Abstract
Phenylacetaldehyde is formed when the xenobiotic and biogenic amine 2-phenylethylamine is inactivated by a monoamine oxidase–catalyzed oxidative deamination. Exogenous phenylacetaldehyde is found in certain foodstuffs such as honey, cheese, tomatoes, and wines. 2-Phenylethylamine can trigger migraine attacks in susceptible individuals and can become fairly toxic at high intakes from foods. It may also function as a potentiator that enhances the toxicity of histamine and tyramine. The present investigation examines the metabolism of phenylacetaldehyde to phenylacetic acid in freshly prepared and in cryopreserved guinea pig liver slices. In addition, it compares the relative contribution of aldehyde oxidase, xanthine oxidase, and aldehyde dehydrogenase in the oxidation of phenylacetaldehyde using specific inhibitors for each oxidizing enzyme. The inhibitors used were isovanillin for aldehyde oxidase, allopurinol for xanthine oxidase, and disulfiram for aldehyde dehydrogenase. In freshly prepared liver slices, phenylacetaldehyde was converted mainly to phenylacetic acid, with traces of 2-phenylethanol being present. Disulfiram inhibited phenylacetic acid formation by 80% to 85%, whereas isovanillin inhibited acid formation to a lesser extent (50% to 55%) and allopurinol had little or no effect. In cryopreserved liver slices, phenylacetic acid was also the main metabolite, whereas the 2-phenylethanol production was more pronounced than that in freshly prepared liver slices. Isovanillin inhibited phenylacetic acid formation by 85%, whereas disulfiram inhibited acid formation to a lesser extent (55% to 60%) and allopurinol had no effect. The results in this study have shown that, in freshly prepared and cryopreserved liver slices, phenylacetaldehyde is converted to phenylacetic acid by both aldehyde dehydrogenase and aldehyde oxidase, with no contribution from xanthine oxidase. Therefore, aldehyde dehydrogenase is not the only enzyme responsible in the metabolism of phenylacetaldehyde, but aldehyde oxidase may also be important and thus its role should not be ignored.
Precision-cut liver slices are now an accepted in vitro system to study xenobiotic metabolism (Lerche-Langrand and Toutain 2000; Panoutsopoulos and Beedham 2005). They can be easily prepared from different animal species by using a similar procedure where a high degree of hepatic lobular architecture of the tissue and intercellular communication are maintained, giving similar conditions to those in vivo (Smith et al. 1986; Parrish, Gandolfi, and Brendel 1995). Cryopreservation of liver slices would greatly facilitate their storage for long periods of time and potentially reducing the number of laboratory animals used. The application of a rapid freezing technique, by direct immersion into liquid nitrogen, preserves both phase I– and phase II–mediated drug metabolism with no significant loss of enzymatic activities (de Graaf and Koster 2003; Martignoni et al. 2004).
Phenylacetaldehyde is formed when the biogenic amine 2-phenylethylamine is inactivated by a monoamine oxidase B–catalyzed oxidative deamination (Wouters 1998). This biogenic amine potentiates the postsynaptic effects of dopamine (Paterson, Jurio, and Boulton 1990; Barroso and Rodriguez 1996) and acts as neuromodulator of catecholamine neurotransmission in the brain (Barroso and Rodriguez 1996). Exogenous 2-phenylethylamine present in various foodstuffs (cheese, chocolate, wine, and beer) can trigger migraine attacks in susceptible individuals (Martin and Behbehani 2001; Millichap and Yee 2003). In addition, it becomes fairly toxic at high intakes and may function as a potentiator that enhances the toxicity of histamine and tyramine (Joosten 1988; Stratton, Hutkins, and Taylor 1991; Taylor 1986).
Exogenous phenylacetaldehyde is found in certain foodstuffs such as honey (Zhou, Wintersteen, and Cadwallader 2002), tomatoes (Tadmor et al. 2002), cheese (Quian and Reineccius 2002), beer (Vesely et al. 2003), and wine (Aznar et al. 2003). Phenylacetaldehyde is then converted to phenylacetic acid by horse liver (Feldman and Weiner 1972) and guinea pig liver (Panoutsopoulos et al. 2004b) aldehyde dehydrogenase (EC 1.2.1.3, aldehyde-NAD(P)+ oxidoreductase) and possibly by bovine milk xanthine oxidase (EC 1.2.3.2, xanthine-oxygen oxidoreductase) and guinea pig liver aldehyde oxidase (EC 1.2.3.1, aldehyde-oxygen oxidoreductase) (Panoutsopoulos and Beedham 2004a).
Aldehyde dehydrogenase catalyzes the irreversible oxidation of exogenous and biogenic aldehydes to the corresponding acids in a NAD+-dependent reaction (Tabakoff, Anderson, and Alivisatos 1973; Pettersson and Tottmar 1982). Although aldehyde dehydrogenase is distributed in all mammalian tissues, its maximum activity is found in the liver (Sladek 2003), with different isozymes in mitochondria (Han and Joo 1991), cytosol (Vallari and Pietruszko 1982), and microsomes (Cho and Joo 1990). However, aldehyde oxidase and xanthine oxidase also catalyze the oxidation of xenobiotic and biogenic aldehydes (Morpeth 1983; Panoutsopoulos and Beedham 2004a). These enzymes are more widely known for their role in the metabolism of heterocyclic compounds (Beedham 1987; Critchley, Rance, and Beedham 1992; Panoutsopoulos and Beedham 2004b) and their relative importance in the metabolism of aromatic aldehydes has largely been ignored. Nevertheless, aromatic aldehydes are known substrates of hepatic aldehyde oxidase and are metabolized to a lesser extent by xanthine oxidase (Morpeth 1983; Panoutsopoulos and Beedham 2004a). Aldehyde oxidase is distributed in most mammalian tissues and mainly in the liver with different isozymes in cytosol and mitochondria (Beedham et al. 1987b).
In this investigation, the enzymatic oxidation of phenylacetaldehyde to phenylacetic acid has been studied in freshly prepared and in cryopreserved guinea pig liver slices. Specific inhibitors were also used in order to determine which aldehyde oxidizing enzymes are involved in the formation of phenylacetic acid from phenylacetaldehyde. The specific inhibitors were isovanillin for aldehyde oxidase (Panoutsopoulos and Beedham 2004a, 2004b; Panoutsopoulos et al. 2004a), allopurinol for xanthine oxidase (Peterson et al. 1990; Panoutsopoulos et al. 2004a), and disulfiram for aldehyde dehydrogenase activity (Lipsky, Shen, and Naylor 2001; Panoutsopoulos et al. 2004a). Guinea pig liver was chosen in this study as aldehyde oxidase has similar substrate specificity to the human liver enzyme and therefore is a good model for in vivo and in vitro screening of human aldehyde oxidase activity (Beedham et al. 1987a, 1990).
MATERIALS AND METHODS
Animals
Dunkin-Hartley female guinea pigs, weighing 450 to 950 g, were used in this study. Animals were fed with FD1 pellets supplemented with ascorbic acid and received hay three times weekly. All animals had free access to food and tap water and were housed in a strictly controlled temperature room (18°C ± 1 °C) with 50% to 55% humidity and a 12-h/12-h light/dark cycle. The animals were handled with humane care in accordance with the National Institutes of Health guidelines (Greek Presidential Decree 1991).
Chemicals
Phenylacetaldehyde, 2-phenylethanol, tetraethylthiuram disulfide (disulfiram), allopurinol, diethylamine,
Preparation of Freshly Prepared and Cryopreserved Guinea Pig Liver Slices
Guinea pigs were killed by cervical dislocation, their livers were immediately excised, and placed in ice-cold Krebs-Henseleit buffer (pH 7.4) containing 2.4 × 10−2 M bicarbonate, which was continuously purged with 95% O2/5% CO2. Freshly prepared liver slices were obtained using a manual slicer according to the method of Panoutsopoulos et al. (2004c). The first and last slices from each liver core were discarded because they contained the liver capsule. The freshly prepared liver slices (each liver slice had a thickness of 0.5 mm) were placed in Krebs-Henseleit buffer pH 7.4 containing 2.4 × 10−2 M bicarbonate at 4°C and saturated with oxygen/carbon dioxide until required. Cryopreserved liver slices were then prepared by direct immersion of the freshly prepared liver slices in liquid nitrogen and kept there until required.
Metabolism of Phenylacetaldehyde with Freshly Prepared and Cryopreserved Guinea Pig Liver Slices
Phenylacetaldehyde (1 × 10−3 M) was incubated with four freshly prepared or cryopreserved liver slices in a total volume of 3 ml Krebs-Henseleit buffer, pH 7.4, the addition of the liver slices indicated the beginning of the experiment. The incubations were carried out at 37°C in a shaking water bath and the medium was oxygenated with 95% O2/5% CO2 initially and every subsequent hour for 5 min, which was sufficient to maintain optimum metabolite production.
Aliquots (0.2 ml) were removed at various time intervals, treated with 0.1 ml of 3.6% perchloric acid, centrifuged for 2.5 min at maximum speed on a Beckman microfuge B and the supernatant was then analyzed by high-performance liquid chromatography (HPLC). Control incubations without the addition of liver slices were also performed. Standard solutions of the aldehyde and its possible metabolites were also analyzed by HPLC. After analysis, the slices were blotted dry and weighed to determine the total weight of liver used in each incubation.
Incubations, up to 180 min, with freshly prepared or cryopreserved liver slices were also performed in the presence of the inhibitors, isovanillin (1 × 10−3 M) for aldehyde oxidase activity (Panoutsopoulos and Beedham 2004a; Panoutsopoulos et al. 2004a), allopurinol (1 × 10−4 M) for xanthine oxidase activity (Peterson et al. 1990), and disulfiram (1 × 10−4 M) for aldehyde dehydrogenase activity (Lipsky et al. 2001). For comparison reasons, the results on the effect of inhibitors in freshly prepared and cryopreserved liver slices have been normalized per 100 mg of liver.
HPLC Analysis of Phenylacetaldehyde and Its Metabolites
Reverse-phase HPLC analysis was carried out using a system supplied from Waters Associates (Northwich, Cheshire, UK), which consisted of a single piston reciprocating 501 pump, a WISP 710B auto-injector, a Lambda-Max 481 LC detector, and a Data Module 740. Chromatographic separation of compounds was achieved using a stainless steel Hypersil ODS column 5 μm (25 cm × 4.6 mm internal diameter) (Shandon Southern Products, Runcorn, Great Britain) with a Waters μBondapak C18 Guard-Pak insert and 30% acetonitrile/70% 2.2 × 10−1 M orthophosphate buffer pH 2.9 containing 1.1 × 10−1 M diethylamine at a flow rate of 1.5 ml/min. The mobile phase was filtered and degassed for 10 min under vacuum before use. The reactions were monitored at 250 nm and 20-μl samples were injected for each analysis. Phenylacetaldehyde and its oxidized metabolites were identified by comparison of their HPLC retention times with those of authentic standards.
Statistical Analysis
Data were expressed as means ± SE. All observations were obtained from six different incubations. The statistical analysis of the results was performed one-way analysis of variance and unpaired Student’s t test.
RESULTS
Metabolism of Phenylacetaldehyde with Freshly Prepared Guinea Pig Liver Slices
In incubations of phenylacetaldehyde (retention time, Rt = 12.2 ± 0.45 min, n = 12) with freshly prepared liver slices, a rapid decrease in aldehyde concentration was observed until it had almost completely disappeared within 45 min. Simultaneously, one main metabolite and two minor metabolites were produced (Figure 1). All three metabolites accounted for most of the aldehyde breakdown. The main metabolite was phenyl-acetic acid (Rt = 7.7 ± 0.4 min, n = 12), which accounted for 55% conversion of phenylacetaldehyde within 45 min and 65% after 90 min. The first minor metabolite was 2-phenylethanol (Rt = 7.5 ± 0.3 min, n = 12), which is formed by reduction of phenylacetaldehyde in liver slices and accounted for 5% after 90 min. The second minor metabolite coeluted with standard mandelic acid (Rt = 3.25 ± 0.4 min, n = 12), which accounted for 27% at 90 min. However, there might be other possibilities of this polar metabolite such as a glycine conjugate, but this compound was not commercially available for comparison. On the other hand, it might be 4-hydroxyphenylacetic acid, which had a similar elution time of 3.4 min.
Effect of Inhibitors on Phenylacetaldehyde Oxidation by Freshly Prepared Guinea Pig Liver Slices
The presence of isovanillin (1 × 10−3 M) inhibited the production of phenylacetic acid by 50% to 55%, although phenyl-acetaldehyde breakdown was inhibited to a greater extent (data not shown). However, there was a small increase in the formation of 2-phenylethanol (Figure 2B ) and mandelic acid (data not shown).
In the presence of allopurinol (1 × 10−4 M), phenylacetic acid production was slightly enhanced (Figure 2A ). In addition, there was a slight enhancement in both the production of 2-phenylethanol (Figure 2B ) and mandelic acid (data not shown).
Disulfiram (1 × 10−4 M) inhibited the production of phenylacetic acid by 80% to 85% (Figure 2A ). However, there was a very significant increase in the production of 2-phenylethanol (Figure 2B ).
Metabolism of Phenylacetaldehyde with Cryopreserved Guinea Pig Liver Slices
In cryopreserved guinea pig liver slices, phenylacetaldehyde rapidly disappeared within 45 min and two metabolites were formed. The major metabolite was phenylacetic acid (Figure 3), which accounted for 50% conversion of phenylacetaldehyde within 45 min and 57% after 90 min. The minor metabolite was 2-phenylethanol (Figure 3), which increased with time to a greater extent than that seen in freshly prepared liver slices (18% at 90 min). However, quantitatively conversion did not occur.
Effect of Inhibitors on Phenylacetaldehyde Oxidation by Cryopreserved Guinea Pig Liver Slices
The presence of isovanillin (1 × 10−3 M) inhibited by about 85% both the production of phenylacetic acid (Figure 4A ) and phenylacetaldehyde breakdown (data not shown). Two other minor metabolites were observed. The first minor metabolite was 2-phenylethanol (Figure 4B ), which remained at the same levels as in the non inhibited incubations. The second minor metabolite was mandelic acid (data not shown), which slowly increased with time.
Allopurinol (1 × 10−4 M) did not inhibit phenylacetaldehyde metabolism, but instead it caused a slight enhancement of phenylacetic acid production (Figure 4A ). However, no other metabolites were seen and likewise no 2-phenylethanol was produced (Figure 4B ).
The presence of disulfiram (1 × 10−4 M) in phenylacetaldehyde incubations caused a 55% to 60% inhibition in the production of phenylacetic acid (Figure 4A ) over the incubation period. In addition, it was shown that upon inhibition with disulfiram there was a very significant increase in the production of 2-phenylethanol (Figure 4B ).
DISCUSSION
2-Phenylethylamine is a naturally occurring biogenic amine that is oxidative deaminated to phenylacetaldehyde by mono-amine oxidase B (Wouters 1998) and is found in many mammalian tissues (Nakajima, Kakimoto, and Sano 1964) including the brain (Henry et al. 1988; Paterson, Jurio, and Boulton 1990). It crosses the presynaptic membrane and potentiates the postsynaptic effects of dopamine (Paterson, Jurio, and Boulton 1990; Barroso and Rodriguez 1996) and may also act as neuromodulator of catecholamine neurotransmission in the brain (Barroso and Rodriguez 1996). Exogenous 2-phenylethylamine is found in certain foodstuffs and has been known to trigger migraine attacks in susceptible individuals, with chocolate being the commonest dietary trigger (Martin and Behbehani 2001; Millichap and Yee 2003). Other foodstuffs that contain particularly high levels of 2-phenylethylamine are cheese (Quian and Reineccius 2002), beer (Vesely et al. 2003), wine (Aznar et al. 2003), and sausage (Stratton, Hutkins, and Taylor 1991).
Although dietary biogenic amines, such as serotonin, 2-phenylethylamine, and tyramine, at levels which are present in food are nontoxic, these become fairly toxic at high intakes (Morrow et al. 1991; Smith 1981). In addition, 2-phenylethylamine may function as a potentiator that enhances the toxicity of histamine and tyramine (Joosten 1988; Stratton, Hutkins, and Taylor 1991; Taylor 1986). Consequently, the consumption of high levels of these biogenic amines can lead to nausea, respiratory distress, hot flashes, sweating, heart palpitations, headaches, bright-red rush, oral burning, and hyper- or hypotension (Bardocz 1995). Individuals with respiratory and coronary problems or those with hypertension or vitamin B12 deficiency are particularly at risk because they are sensitive to lower doses of biogenic amines (Bardocz 1995). People with gastrointestinal problems (gastritis, irritable bowel syndrome, Crohn’s disease, stomach and colonic ulcers) are also at risk because the activity of oxidases in their intestines is usually lower than that in healthy individuals (Bardocz 1995). In women, there is a premenstrual decrease in the activity of monoamine oxidase B and this can also be a problem (Bardocz 1995). Individuals on any medication that acts as a blocker of monoamine oxidase and diamine oxidase activity can also be affected, because such drugs prevent the elimination of amines and if they ingest elevated levels of dietary biogenic amines can cause a fatal reaction (Stockley 1993). These monoamine oxidase and diamine oxidase inhibitors include painkillers and drugs used for the treatment of stress and depression, and also of Alzheimer’s and Parkinson’s diseases.
Therefore, because of the toxicological effects that the parent compound 2-phenylethylamine may possess, it is useful to study the metabolism of phenylacetaldehyde by oxidizing enzymes. The present investigation compares the relative contribution of aldehyde oxidase, xanthine oxidase, and aldehyde dehydrogenase in the oxidation of phenylacetaldehyde in guinea pig liver slices.
The results in this study have shown that the major metabolite of phenylacetaldehyde, in freshly prepared and cryopreserved liver slices, is phenylacetic acid (Figure 5), with small amounts of 2-phenylethanol (Figure 5) and mandelic acid (in freshly prepared liver slices) also being produced. 2-Phenylethanol could be formed by reduction of phenylacetaldehyde by alcohol dehydrogenase. Incubations, under identical conditions, with homovanillamine, 5-hydroxytryptamine, vanillin, or isovanillin also resulted in formation of their corresponding alcohol derivatives (Beedham et al. 1995; Panoutsopoulos and Beedham 2005).
The effects of inhibitors tested, in freshly prepared liver slices, showed that disulfiram inhibited phenylacetaldehyde oxidation by 80% to 85%, whereas isovanillin inhibited to a lesser extent (50–55%) and allopurinol had no effect. In fact, allopurinol consistently caused a small enhancement in phenylacetic acid production. This shows that aldehyde dehydrogenase is the predominant enzyme that catalyzes phenylacetaldehyde oxidation with a lower participation from aldehyde oxidase and no contribution from xanthine oxidase. However, in cryopreserved liver slices, the contribution of these enzymes in phenyl-acetaldehyde metabolism changed, as phenylacetic acid production was inhibited by isovanillin (85%) and to a lesser extent by disulfiram (55%). This is probably due to the low aldehyde dehydrogenase activity that is present in cryopreserved liver slices (about 70% lower) compared to freshly prepared liver slices (Panoutsopoulos and Beedham 2004c). However, the metabolism of phenylacetaldehyde was similar to that seen in freshly prepared guinea pig liver slices, indicating that aldehyde oxidase is involved and therefore it has a prominent role in the oxidation of phenylacetaldehyde. Allopurinol once again caused a small enhancement in the production of phenylacetic acid.
A similar enhancement in acid production was seen when vanillin (Panoutsopoulos and Beedham 2005) was incubated with freshly prepared guinea pig liver slices in the presence of allopurinol. This enhancement may be due to xanthine dehydrogenase, which is present in liver slices. Xanthine oxidase differs from xanthine dehydrogenase in the presence of a sulphydryl group (Kaminski and Jezewska 1982). Conversion of xanthine oxidase from dehydrogenase (D form) into an oxidase (O form) occurs upon oxidation or binding of sulphydryl groups by several oxidizing agents or ligands (della Corte and Stripe 1972; Waud and Rajagopalan 1976). This may increase the formation of phenylacetic acid.
The inhibition of aldehyde dehydrogenase by disulfiram caused a very significant increase in 2-phenylethanol production. Because disulfiram inhibits aldehyde dehydrogenase activity, it blocks the reaction toward phenylacetic acid and therefore results in the accumulation of phenylacetaldehyde which is reduced by NADH and alcohol dehydrogenase (Figure 5).
The oxidation of phenylacetaldehyde has been previously shown with aldehyde dehydrogenase, aldehyde oxidase, and xanthine oxidase in separate isolated enzyme preparations under identical incubation and chromatographic conditions (Panoutsopoulos et al. 2004b). Aldehyde dehydrogenase was found as the predominant enzyme involved in phenylacetaldehyde oxidation with aldehyde oxidase playing a less prominent role. Although phenylacetaldehyde was converted to its acid by high amounts (3.8 mg) of bovine milk xanthine oxidase, it had little effect when low amounts (0.0475 mg) of xanthine oxidase were used. This low amount of xanthine oxidase is equivalent to xanthine oxidase, which is present in guinea pig liver. Therefore, xanthine oxidase does not contribute to the oxidation of phenylacetaldehyde due to low amounts being present in guinea pigs (Panoutsopoulos et al. 2004b).
Phenylacetaldehyde has been shown as a substrate of aldehyde dehydrogenase (ALDH)-1 with K m value of 1.5 × 10−6 M (Pietruszco 1989) and ALDH-2 with K m values of 0.6 × 10−6 M (Pietruszco 1989) and 2.9 × 10−8 M (Klyosov 1996). Reaction of phenylacetaldehyde with rat brain mitochondrial aldehyde dehydrogenase has been reported by Weiner and Ardelt (1984) who found a K m value of 1.3 × 10−5 M. Oxidation of phenylacetaldehyde to phenylacetic acid, via guinea pig liver aldehyde oxidase, has also been previously reported (K m = 5.3 × 10−5 M, V max = 0.44 μmol/min/mg protein and K s = 5.0 ml/min/mg protein) using ferricyanide as an electron acceptor (Panoutsopoulos et al. 2004b). These results indicate that phenylacetaldehyde is also an excellent substrate of aldehyde oxidase and this enzyme can therefore play a prominent role in the metabolism of phenyl-acetaldehyde.
In conclusion, it would appear that aldehyde oxidase not only contributes to the metabolism of aldehydes derived from drugs, xenobiotics, foods, and flavorings, but also the metabolism of the intermediate aldehyde derivatives from biogenic amines, such as 2-phenylethylamine.