Registration Dossier

Administrative data

Endpoint:
cytotoxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not reported
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Study not conducted or similar to an OECD method, no information on GLP. Study does not cover any specific REACH endpoint.

Data source

Reference
Reference Type:
publication
Title:
1-BROMOALKANES AS NEW POTENT NONTOXIC GLUTATHIONE DEPLETORS IN ISOLATED RAT HEPATOCYTES
Author:
Khan. S. and O'Brien. P.J.
Year:
1991
Bibliographic source:
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol. 179, No. 1, 1991, pp 436-441

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The effect of 1-bromoalkanes on intracellular glutathione (GSH) was studied in freshly isolated rat hepatocytes.
GLP compliance:
not specified
Type of method:
in vitro
Endpoint addressed:
not applicable

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Tests carried out on a variety of 1-bromoalkanes at the highest purity commercially available.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male

Results and discussion

Details on results:
1-Bromoalkanes at micromolar concentrations depleted intracellular GSH levels markedly after addition to isolated rat hepatocytes. The degree of GSH depletion increased with increasing chain length of bromoalkanes (C2-C7). Thus, 1-bromohexane and 1-bromoheptane at 100µM were the most effective at depleting intracellular GSH levels (83% and 87% respectively) in isolated hepatocytes after 30 minutes. In the absence of cytosolic GSH-S-transferases, GSH reacted much more slowly with bromoalkanes. This indicates that the substrate specificity for the GSH-S-transferases catalysing the GSH depletion is determined by the chain length and lipophilicity of the 1-bromoalkane.
Other 1-bromoalkanes were also compared for their effectiveness at inducing cytotoxicity towards isolated hepatocytes. Cytotoxicity increased with the chain length of 1-bromoalkanes with bromohexane being the most cytotoxic. Bromooctane and bromoheptane were much less cytotoxic. This presumably indicates that high concentrations of bromohexane can modify membrane macromolecules by direct alkylation or via peroxidation in the GSH depleted cell which results in cytotoxicity.

Any other information on results incl. tables

Table 1. Intracellular GSH Depletion by 1-Bromoalkanes in Isolated Rat Hepatocytes

 

GSH content (nmol / 106cells)

1 min

10 min

30 min

60 min

Control

63.2 ± 4.6

62.5 ± 5.1

63.1 ± 4.8

60.0± 4.8

1-bromopropane (100 µM)

58.4 ± 4.1

52.3 ± 3.6

46.4 ± 3.2

40.8 ± 2.9

Diethyl malenate (350 µM) (positive control)

50.8 ± 4.6

36.2 ± 4.2

21.8 ± 2.5

15.6 ± 1.4

The results show that 1 -bromopropane is adept at depleting GSH.

Table 2. 1-Bromoalkanes Induced Cytotoxicity Torwards Isolated Rat Hepatocytes

 

Trypan blue uptake (%) at time

0.5 h

1.0 h

2.0 h

3.0 h

Control

17 ± 2

18 ± 1

20 ± 3

23 ± 2

1-bromopropane (5 mM)

24 ± 3

29 ± 2

30 ± 3

36 ± 5

Diethyl malenate (0.7 mM) (positive control)

35 ± 3

40 ± 3

43 ± 4

48 ± 4

Results are expressed as an average of three experiments ± SD.

Applicant's summary and conclusion

Conclusions:
In conclusion, this study demonstrates that 1-bromoheptane is the most effective GSH-S-transferase substrate tested, the least cytotoxic and therefore can be used as a tool to modulate cellular GSH levels in isolated hepatocytes. It is also clear that GSH depleted cells have adequate antioxidant and enzymic systems to counteract physiological oxidative stress. The cytotoxicity and lipid peroxidation following GSH depletion by the GSH-S-transferase substrates used by other investigators may indicate that metabolism of these substrates results in radical formation and lipid peroxidation.
Executive summary:

The effect of 1-bromoalkanes on intracellular glutathione (GSH) was studied in freshly isolated rat hepatocytes. Treatment of cells with bromoalkanes depleted cellular GSH levels without causing cytotoxicity. The extent of GSH depletion was directly proportional to the concentration and increasing chain length of 1-bromoalkanes (C2-C7). Bromoheptane (100 µM) depleted GSH by 87% in 30 mins which remained depleted for the 4 hr study period without causing cytotoxicity. A 30 fold higher concentration of bromoheptane was required before cytotoxicity ensued. Bromoheptane would therefore be particularly useful for studying the role of GSH in modulating xenobiotic cytotoxicity.