Allyl alcohol

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data taken from a peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

The biotransformation of allyl alcohol and acrolein in rat liver and lung preparations.

Author:

Patel, J.M., Wood., J.C., Leibman, K.C.

Year:

1980

Bibliographic source:

Drug metabolism and disposition 8: 305-308

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study identifies and characterises the pathways of biotransformation of allyl alcohol by rat liver and lung preparations.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Allyl alcohol

EC Number:

203-470-7

EC Name:

Allyl alcohol

Cas Number:

107-18-6

Molecular formula:

C3H6O

IUPAC Name:

prop-2-en-1-ol

Details on test material:

Allyl alcohol was obtained from Eastman Kodak Corp. (Rochester, N.Y.). The purity of allyl alcohol used is not known.

Radiolabelling:

no

Test animals

Species:

rat

Strain:

other: Holtzman

Sex:

male

Details on test animals or test system and environmental conditions:

Male Holtzman rats (approximately 120-130 g body weight) were pretreated with 75/mg/kg sodium phenobarbital for 3 days. From these rats, liver cytosol and microsomes were isolated. In addition lung cytosol and microsomes were also isolated.

Administration / exposure

Route of administration:

other: in vitro test

Vehicle:

not specified

Details on exposure:

Experiments carried out in vitro using reconstituted lyophilised liver or lung 9000g supernatant fractions.

Duration and frequency of treatment / exposure:

See 'Details on study design' field.

Doses / concentrations

Remarks:

Doses / Concentrations:
See 'Details on study design' field.

Control animals:

other: in vitro control used

Details on study design:

Metabolism of allyl alcohol to acrolein: studied using a reaction mixture containing 1.0 mM NAD+, 5.0 mM MgSO4, 5.0 mM allyl alcohol and reconstituted lyophilised liver or lung 9000g supernatant fraction (equivalent to 45mg of protein or 15 or 25 mg of microsomal or cytosolic protein respectively) in 3.0 ml of 0.1M Tris-HCl buffer (pH 7.5). 0.5 mM pyrazole was used to inhibit alcohol dehydrogenase. Each reaction flask was incubated at 37°C for 45 minutes. After incubation each reaction flask was placed into an icebath and 1.0 ml of 70% HClO4 added. This mixture was then centrifuged (1400 rpm for 10 minutes) and the supernatant was mixed with 5.0ml of solution containing 0.1% 2,4-dinitrophenylhydrazine (DNPH) in 0.2 N HCl and kept overnight. The acrolein-DNPH derivative formed was extracted in 10.0 ml of HPLC-grade chloroform. The extract was washed with 2 N HCl and distilled water to remove excess DNPH. The acrolein was concentrated to dryness using nitrogen. Dried samples were dissolved in methylene chloride to be analysed by HPLC.

Metabolism of allyl alcohol to acrylic acid: Studied using a reaction mixture consisting of 1.0 mM NAD+, 5.0 mM MgSO4, 5.0 mM allyl alcohol and reconstituted lyophilised liver or lung 9000g supernatant fraction (equivalent to 45mg of protein or 15 or 25mg of cytosolic or microsomal protein respectively in 3.0ml of 0.1M Tris-HCl buffer (pH 7.5). 0.5 mM disulfram was used for aldehyde hydrogenase inhibition. Each reaction flask was incubated at 37°C for 45 minutes and then terminated by the addition of 1.5ml of 6N HCL.

Metabolism of acrolein to acrylic acid: Studied using a reaction mixture consisting of 1.0 mM NAD+ or NADP+, 5.0 mM MgSO4, 5.0 mM acrolein and reconstituted lyophilised liver or lung 9000g supernatant fraction (equivalent to 45mg of protein or 15 or 25mg of cytosolic or microsomal protein respectively in 3.0ml of 0.1M Tris-HCl buffer (pH 7.5). 0.5 mM disulfram was used for aldehyde hydrogenase inhibition. Each reaction flask was incubated at 37°C for 45 minutes and then terminated by the addition of 1.5ml of 6N HCL. Each reaction flask was centrifuged (1400 rpm for 10 minutes) and the acrylic acid extracted from the supernatant using 10ml ether. The ether layer was concentrated to 10-15 ul using nitrogen and subsequently added to 0.8ml of HPLC grade methylene chloride. 200 ul of a 1.0 mM solution of PNBDI in methylene chloride and heated at 80°C for 2 hours. This was then analysed by HPLC.

Formation of glycidol and glycidaldehyde (microsomal oxidation products of allyl alcohol and acrolein respectively): 3.0 mM of allyl alcohol or acrolein were incubated with 10 or 15mg of liver or lung microsomal protein in a 3.0ml incubation mixture. After incubation for 5 minutes at 37°C, the reaction flasks were placed into an icebath for 18 minutes after the addition of the substrate. Methanol was added to each reaction flask for deproteinisation and centrifuged (at 1400 rpm for 10 minutes). The supernatant was filtered through a 0.45u Millipore filter and the filtrate dried under an airstream. The residue was extracted using ethyl acetate and after centrifugation the ethyl acetate layer was concentration under nitrogen and analysed using gas and thin-layer chromatography.

Hydration of glycidol and glycialdehyde by liver microsomal epoxide hydrase: acrolein was incubated with liver and lung microsomes at 37°C for 60 minutes. Additionally 0.1 mM glycialdehyde was incubated with 10 or 15 mg of liver or lung microsomal protein respectively in 3.0 ml volumes (also containing 0.1 M Tris-HCl buffer) at 37°C for 60 minutes. Each reaction was terminated by the addition of methanol. Each reaction flask was centrifuged (1400 rpm for 10 minutes) and the supernatant evaporated under an airstream. 1.0 ml of distilled was added to the residue and once again centrifuged. The supernatant from this was used in the determination of glyceraldehydes content by a spectrophotometric method.

Formation of glycerol or glycidol by liver microsomes with allyl alcohol: Two incubation mixtures were used. Incubation mixture A contained: 0.75 mM nicotinamide, 0.5 mM MgSO4, 60 uM EDTA, 30 uM ATP, 0.25 mM glucose 6-phosphate, 0.1 mM NADP+, 10 units of glucose 6-phosphate dehydrogenase, 100 mg of liver microsomal proteins and 5.0 mM allyl alcohol. Incubation mixture B contained: 1.0 mM glycidol and 15 mg of liver or lung microsomal protein in a volume of 5.0 ml (containing 0.1 M Tris-HCl buffer). Both incubation mixed were incubated along with a control (containing no substrates) at 37C for 60 minutes. 200 g of NaCl was added to mixture A and 15 g to mixture B to terminate the reactions. The contents of mixtures A and B were extracted using 150 ml and 10 ml isopropyl alcohol respectively. Mixtures were centrifuged (200- rpm for 15 minutes) and the isopropyl alcohol layer was removed and evaporated to dryness under an airstream. 20 ml and 2 ml of isopropyl alcohol were added to residues A and B and once again centrifuged. The isopropyl layer was removed and evaporated to dryness. The residues were dissolved into 0.2 ml of pyridine and analysed by modified HPLC and mass spectrometry.

Results and discussion

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Acrolein, acrylic acid, glycidol, glycidaldehyde and glyceraldehyde.

Any other information on results incl. tables

The metabolic conversion of allyl alcohol to acrolein (table 1) and further metabolism to acrylic acid has been confirmed in liver 9000g supernatant fractions and cytosol in the presence of NAD+. Allyl alcohol metabolism to acrolein was not seen when NAD+ was replaced with NADP+ or NADPH or in liver microsomes with NAD+. 80% of allyl alcohol was metabolised to acrolein when liver 9000g supernatant and cytosol fractions were used. In addition, when lung fractions are used, allyl alcohol does not appear to be metabolised to acrolein.The presence of pyrazole appeared to inhibit the metabolism of allyl alcohol to acrolein in both 9000g and cytosol fractions.

 

Acrolein which was formed in allyl alcohol metabolism was further metabolised to acrylic acid (however, only 15% of the acrolein was metabolised to acrylic acid). The presence of pyrazole seemed to affect the formation of acrylic acid. 

Metabolism of acrolein to acrylic acid (see table 2) was seen in liver 9000g supernatant, cytosolic and microsomal preparations (in presence of NAD+ or NADP+). Acrylic acid was not present when NADPH was used in the liver incubation mixture or if lung preparations were used.

Incubation of allyl alcohol and acrolein with liver and lung microsomes (with NADPH), revealed that glycidol and glycidaldehyde were formed.

Hydration of these epoxides glycidol and glycidalehyde to glycerol and glyceraldehyde respectively were seen when incubated with liver or lung microsomal preparations. (see table 3). When boiled microsomes were used, neither glycerol or glyceraldehyde were formed.

Table 1. Metabolism of allyl alcohol to acrolein and acrylic acid by rat liver fractions.

Metabolite production  (nmol/min/mg protein)	Acrolein		Acrylic acid
	9000g	Cytosol	9000g	Cytosol
No additions	0.0 +/- 0.0	0.0 +/- 0.0	0.0 +/- 0.0	0.0 +/- 0.0
NAD+ added	45.4 +/- 4.8	36.2 +/- 3.9	4.3 +/- 0.5	3.1 +/- 0.2
NAD+ & pyrazole (0.5 mM)	13.7 +/- 1.2*	11.0 +/- 1.2 *	0.0 +/- 0.0	0.0 +/- 0.0
NAD+ & disulfiram (0.5 mM) added	40.8 +/- 3.0	31.2 +/- 3.6	1.2 +/- 0.3 *	0.9 +/- 0.1*

* Significantly different from uninhibited reactions (p < 0.01)

Table 2. Metabolism of acrolein to acrylic acid by rat liver fractions

	Acrylic acid (nmol/min/mg protein)
	9000g	Cytosol	Microsomes
No additions	0.0	0.0	0.0
NAD+ added	15.6 +/- 1.3	11.1 +/- 0.8	4.0 +/- 0.2
NAD+ + disulfram (0.5mM) added	6.2 +/- 0.8*	3.2 +/- 0.2*	0.0 +/- 0.0*
NADP+ added	4.8 +/- 0.3	3.2 +/- 0.4	1.5 +/- 0.1
NADP+ + disulfram (0.5mM) added	0.9 +/- 0.1*	0.0 +/- 0.0*	0.0 +/- 0.0*

* Significantly different from uninhibited reactions (p < 0.01)

Table 3. Liver and lung microsomal hydration of glycidol and glycidaldehyde

	Liver		Lung
	Glycerol	Glyceraldehyde	Glycerol	Glyceraldehyde
	umol/mg protein
No additions	0.0	0.0	0.0	0.0
NADPH added	0.0	0.0	0.0	0.0
Allyl alcohol added	0.0	-	0.0	-
Acrolein added	-	0.0	-	0.0
Allyl alcohol + NADPH added	0.32	-	-	-
Acrolein + NADPH added	-	1.33	-	1.02
Glycidol added	0.44	-	0.15	-
Glycidaldehyde added	-	1.55	-	1.2
Glycidol added#	0.0	-	0.0	-
Glycidaldehyde added#	-	0.0	-	0.0

# Boiled microsomes were used

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): other: Biotransformation pathways determined
This study demonstrated metabolic conversion of allyl alcohol to acrolein in liver fractions (cytosol, 9000g supernatant) but not liver microsomes. In liver and lung microsomes, allyl alcohol is converted to glycidol; further metabolism of acrolein to glyceraldehyde or acrylic acid, glycidol to glycerol is microsome-mediated.