Registration Dossier

Administrative data

Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not reported.
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: The study was conducted using a novel methodology and examining a specific effect. Insufficient information exists to allow for suitable comparison with other studies and literature papers, therefore no reliability assessment could be conducted.

Data source

Reference
Reference Type:
publication
Title:
Modifications in the activity of some respiratory enzymes in dinitrobutylphenol poisoning.
Author:
Pallade S and Goldstein I.
Year:
1963
Bibliographic source:
Med. Lavoro., Vol. 54, No. 8-9, pp. 578-581.

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The test material was administered by subcutaneous injection every 3 days for a month. Animals were then sacrificed and the activity of cytochrome oxidase and succino-dehydrogenase was determined in the cerebral substance.
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
not applicable

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
No information on test animals is reported in the original text.

ENVIRONMENTAL CONDITIONS
No details on environmental conditions.

Administration / exposure

Route of administration:
subcutaneous
Vehicle:
unchanged (no vehicle)
Details on exposure:
Poisoning was performed by administering 24mg/kg of dinitro-sec-butylphenol by subcutaneous injection (which represented half the LD50) every 3 days for a month
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data on analytical verification of doses was reported in the original text.
Duration of treatment / exposure:
1 month
Frequency of treatment:
every 3 days
Post exposure period:
not applicable to test methodology.
Doses / concentrations
Remarks:
Doses / Concentrations:
24mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
20 (sex not specified)
Control animals:
yes
Details on study design:
The animals were sacrificed at the end of the poisoning period. The activity of cytochrome oxidase and of succino-dehydrogenase was determined in the cerebral substance. After rapid sampling, the cerebral hemispheres were placed in a phosphate buffer at pH 7.4 and cleaned from vessels and meniges. Using a Potter-Elvehjm homogeniser, the cerebral substance was suspended in the phosphate buffer in a proportion of 4%. The C cytochrome necessary for analyses was prepared in the laboratory from bovine cardiac muscle. Enzyme activity was determined with the Warburg apparatus at a temperature of 37ºC according to the Schneider-Potter method, adapted by Pigareva and Cetverikova. Oxygen consumption was recorded every 15 minutes for 60 minutes.

By comparing values of the activity of cerebral succino-dehydrogenase and cerebral cytochrome oxidase in poisoned animals with those of the controls, a series of differences were observed.

Examinations

Examinations:
Enzyme activity was determined with the Warburg apparatus at a temperature of 37ºC according to the Schneider-Potter method, adapted by Pigareva and Cetverikova. Oxygen consumption was recorded every 15 minutes for 60 minutes.
By comparing values of the activity of cerebral succino-dehydrogenase and cerebral cytochrome oxidase in poisoned animals with those of the controls, a series of differences were observed.
Positive control:
not applicable to test methodology.

Results and discussion

Details on results:
The activity of cerebral succino-dehydrogenase was reduced with poisoned rats.
This difference between controls and poisoned animals was progressively accentuated, being evidenced from the statistical point of view by positive “t” values for the last moments of the determination (graph 1).
The activity of cerebral cytochrome oxidase in poisoned animals exhibited on the contrary higher values than with the controls. “t” values were positive here for all moments of the determination (graph. 2).
The results obtained show a modification in the activity of the succinate-oxidase system in animals poisoned by dinitro-sec-butylphenol. As far as we know, the activity of the succinate-oxidase system could be summarised as follows: succino-dehydrogenase makes hydrogen unstable on the substratum represented by succinic acid and catalyses its transport to b, c, a cytochromes, which undergo reduction in the same order: cytochrome oxidase catalyses a transfer of electrons starting from the cytochromes to molecular oxygen and water and carbon dioxide appear as the final degradation products. The cytochromes act as intermediate oxido-reduction systems that exert their function by the action of dehydrogenases that labilise hydrogen of the substratum and by the action of cytochrome oxidase that labilises molecular oxygen. The increase in cytochrome oxidase activity during poisoning with DNBP, and thus by a substance increasing metabolism, proves that this enzyme participates in the process of increasing cellular metabolism. According to our results, it may therefore be considered that the route having cytochromes as the intermediate ring, may represent one of the modalities intensifying the oxido-reduction processes characteristic of poisoning by dinitrophenols. It is well-known that the body possesses several routes for the oxido-reduction of tissue with which the cytochrome system participates. Thus, as we have pointed out, the most studied route in poisoning by DNBP has been that which contains pyridine-nucleotide and flavoproteins as the intermediate ring and it has been concluded that hydrogen transfer between these rings is blocked. Inhibition of succino-dehydrogenase, indicated in this work, shows that there is also blocking in the context of this succinate-dehydrogenase system. In addition, this enzyme that is particularly sensitive to toxic action has also been found to be modified by poisoning by other substances.

The results presented above provide us with supplementary information concerning the mechanism of poisoning by dinitrophenol in that the toxic action is also exerted on enzyme systems other than those indicated in the literature. Consequently, the conclusion is reached that disturbance of cellular respiratory processes is wider, affecting several physiological mechanisms, among which are also cytochrome oxidase and succino-dehydrogenase.

Applicant's summary and conclusion

Conclusions:
The results obtained, namely an increase in cytochrome oxidase activity and a reduction in succino-dehydrogenase activity, suggest that enzymatic systems other than the ATP system may contribute to the pathogenesis of dinitrophenol poisoning.
Executive summary:

According to several authors, the characteristic increase in cellular metabolism observed in dinitrophenol poisoning, may be attributed to an inhibition in the synthesis of ATP or to alterations in its phosphorylation.

The authors have estimated the activities of cytochrome oxidase and succino-dehydrogenase in the brains of rats intoxicated with dinitrobutylphenol and also of control rats.

The results obtained, namely an increase in cytochrome oxidase activity and a reduction in succino-dehydrogenase activity, suggest that enzymatic systems other than the ATP system may contribute to the pathogenesis of dinitrophenol poisoning.