diet (control), 10 µg Zn/g diet (marginally zinc deficient, MZD), or 7 µg Zn/g diet (zinc deficient, ZD) as zinc carbonate. We have previously shown that this moderate level of zinc deficiency does not affect food intake or pup birth weight (Chowanadisai et al. 2004)*.
*References:
- Reeves P. G., Nielsen F. H. and Fahey G. C. Jr (1993) AIN-93 purified diets of laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123, 1939-1951.
- Chowanadisai W., Kelleher S. L. and Lönnerdal B. (2005) Zinc deficiency is associated with increased brain zinc import and LIV-1 expression and decreased ZnT-1 expression in neonatal rats. J. Nutr. 135, 1002–1007.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Zinc levels were confirmed by atomic absorption spectrophotometry (Clegg et al. 1981)*.

*References:
- Clegg M. S., Keen C. L., Lönnerdal B. and Hurley L. S. (1981) Influence of ashing techniques on the concentration of trace elements in animal tissues. Biol. Trace Elem. Res. 3, 107–115.

Duration of treatment / exposure:

Rats were dosed from 70 days prior to conception through weaning at PN20. On PN20, pups were weaned onto either their maternal diet (control, marginal zinc deficient, or zinc deficient) or control diet (marginal zinc deficient to control, zinc deficient to control) until PN65.

Frequency of treatment:

ad libitum, via diet

Duration of test:

not specified

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 other: µg Zn/g diet

Remarks:

control diet

Doses / concentrations 2

Dose / conc.:

10 other: µg Zn/g diet

Remarks:

marginally zinc deficient diet

Doses / concentrations 3

Dose / conc.:

7 other: µg Zn/g diet

Remarks:

zinc deficient diet

No. of animals per sex per dose:

not specified

Control animals:

yes

Details on study design:

EXPERIMENTAL GROUPS:
Pups were sacrificed and examined at PNs 2, 11, and 65 in order to investigate on effects of prenatal zinc deficiency, neonatal zinc deficiency, and long-term consequences of early zinc deficiency, respectively.
- PND2: N=18/diet, n=6/diet for NDMA receptor, NGF, or BDNF protein levels or brain mineral analysis; n=6/diet for NMDA receptor mRNA expression; n=6/diet for mineral analysis; n=2/diet for immunohistochemistry. Only one to two pups/litter were used for analysis.
-PND11: N=18/diet; n=6 brains and hippocampi/diet for NMDA receptor, NGF, BDNF, or PS-NCAM protein levels or mineral analysis; n=6/diet for NMDA receptor mRNA expression analysis; n=6/diet for mineral analysis. Only one to two pups/litter were used for analysis.
- PND65: n=6/diet for NMDA receptor protein expression; n=6/diet for mineral analysis. Only one to two pups/litter were used for analysis.

ANALYSIS OF ZINC LEVELS:
Plasma and whole brain zinc levels were analysed by flame atomic absorption spectrophotometry.

ANALYSIS OF mRNA LEVELS:
Total brain RNA was isolated, and the mRNA fraction was isolated. The NMDA receptor subunit mRNA (NR1, NR2A, and NR2B) levels were analysed via reverse-transcription PCR (RT-PCR).

ANALYSIS OF PROTEIN LEVELS:
- Total brain and hippocampal protein were extracted. NMDR subunit and polysialic acid-neural cell adhesion molecule (PSA-NCAM) protein levels were determined via Western blot analysis.
- Total brains were homogenised, centrifuged, the nuclear pellet discarded, and the protein content was measured. Afterwards protein levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NFG) were determined via ELISA (commercial kit).

IMMUNOHISTOCHEMISTRY
Brains were dissected, fixed, embedded in paraffin, sectioned, and stained using a PSA-NCAM antibody in combination with DAB and counterstained with hematoxylin.

Statistics:

Statistical comparisons were made using one-way ANOVA with a Tukey-Kramer post hoc test. Significant effect of diet at each age was determined at p < 0.05.

Results and discussion

Effect levels

Remarks on result:

not determinable because of methodological limitations

Observed effects

BODY WEIGHT DATA:
- There was no significant effect of diet on weight at PN65.

ANALYSIS OF ZINC LEVELS:
- At PN11, plasma zinc concentration was significantly lower in pups from dams fed ZD (7 µg Zn/g diet) and MZD (10 µg Zn/g diet) compared with control rats (25 µg Zn/g diet). At PN65, plasma zinc concentration was significantly lower in rats weaned onto their maternal diet compared with control rats, whereas plasma zinc concentrations in zinc-deficient rats weaned onto control diet were not significantly different from controls.
- There was no significant effect of diet on brain zinc concentration at PN2, PN11, or in rats weaned onto their maternal or control diet at PN65.

ANALYSIS OF mRNA LEVELS:
- At PN2, pups from ZD dams had lower whole brain NR1, NR2A and NR2B mRNA levels compared with control pups.
- At PN11, pups from ZD dams had lower whole brain NR2A and NR2B mRNA levels, whereas rats fed MZD had lower NR2A mRNA levels compared with control pups.

ANALYSIS OF PROTEIN LEVELS:
- At PN2, pups from ZD dams had lower whole brain NR1, NR2A and NR2B protein levels compared with control pups. Pups from dams fed MZD had lower NR1 and NR2A protein levels compared with control pups. At PN11, pups from ZD dams had lower whole brain NR1, NR2A and NR2B protein levels, whereas pups from dams fed MZD had lower NR1, NR2A and NR2B protein levels compared with control pups. At PN65, zinc-deficient rats, regardless of postweaning diet, had lower whole brain NR1 protein levels compared with control rats.
- At PN11, pups from ZD dams had lower hippocampus NR1, NR2A and NR2B protein levels, whereas rats fed MZD had lower hippocampus NR1 and NR2A protein levels compared to control pups.
- At PN2, pups from dams fed MZD and ZD had lower brain NGF concentrations compared to
control pups. At PN11, pups from dams fed control diet had higher brain NGF concentrations compared to MZD and ZD pups. Pups from dams fed ZD had lower brain BDNF concentration compared with control pups at PN11.
- At PN11, PSA-NCAM protein expression in the hippocampus was significantly lower in pups from dams fed MZD and ZD.

IMMUNOHISTOCHEMISTRY
More PSA-NCAM immunoreactive cells were observed in the granule cell layer of dentate gyrus in the hippocampus of control rats compared to ZD and MZD rats.

Applicant's summary and conclusion

Conclusions:

In this non-guideline, non-GLP mechanistic developmental neuro-toxicity study (Chowanadisai et al., 2005) in rats, the effects of maternal and early zinc deficiency on NMDA receptor expression and neurotrophins was evaluated. Female Sprague-Dawley rats were dosed with control diet (25 µg Zn/g diet), marginally zinc deficient diet (10 µg Zn/g diet), or zinc deficient diet (7 µg Zn/g diet) from 70 days prior to conception through weaning at PN20. On PN20, pups were weaned onto either their maternal diet (control, marginal zinc deficient, or zinc deficient) or control diet (marginal zinc deficient to control, zinc deficient to control) until PN65. Pups were sacrificed and examined at PNs 2, 11, and 65 in order to investigate on effects of prenatal zinc deficiency, neonatal zinc deficiency, and long-term consequences of early zinc deficiency, respectively. The brains of the pups were examined for NMDA receptor subunit NR1, NR2A, and NR2B mRNA and protein levels, polysialic acid-neural cell adhesion molecule (PSA-NCAM) protein levels, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NFG) protein levels, and zinc levels in brain and plasma. Moreover, PSA-NCAM expression in brain tissue was examined via immunohistochemistry.

At PN11, plasma zinc concentration was significantly lower in pups from dams fed ZD (p < 0.01) and MZD (p < 0.05) compared with control rats. At PN65, plasma zinc concentration was significantly lower in rats weaned onto their maternal diet (p < 0.05) compared with control rats, whereas plasma zinc concentrations in zinc-deficient rats weaned onto control diet were not significantly different from controls. There was no significant effect of diet on brain zinc concentration at PN2, PN11, or in rats weaned onto their maternal or control diet at PN65.
At PN2, expression of whole brain NMDA receptor subunits NR1, NR2A, and NR2B was lower in pups from dams fed ZD and MZD compared to controls, as analyzed by using relative RT–PCR and immunoblotting. At PN11, whole brain and hippocampi NR1, NR2A, NR2B and PSANCAM (polysialic acid–neural cell adhesion molecule) expression and the number of PSA-NCAM immunoreactive cells were lower in pups from dams fed ZD compared to controls. Whole brain brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) concentrations were lower in pups from dams fed ZD or both low zinc diets, respectively. Whole brain NR1 expression remained lower in previously zinc-deficient rats at PN65.

This mechanistic developmental neuro-toxicity study in rats supports the evidence of an impairment of maternal zinc deficiency [10 µg Zn/g diet (marginally zinc deficient), or 7 µg Zn/g diet (zinc deficient)] on the development of offspring brain in comparison to a control diet containing 25 µg Zn/g in the diet.

Although a relevant route of exposure was chosen and two deficient diets were used, and the methods and results are described appropriately, the results can only be regarded as supportive, because this study represents a mechanistic study to address a very specific endpoint of neuro-toxicity and no regulatory relevant endpoints were addressed and is thus an unsuitable system in a regulatory context.