N,N-dimethylpyridin-4-amine

Neurotoxicity

Administrative data

Endpoint:

neurotoxicity

Remarks:

other: in vitro mechanistic study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Peer-reviewed, scientifically valid study of mechanism of action in vitro.

Data source

Reference

Reference Type:

publication

Title:

Pyridine derivatives stimulate phosphatidylcholine secretion in primary cultures of rat type II pneumocytes

Author:

Kai H, Murahara K, Isohama Y, et al.

Year:

1996

Bibliographic source:

J Pharm Pharmacol 48:53-56

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Mechanistic study of cells in vitro

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dimethylpyridin-4-amine

EC Number:

214-353-5

EC Name:

N,N-dimethylpyridin-4-amine

Cas Number:

1122-58-3

Molecular formula:

C7H10N2

IUPAC Name:

N,N-dimethylpyridin-4-amine

Constituent 2

Reference substance name:

N,N-Dimethyl-4-pryridinamine

IUPAC Name:

N,N-Dimethyl-4-pryridinamine

Details on test material:

obtained from Sigma Chemical Co., St. Louis, MO. No data on purity.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Type II pneumocytes were isolated from male (180-200 g) pathogen-free Wistar rats (Kyudo Farm, Fukuoka, Japan) under anesthesia with Na pentobarbitone (25 mg/kg i.p.) The lung lavage, trypsin digestion, mechanical dissociation and plating of cells on bacteriological plastic dishes coated with IgG were according to a modificaiton of the method of Dobbs, et al, 1986. Cells were grown in DMEM supplemented with 10% FBS, penicillin, streptomycin, and were plated in 24-well tissue culture plates in 5% CO2 in water-saturated atmosphere for 18 h. Methyl-3H choline was added as a radiolabel tracer. The purity of the type II pneumocytes was 95% +/1 3%. The viability of the pneumocytes was 98 +/1 2% as judged by trypan blue exclusion test.

Administration / exposure

Route of administration:

other: in vitro

Vehicle:

other:

Details on exposure:

The test material was added to cell growth media at a concentration of 3 mM or 0.3 mM. The higher concentration was sufficient to induce biological effects in several electrophysiological studies.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Up to 90 min

Frequency of treatment:

Once

Doses / concentrations

Remarks:

Doses / Concentrations:
0.3 adn 3 mM
Basis:
other: final concentration in cell culture media

No. of animals per sex per dose:

not applicable

Details on study design:

The secretion of phosphatidylcholine into cell media was determined according to the method of Rice and Singleton, 1987. The test materials were added to freshly rinsed cells 30 m prior to addition of phosphatidylcholine secretion stimulants. The incubation with the test materials continued for 90 m, after which medium was aspirated, cells lysed with 2 ml ice cold 0.05% Triton X-100 solution and lipids were extracted from both cells and medium with chloroform and methanol according to Folch, et al, 1957. Phosphatidylcholine was separated from other lipids by TLC (Miyata et al, 1987) and radioactivity was measured with a liquid scintillation counter after the addition of Aquasol II to each sample. Secretion was expressed as the % secreted into the medium compared to total in the cell and medium. The degree of stimulation was expressed at the percentage of the stimulated secretion compared to unstimulated secretion. Cellular integrity was assays by the rate of lactate dehydrogenase release into the medium, using LDH kits (Nippon Shoji Co., Ltd). K+-channel investigation included used of sphingosine, phorbol 12-myristate-13-acetate (PMA) and BAPTA/AM in ethanol or DMSO; these solvents (ethanol and DMSO) were added to cell media of corresponding control groups.

Examinations

Observations and clinical examinations performed and frequency:

not applicable

Specific biochemical examinations:

not applicable

Neurobehavioural examinations performed and frequency:

Electrophysiolocial studies of cultured type II pneumocytes (not further defined)

Sacrifice and (histo)pathology:

Not applicable

Other examinations:

Not applicable

Positive control:

Not applicable

Statistics:

Not applicable

Results and discussion

Results of examinations

Details on results:

Toxicity was not observed in the cultured cells, as lactate dehydrogenase release did not exceed 1% of the total cellular content. At 3 mM concentration, which was sufficient to induce biological effects in several electrophysiological studies of pneumocytes, DMAP stimulated secretin of phosphatidylcholine. 4-Aminopyridine (4AP) and 4-pyrolidinopyridine.also showed this effect, and the mechanism of the effect was examined in detail using 4AP. Intracellular binding of calcium ion (Ca++) suppressed the stimulatory effect of 4AP, as did inhibiors of Ca++-dependent calmodulin kinase and Ca++-dependent protein kinase C.

Effect levels

Dose descriptor:

other: Secretion of phosphatidyl choline

Effect level:

3 other: mM

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: DMAP stimulated phosphatidylcholine secretion from pneumocytes

Remarks on result:

other:

Applicant's summary and conclusion

Conclusions:

DMAP and 4-aminopyridine both stimulated the secretion of phosphatidylcholine from cultured rat type II pneumocytes in vitro. This is consistent with blockage of K+ channels, which activates the secretory process through an increase in intracellular calcium ions.