Potassium 1,1,2,2,3,3,4,4,4-nonafluorobutane-1-sulphonate

Specific investigations: other studies

Administrative data

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2011

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Data source

Reference

Reference Type:

publication

Title:

The inhibition of human and rat 11B-hydroxysteroid dehydrogenase 2 by perfluoroalkylated substances

Author:

Zhao, B.; Lian, Q.; Chu, Y.; Hardy, D.; Xiao-Kun, L.; Ge, R.

Year:

2011

Bibliographic source:

Journal of Steroid Biochemistry and Molecular Biology. 125: 143-147.

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: The inhibition of human and rat 11beta-hydroxysteriod dehydrogenase by PFBS was evaluated in isolated liver and kidney microsomes.

- Short description of test conditions: Rat kidney was homogenized in 0.01mM phosphate buffered saline (PBS, pH 7.4) containing 0.25M sucrose, and nuclei and large cell debris were removed by centrifugation at 700×g at 4 ◦C for 30 min. The post-nuclear supernatants were centrifuged at 14,500×g at 4 ◦C for 30 min to remove mitochondria, and the resulting supernatants were further centrifuged twice at 105,000×g at 4 C to collect microsomes. The resultant microsomal pellets were resuspended. Protein contents were measured by Bio-Rad Dye Reagent Concentrate according to manufacturer’s instructions. The concentration of microsome protein was adjusted to 2mg/ml. Microsomes were used for measurement of 11beta-HSD2 activities.

The oxidation of CORT by 11beta-HSD2 was determined in a mixture containing 0.2–0.5 uM CORT (plus 30,000cpm [3H]-CORT), 0.2mM NAD+, 10mM DTT and 2% ethanol in 0.1mM potassium phosphate buffer (pH 7.2, 250 uL total volume) at 37 C. Because 11beta-HSD2 in kidney microsomes is present as a dimer, 10 mM DTT (a reducing agent) was added to reaction buffer to disrupt kidney microsomes and the disulfyl group of 11beta-HSD2 to convert 11beta-HSD2 to as a monomer to increase its activity. Reactions were initiated by the addition of microsomes and NAD+ and terminated by the addition of 2ml ice-cold ether. The steroids were extracted by vigorous mixing for 1min, and the organic layer was dried under nitrogen. The extraction efficiency for CORT and 11DHC were 105.8±8.8% (mean±SD) for CORT and 104.2±14.8% for 11DHC. The steroids were separated chromatographically on thin layer plates in chloroform and methanol (90:10, v/v), and the radioactivity was measured using a scanning radiometer (System AR2000, Bioscan Inc., Washington, DC). The percentage conversion of CORT to 11DHC and was calculated by dividing the radioactive counts identified as 11DHC by the total counts associated with CORT plus 11DHC as previously described [16], and the velocity of the 11beta-HSD2 was calculated according to the percentage of substrate conversion, substrate concentration, enzyme amount and reaction time. The formation of product was determined at 4 time points within the linear portion of the reaction.

Inhibition of the oxidation of CORT catalyzed by 11beta-HSD2 was measured using varying concentrations of each inhibitor with the substrate concentrations set to about 2× Km to calculate the half maximal inhibitory concentration (IC50) values. The mode of inhibition was assayed by adding different fixed CORT concentrations in the presence of various concentrations of each inhibitor. Initial velocity data were fit to competitive, noncompetitive, uncompetitive, and mixed inhibition modes. The inhibition constant Ki was determined.

- Parameters analysed / observed: 11beta-HSD2 enzyme activity and inhibition were determined.

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

other: Inhibition of human and rat 11beta-hydroxysteriod dehydrogenase.

Test material

Test material information

Constituent 1

Reference substance name:

Potassium 1,1,2,2,3,3,4,4,4-nonafluorobutane-1-sulphonate

EC Number:

249-616-3

EC Name:

Potassium 1,1,2,2,3,3,4,4,4-nonafluorobutane-1-sulphonate

Cas Number:

29420-49-3

Molecular formula:

C4HF9O3S.K

IUPAC Name:

potassium 1,1,2,2,3,3,4,4,4-nonafluorobutane-1-sulfonate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sigma Aldrich
- Expiration date of the lot/batch: No data
- Purity test date: No data

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Rats were utilized to isolate kidney microsomes.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CHarles River Laboratories
- Age at study initiation: No data
- Weight at study initiation: 250-300 g
- Fasting period before study: No data
- Housing: No data
- Diet (e.g. ad libitum): No data
- Water (e.g. ad libitum): No data
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: No data

Administration / exposure

Route of administration:

other: Rat and human kidney microsomes were utlized to isolate 11beta-HSD2 enzymes.

Vehicle:

not specified

Details on exposure:

Upon incubation with PFBS, 11beta-HSD2 activity was measured by the conversion of corticosterone to 11-dehydrocorticosterone in the presence of increasing concentrations of PFBS up to 250 uM.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Not specified.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: uM

Doses / concentrations 2

Dose / conc.:

50 other: uM

Doses / concentrations 3

Dose / conc.:

250 other: uM

Details on study design:

See 'Principles of Method' section.

Results and discussion

Details on results:

PFBS generated 1.25 +/- 2.56% inhibition of 11beta-HSD2 activity at the highest dose tested (250 uM).

Applicant's summary and conclusion

Conclusions:

PFBS generated 1.25 +/- 2.56% inhibition of 11beta-HSD2 activity at the highest dose tested (250 uM). At concentrations up to 250 mM, K+PFBS did not statistically significantly impact 11beta-HSD2 activity.

Executive summary:

The inhibition of human and rat 11beta-hydroxysteriod dehydrogenase 2 by PFBS was evaluated in isolated kidney microsomes. Human microsomes were purchased. Rat kidney from male Sprague Dawley rats was homogenized in 0.01mM phosphate buffered saline (PBS, pH 7.4) containing 0.25M sucrose, and nuclei and large cell debris were removed by centrifugation at 700×g at 4 ◦C for 30 min. The post-nuclear supernatants were centrifuged at 14,500×g at 4 ◦C for 30 min to remove mitochondria, and the resulting supernatants were further centrifuged twice at 105,000×g at 4 C to collect microsomes. The resultant microsomal pellets were resuspended. Protein contents were measured by Bio-Rad Dye Reagent Concentrate according to manufacturer’s instructions. The concentration of microsome protein was adjusted to 2mg/ml. Microsomes were used for measurement of 11beta-HSD2 activities. The oxidation of CORT by 11beta-HSD2 was determined in a mixture containing 0.2–0.5 uM CORT (plus 30,000cpm [3H]-CORT), 0.2mM NAD+, 10mM DTT and 2% ethanol in 0.1mM potassium phosphate buffer (pH 7.2, 250 uL total volume) at 37 C. Because 11beta-HSD2 in kidney microsomes is present as a dimer, 10 mM DTT (a reducing agent) was added to reaction buffer to disrupt kidney microsomes and the disulfyl group of 11beta-HSD2 to convert 11beta-HSD2 to as a monomer to increase its activity. Reactions were initiated by the addition of microsomes and NAD+ and terminated by the addition of 2ml ice-cold ether. The steroids were extracted by vigorous mixing for 1min, and the organic layer was dried under nitrogen. The extraction efficiency for CORT and 11DHC were 105.8±8.8% (mean±SD) for CORT and 104.2±14.8% for 11DHC. The steroids were separated chromatographically on thin layer plates in chloroform and methanol (90:10, v/v), and the radioactivity was measured using a scanning radiometer (System AR2000, Bioscan Inc., Washington, DC). The percentage conversion of CORT to 11DHC and was calculated by dividing the radioactive counts identified as 11DHC by the total counts associated with CORT plus 11DHC as previously described [16], and the velocity of the 11beta-HSD2 was calculated according to the percentage of substrate conversion, substrate concentration, enzyme amount and reaction time. The formation of product was determined at 4 time points within the linear portion of the reaction. Inhibition of the oxidation of CORT catalyzed by 11beta-HSD2 was measured using varying concentrations of each inhibitor with the substrate concentrations set to about 2× Km to calculate the half maximal inhibitory concentration (IC50) values. The mode of inhibition was assayed by adding different fixed CORT concentrations in the presence of various concentrations of each inhibitor. Initial velocity data were fit to competitive, noncompetitive, uncompetitive, and mixed inhibition modes. The inhibition constant Ki was determined. PFBS generated 1.25 +/- 2.56% inhibition of 11beta-HSD2 activity at the highest dose tested (250 uM). At concentrations up to 250 mM, K+PFBS did not statistically significantly impact 11beta-HSD2 activity.