By O’Brien, Peter J; Siraki, Arno G; Shangari, Nandita
Aldehydes are organic compounds that are widespread in nature. They can be formed endogenously by lipid peroxidation (LPO), carbohydrate or metabolism ascorbate autoxidation, amine oxidases, cytochrome P-450s, or myeloperoxidase-catalyzed metabolic activation. This review compares the reactivity of many aldehydes towards biomolecules particularly macromolecules. Furthermore, it includes not only aldehydes of environmental or occupational concerns but also dietary aldehydes and aldehydes formed endogenously by intermediary metabolism. Drugs that are aldehydes or form reactive aldehyde metabolites that cause side-effect toxicity are also included. The effects of these aldehydes on biological function, their contribution to human diseases, and the role of nucleic acid and protein carbonylation/oxidation in mutagenicity and cytotoxicity mechanisms, respectively, as well as carbonyl signal transduction and gene expression, are reviewed. Aldehyde metabolic activation and detoxication by metabolizing enzymes are also reviewed, as well as the toxicological and anticancer therapeutic effects of metabolizing enzyme inhibitors. The human health risks from clinical and animal research studies are reviewed, including aldehydes as haptens in allergenic hypersensitivity diseases, respiratory allergies, and idiosyncratic drug toxicity; the potential carcinogenic risks of the carbonyl body burden; and the toxic effects of aldehydes in liver disease, embryo toxicity/ teratogenicity, diabetes/hypertension, sclerosing peritonitis, cerebral ischemia/neurodegenerative diseases, and other aging- associated diseases.
Keywords Aldehydes, Aldehyde Metabolism, Aldehyde-Metabolizing Enzymes, DNA and Protein Carbonylation, Carbonyl Body Burden, α- Oxoaldehdye
SUMMARY
Although there have been various reviews on particular aldehydes that are of potential carcinogenic concern, such as formaldehyde (CEPA, 2001; Collins and Lineker, 2004; Heck et al., 1990; IARC, 2004; IPCS, 2002b; Liteplo and Meek, 2003), acetaldehyde (CEPA, 2000b; IARC, 1999; Liteplo and Meek, 2003) crotonaldehyde (IARC, 1995a), furfural (IARC, 1995c; IPCS, 2000), and acrolein (CEPA, 2000b; IARC, 1995b; IPCS, 2002a), this review compares many aldehydes and their reactivity to biomolecules particularly macromolecules. Furthermore, it includes not only aldehydes of environmental or occupational concerns but also dietary aldehydes and aldehydes formed endogenously by intermediary metabolism. Also included are drugs that are aldehydes or that form reactive aldehyde metabolites that cause side-effect toxicity. The effects of these aldehydes on biological function, their contribution to human diseases, and the role of nucleic acid and protein carbonylation/ oxidation in mutagenicity and cytotoxicity mechanisms, respectively, as well as carbonyl signal transduction and gene expression, are reviewed. Their metabolic activation and detoxication by aldehyde- metabolizing enzymes are also reviewed, as well as the toxicological and anticancer therapeutic effects of metabolizing enzyme inhibitors. Some of these enzymes are genetically polymorphic so that the susceptibility of the individual to diseases associated with aldehyde accumulation (e.g., diabetes) could be affected. Finally, the human health risks from clinical and animal research studies are reviewed, including aldehydes as haptens in allergenic hypersensitivity diseases, respiratory allergies, and idiosyncratic drug toxicity.
1. SOURCES OF ALDEHYDES
a. Natural and Anthropogenic Aldehydes: Urban, Rural, and Indoor
A trace amount of formaldehyde is present in the air as a result of photochemical oxidation (hydroxyl radicals) of hydrocarbons, for example, methane naturally present in the atmosphere (Atkinson, 1990; Riedel et al., 1999). In rural air, terpenes and isopropene emitted by foliage also react with hydroxyl radicals to form formaldehyde (Atkinson, 1990). In urban areas, motor vehicle exhaust became an important source of aldehydes in air both through direct emission of aldehydes and through the emission of hydrocarbons, which in turn were converted to aldehydes through photochemical oxidation reactions(Cecinato et al., 2002; Destaillats et al., 2002; Grosjean et al., 1996; Maldotti et al., 1980; Rao et al., 2001). The most recent Urban Air Toxics Study results posted on the U.S. Environmental Protection Agency (ERA) web site include formaldehyde, acetaldehyde, and acrolein as significant contributors to the summed risk values for mobile sources of air toxins. These aldehyde air toxins are also regulated under the Canadian Environmental Protection Act (CEPA, 2000a, 2000b). The largest carbonyl emissions were attributed to the oxidation and ring breaking of benzene, toluenes, ethylbenzene, and xylenes in the internal combustion engine or in the exhaust and can be divided into the following four carbonyl classes whose chemical structures are shown in Figure 1:
1. Alkanals: formaldehyde, acetaldehyde, valeraldehyde, hexanal, heptanal, nonanal, dodecyl aldehyde.
2. Alkenals: acrolein, crotonaldehyde, methacrolein.
3. Aromatic aldehydes: benzaldehyde, m-tolualdehyde, 2,5- dimethylbenzaldehyde, 3-hydroxybenzaldehyde.
4. α-Oxoaldehydes: glyoxal, glycolaldehyde, glyoxylic acid.
With the increasing use of alternate and reformulated automotive fuels, an additional source of aldehyde emission is now in evidence. Depending on the type of oxygenates (ethanol, methanol, or methyl tert-butyl ether) added to the automotive fuels, increased amount of formaldehyde or acetaldehyde are now emitted in automobile exhaust(Gaffney and Marley, 2000; Kirchstetter et al., 1996; Maejima et al., 1992). Grosjean et al. (2002) attributed the change in ambient acetaldehyde/ formaldehyde ratio to a change in reliance on ethanol as vehicle fuel in Brazil. The most abundant carbonyls expressed as percentages of Los Angeles air carbonyl content on a parts per billion basis are formaldehyde (24%), acetaldehyde (18%), glyoxal/methylglyoxal (8%), acetone (7%), and acrolein (5%). Furthermore, the two glyoxals exhibit midafternoon maxima probably because they are photochemical oxidation products of aromatic and olefinic hydrocarbons (Ghilarducci and Tjeerdema, 1995; Grosjean et al., 1996; Zervas et al., 2002). Other sources of aldehydes in the air include emissions from forest fire and agriculture burns(Dost, 1991; Materna et al., 1992), and incinerators (Dempsey, 1990). Coal- based power plants (Sverdrup et al., 1994) and diesel engines (Wheeler et al., 1980) also emit aldehydes into the air.
Aldehydes are present in residential and workplace environments, and are detectable in confined spaces such as spacecrafts and airline cabins (James, 1997; NRC, 2002). Aldehyde levels in indoor air were 4- to 10-fold higher than those of outdoor air (Liteplo and Meek, 2003; Shah and Singh, 1988; Willis et al., 2002; Zhang and He, 1994), indicating that indoor sources of aldehydes are from local emissions. The major sources of indoor formaldehyde and acetaldehyde were from furniture, carpets, particle boards, fabrics, and paints (Brown, 1999; Kelley et al., 1966; Pickrell et al., 1983). Cooking fumes contained a variety of aldehydes including formaldehyde, acetaldehyde and acrolein (Lane and Smathers, 1991; Svensson et al., 1999). Woodburning stoves and fireplaces are also a source of aldehyde in homes (Lipari et al., 1984; Ramdahl et al., 1982). Formaldehyde and acetaldehyde were also generated in the indoor environment by the action of ozone on anthropogenic hydrocarbons (Weschler and Shields, 1997). Cigarette smoke is an important source of indoor aldehydes (Mansfield et al., 1977; Rickert et al., 1980). Mainstream smoke analysis of cigarette smoke for aldehydes showed that acetaldehyde constituted the major component at 709 g/ cigarette, followed by acrolein (82), formaldehyde (54), crotonaldehyde (15) (Smith and Hansen, 2000a), diacetyl (207), and methylglyoxal (38) (Saint-Jalm et al., 1980). Sidestream smoke as a result of lower-temperature pyrolysis contains about 12-fold more acrolein than mainstream smoke. Furthermore, the charcoal filters adsorbed the acrolein by 50-80% and decreased the smoker’s exposure (Ghilarducci et al., 1995). Formaldehyde is present in the air of hospitals, mortuaries, and laboratories, where it is used in tissue preservatives, disinfectants, and embalming fluids (WHO, 1989). Dermal and inhalation exposure to formaldehyde is also possible in cosmetic and hair salons, where it is used as a fumigant (Ackland et al., 1980; Olcerst, 1999). Dermal and inhalation exposure to aldehydes may also occur through contact with a wide range of consumer products, such as fabrics, cleaning agents, sun-tan lotions, and many cosmetic products containing formaldehyde or formaldehyde-releasing substances (Flyvholm and Anderson, 1993; WHO, 1989).
FIG. 1. Structures of the most common aldehydes.
Formaldehyde levels have been reported to be as high as 1.38 mg/ L and 6.8 mg/L in rainwater (Kitchens et al., 1976) and fog water (Muir, 1991), respectively, but are generally
Formaldehyde is also generated in vivo from numerous xenobiotics and pharmaceuticals, such as hexamethylphosphoramide bis(chloromethyl)ether, diphenylhydramine, and codeine, to name a few (Dahl and Hadley, 1983; Heck, 1989; Heck et al., 1990). Industrial chemicals such as methanol and methyl tert-butyl ether, a gasoline oxygenater (Hutcheon et al., 1996), are also precursors of formaldehyde. The food preservative hexamethylenetetramine releases formaldehyde and ammonia in the acidic environment of the stomach (WHO, 1974).
b. Endogenous Aldehydes Formed During Biomolecule Metabolism
i. Lipid Peroxidation (LPO)
The accumulation of DNA damage and DNA adducts is believed to play a significant role in genetic diseases, including cancer. Recently, it has been shown that the background levels of exocyclic propano/etheno-DNA adducts in tissues from unexposed humans arise from the endogenous alkenals, hydroxyalkenals, dialdehydes, and alkanal products of lipid peroxidation (LPO) decomposition (Bartsch, 1999). Furthermore, these adducts were increased in subjects ingesting high dietary ω-6 polyunsaturated fatty acids and decreased in subjects consuming vitamin E supplements or having an increased vegetable intake. There is therefore increasing interest in these adducts as possible biomarkers of cancer risk (Hagenlocher et al., 2001). These DNA adducts were also increased in patients with copper storage disease (Wilson’s disease) or iron storage disease (hemochromatosis) associated with liver cancer (Nair et al., 1999). Endogenous carbonylated proteins were also increased in the liver during LPO induced by ethanol or carbon tetrachloride. Using malondialdehyde antibodies, the major adduct was identified as a malondialdehyde adduct with cytochrome oxidase subunit IV (Chen et al., 2000). Cytochrome oxidase was also inactivated and could contribute to subsequent cytotoxicity. This and other LPO aldehyde decomposition products are believed to be responsible for many endogenous protein adducts. Endogenous carbonylated proteins have been shown to increase during aging as well as in various pathological states, including premature diseases, muscular dystrophy, rheumatoid arthritis, metal storage diseases, and atherosclerosis (Chevion et al., 2000). A marked increase in plasma/ urinary aldehydes (by products of LPO and their metabolites) occurred in patients with childhood cancer (Yazdanpanah et al., 1997) or alcoholic liver disease (Aleynik et al., 1998). Plasma heptanal levels increased from 0.1 to 3.5 M (Yazdanpanah et al., 1997).
Besides endogenous LPO products, other contributors to the body carbonyl burden are ingested exogenously and include drugs and environmental agents that undergo biotransformation to form reactive aldehyde metabolites or induce LPO. For instance, there was an increase in plasma LPO derived prostanoids (F^sub 2^-isoprostanes) in smokers (Morrow et al., 1995). Animal experiments also showed increased plasma/urinary aldehydes in rats/mice with iron overload (Bartfay et al., 1999) or following the administration of dioxin, paraquat, carbon tetrachloride, endrin (Shara et al., 1992), or dichromate (Bagchi et al., 1995b). Doxorubicin administered to rats also increased aldehyde levels in the heart and plasma before cardiotoxicity ensued, and both events were prevented with carnitine (Luo et al., 1999). Doxorubicin (10 mg/kg) increased the urinary excretion of formaldehyde from 48 to 158 nmol/kg body weight, acetaldehyde from 2 to 14 nM/kg, and malondialdehyde from 4 to 17 nmol/kg 6 h later. Aldehyde levels were still high 72 h later (Bagchi et al., 1995a). Plasma cytotoxic alkenals (frans-2- heptenal, 4-hydroxynonenal [4-HNE], irani-2-nonenal) were increased up to 3.5-fold (Luo et al., 1999). Formaldehyde, is also formed by oxidative stress (e.g., from polyamine oxidation by ROS) and is increased in tumors. Furthermore it also plays a role in the binding of anthracycline or mitoxantrone anticancer drugs to DNA in vivo (Kato et al., 2000).
Dicarbonyls or oxoaldehydes are of particular concern because of their propensity to covalently cross-link proteins as a result of forming Amadori intermediate products (Figure 1) and advanced glycation end-products (AGEs). Cross-linking of long lived proteins have been found in vivo and were associated with the pathophysiologies of aging and the long-term complications of diabetes and atherosclerosis. The dicarbonyl content of fresh human urine was reported to be glyoxal (41 M), diacetyl (2.1 M), and methylglyoxal (1.5 M) (Espinosa-Mansilla et al., 1998), whereas the concentrations in blood were glyoxal (211 pmol/g), methylglyoxal (80 pmol/g), and lower levels of 3-deoxyglucosone (Thornalley, 1998). The early glycation adduct fructosamine, an Amadori product, is about 140 M in plasma and was increased two- to threefold in diabetes (Thornalley et al., 1999). Plasma glyoxal and methylglyoxal and their derived AGEs and advanced lipoxidation end-products (ALEs) levels were also increased (more than three- to fourfold) in diabetics or patients with renal disease (Odani et al., 1998). A sevenfold increase of deoxyglucosone (to 11 M) was found in the serum of continuous ambulatory peritoneal dialysis patients (Tsukushi et al., 1999). These levels may appear low, but these oxoaldehydes have high reactivity to proteins and nucleic acids.
ii. Carbohydrate or Ascorbate Autoxidation
Glyoxal, unlike methylglyoxal, is a major lipid oxidative degradation product. Glyoxal is also a major DNA oxidative degradation product formed from the oxidation of deoxyribose C4’/ C5′ carbons by ROS and causing a DNA strand break. The amount of glyoxal formed was 17-fold that of 8-OHdeoxyguanosine (Murata- Kamiya et al., 1997). The glyoxal released could then form adducts with neighboring DNA guanine (discussed later). Glyoxal was the only dicarbonyl formed during glucose autoxidation under physiological conditions, presumably formed from the cleavage of the C2-C3 bond of glucosone or another intermediate. Arabinose was also found and presumably arose from the cleavage of the C1-C2 bond of glucose. The AGEs formed with ribonuclease were N^sub ε^- carboxymethyllysine (CML) for glyoxal and pentosidine for arabinose (Wells-Knecht et al., 1995). Dicarbonyl products formed from fructosyl-lysine autoxidation were glyoxal > 3-deoxyglucosone > methylglyoxal (Thornalley et al., 1999). Ascorbate autoxidation is also believed to contribute to age-induced cataractogenesis, which is associated with a buildup of proteins glycated by oxidized catabolites of endogenous ascorbate present in the lens (up to 2 mM). As shown in Figure 2a, the reactive catabolites implicated include diketo compounds and L-erythrulose (Simpson et al., 2000).
FIG. 2a. Endogenous glyoxal formation by autoxidation of glycolaldehyde formed by (left) sorbitol metabolism or (right) ascorbate oxidative degradation or a sugars or DNA deoxyribose or unsaturated fatty acids.
Glyoxal is also formed by the transition-metal-catalyzed autoxidation of the ene-diol tautomer of glycolaldehyde (Benov and Fridovich, 1998) (Figure 2b), hydroxypyruvaldehyde, another oxoaldehyde, is rapidly formed by the autoxidation of the ene-diol tautomers of glyceraldehyde or dihydroxyacetone (Thornalley et al., 1984). Superoxide and hydrogen peroxide are also formed and likely cause the oxyhemoglobin oxidation and cytochrome c reduction that also occurs (Thornalley, 1985). These dicarbonyls are the most reactive carbonyls, as at micromolar concentrations they cross-link proteins, glycate proteins, form AGE, and inactivate enzymes, in contrast to glucose, which requires 20 mA/and very long autoxidation times to cause protein carbonylation and enzyme inactivation (Ukeda et al., 1997).
FIG. 2b. Endogeneous glycolaldehyde and glyoxal metabolic pathways. AKR, aldo-ketoreductase; HPR, hydroxypyruvate reductase; LDH; lactate dehydrogenase.
iii. Carbohydrate Metabolism
Methylglyoxal, unlike glyoxal, is formed enzymatically from the triose phosphate intermediates (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate) produced during the glycolytic metabolism of glucose or from the metabolism of ketone bodies or threonine (Thornalley, 1996). It has been estimated from the triose phosphate and isomerase concentrations that 0.4 mM methylglyoxal is formed per cell per day (Richard, 1991). In Figure 3a, we also show that triose phosphate (i.e., dihydroxyacetone [DHAP] and glyceraldehyde 3-phosphate) formation by the pentose phosphate pathway could also be responsible for methylglyoxal formation from xylitol, ribose, and deoxyribose, which are much more effective than glucose. The endogenous rate of methylglyoxal formation has been estimated at about 120 mol/day, i.e., 0.1% of the glucotriose flux (Thornalley, 1996). Methylglyoxal formation markedly increased when hepatocytes were incubated with glyceraldehyde. Methylglyoxal formation from xylitol also markedly increased when glycolysis was inhibited with fluoride and diamide (Sato et al., 1980). Methylglyoxal formation also increased when glyceraldehyde dehydrogenase underwent oxidative inactivation by ROS generated by hyperglycemia or was inactivated by increased cellular NADH levels (metabolic pseudohypoxia) (Brownlee, 2\001). Cellular methylglyoxal levels were decreased by lowering triose phosphate levels by maximizing transketolase activity with thiamine therapy (Thornalley et al., 2001). On the other hand, thiamine deficiency increased tissue and plasma methylglyoxal levels (and its metabolite glyoxylic acid) (Liang, 1960; Vogt-Moller, 1931). 3-Deoxyglucosone may also be formed as a hydrolysis product of fructose 3-phosphate, a metabolite formed from fructose catalysed by fructose-3-kinase. The fructose of lens and heart could be formed from glucose via the polyol pathway implicated in diabetic complications.
FIG. 3a. Endogenous methylglyoxal and glyoxal formation.
FIG. 3b. Endogenous methylglyoxal metabolism.
It is generally thought that glyoxal is formed almost exclusively nonenzymically (e.g., via oxidation of Amadori products or lipids) and is not formed by intermediary metabolism, unlike methylglyoxal. As shown in Figure 2a, glycolaldehyde is formed by intermediary metabolism from hydroxypyruvate (from glyceraldehyde, fructose, and sorbitol) or D-xylulose 1-phosphate (from xylitol) or glyoxylate (from serine, glycine, and hydroxyproline). Fructose in some tissues could also be formed from sorbitol catalyzed by aldose reductase (the polyol pathway). However, glyoxal was formed from glycolaldehyde by reactive oxygen species (ROS) (Benov and Fridovich, 1998; Okado-Matsumoto and Fridovich, 2000; Vogt-Moller, 1931). In Figure 2b a pathway is therefore proposed for the formation of glyoxal from glyceraldehyde via glycolaldehyde autoxidation, and the identity of the metabolizing enzymes involved is outlined for the first time.
iv. Amine Oxidases-Catalyzed Metabolic Activation
FAD-dependent oxidases include tissue monoamine oxidase (MAO), located in the mitochondrial outer membrane and inhibited by pargyline, quinacrine, or deprenyl, and polyamine oxidase (PAO), located in the peroxisomes and cytosol, and is inhibited by quinacrine or CGP 48664. MAO exists as two isoforms, A (e.g., serotonin substrate) and B (phenethylamine, dopamine substrates), and catalyzed the deamination of tyramine and amine neurotransmitters such as tryptamine, resulting in the formation of H^sub 2^O^sub 2^, NH^sub 3^, and phenylacetaldehyde (Figure 4a). The latter two products are detoxified by the mitochondria. Succinic semialdehyde is formed during the MAO-catalyzed metabolism of the neurotransmitter gamma-aminobutyric acid.
FIG. 4a. Formation of formaldehyde and acetaldehyde by serum amine oxidase (SAO).
In contrast, PAO utilizes diamine and polyamine substrates and forms H^sub 2^O^sub 2^ (detoxified by peroxisomal catalase) but not NH^sub 3^ when it catalyzes the oxidation of spermine to form spermidine and 3-aminopropanal. Acetylspermine and diacetylspermine are even better substrates. The polyamines and PAO are ubiquitous in cells (including erythrocytes) and are involved in cellular growth, differentiation, and carcinogenesis. 3-Aminopropanal is a potent lysomotropic neurotoxin (particularly neurotoxic to neurons) that is formed from polyamines during cerebral ischemia. 3-Aminopropanal and acrolein (spontaneously generated by aminopropanal, Figure 4b) are considered to be the major cytotoxic products of brain injury (Li et al., 2003; Seller, 2000). Hepatic PAO was also induced fourfold in vivo by liver iron loading, and the increased aminopropanal formation by spermine oxidation may have caused the increased LPO and hepatotoxicity (Tipnis et al., 1997).
The copper-dependent amine oxidases such as plasma amine oxidases and diamine oxidases are inhibited by copper chelating agents, semicarbazide, or aminoguanidine, and are thought to “detoxify” xenobiotic amines but form toxic aldehydes, H^sub 2^O^sub 2^, and NH^sub 3^ (depending on the substrate), which have been implicated in vascular damage and advanced protein aggregation found in vascular disorders, such as diabetic complications, atherosclerosis, Alzheimer’s disease, and aging. They include tissue diamine oxidase (DAO) and serum amine oxidase (SAO). DAO is mostly found in the intestinal mucosa and kidney but is also induced by putrescine or injury in the heart, liver, and brain. It utilizes the diamines putrescine and cadaverine as well as spermine to form aminoaldehydes. Acrolein was formed from spermidine, and 3- aminopropanal/malondialdehyde was formed from 1,3-propanediamine (Seller, 2000). SAO exists as membrane or soluble forms in the vascular system for example, the cardiovascular smooth muscle cells, kidney cells, cartilage, and adipocytes, and contain topa quinone as a cofactor. Serum SAO is increased in patients with diabetic complications, vascular disorders, and heart disease. They catalyze the oxidative deamination of the polyamines spermine, spermidine, or the industrial chemical allylamine in the plasma to form the highly toxic acrolein. The aminoaldehyde first formed from spermine undergoes an oxidative deamination to form a dialdehyde. Both the aminoaldehyde or dialdehyde products can then be oxidatively detoxified by aldehyde dehydrogenases or can undergo spontaneous β-elimination to form putrescine and acrolein (Seiler, 2000). The latter is most likely to occur in the extracellular space, as both SAO and polyamines are found in the plasma, unlike aldehyde dehydrogenase. Spermine is synthesized in all eukaryotic cells and is involved in a variety of cellular processes, including the control of cell growth. Rapidly dividing cells such as tumors, bone marrow, and intestinal epithelial cells have elevated levels of poly amines due to enhanced polyamine synthesis by ornithine decarboxylase/S-adenosylmethionine decarboxylase and increased uptake of poly amines. Polyamine uptake is mediated by a transporter, which is also under feedback inhibition by polyamine. Furthermore, polyamine analogs are now being developed as anticancer agents as they underwent an unregulated rapid massive intracellular and intramitochondrial accumulation resulting in tumor cell apoptosis, which was prevented by an amine oxidase inhibitor (Hu and Pegg, 1997). Formaldehyde and methylglyoxal were also formed by SAO from methylamine, a metabolite of epinephrine, sarcosine, creatinine, and also a gut bacteria product. Methylglyoxal was also formed by SAO from aminoacetone, a decarboxylation product of 2- amino-3-keto-butyrate formed by the mitochondrial metabolism of threonine. The formation of acrolein by SAO in the coronary artery was suggested to cause the coronary artery contraction resulting in myocardial necrosis that is induced by allylamine (Conklin et al., 2001). Plasma SAO and methylglyoxal were also increased by the uncontrolled hyperglycemia of diabetics. It has also been proposed that excessive methylglyoxal/formaldehyde formation by SAO from aminoacetone/methylamine causes vascular endothelial cell protein cross-linking and cell injury associated with diabetic atherosclerosis (Yu et al., 1997). Plasma SAO was also increased in patients with congestive heart failure. Human placental SAO has been cloned and sequenced with an identity of about 60% for the cDNA sequence of human kidney amine oxidase. It has two subuits each containing one atom of Cu and one molecule of covalently bound topa quinone (Zhang and Mclntire, 1996). Another member of the SAO family recently discovered is the ectoenzyme vascular adhesion protein-1, an adhesion protein with a dual function that is found on the endothelial cell surface and rapidly deaminates methylamine and aminoacetone (Smith et al., 1998). Spermine oxidized by serum amine oxidase was cytotoxic to tumor cells particularly at 42C, a cytotoxic effect that was attributed to the acrolein formed (Agostinelli et al., 1994). Amine oxidase inhibitors usually prevent the cytotoxicity of polyamine oxidation products. Aminoguanidine is currently the only DAO, SAO inhibitor used in the clinic.
FIG. 4b. Amine oxidases-catalyzed metabolic activation. DAO = diamine oxidase; PAO = polyamine oxidase; SAO = serum amine, oxidase.
v. Cytochrome P-450 Catalyzed Metabolic Activation
Endogenous formaldehyde and and aldehydes with one less carbon atom than the initial alcohol substrate (eg., acetaldehyde) were formed by the cytochrome P-450 (P450) (CYP2E1) catalyzed oxidation of glycerol, ethylene glycol, 1,2-propane diols, or polyhydroxylated alcohols containing vicinal diols likely mediated by a ferryl-type oxidant species involving a radical mechanism or the homolytic cleavage of a dioxetane intermediate (Clejan et al., 1992; Kukielka et al., 1995). CYP2E1 likely catalyzed methylglyoxal formation in vivo from ketone bodies, as acetone can be hydroxylated to acetol (hydroxyacetone), which is then hydroxylated to methylglyoxal (Bondoc et al., 1999). Ketosteroids were also formed during steroid metabolism, and ketoprostaglandins were formed during prostaglandin metabolism.
vi. Myeloperoxidase-Catalzed Metabolic Activation
Plasma contains myeloperoxidase (MPO) and H^sub 2^O^sub 2^ (released from activated neutrophils at sites of inflammation), as well as chloride (0.1 M) and α-amino acids (4-5 mM). Peroxidase/ H^sub 2^O^sub 2^-catalyzed protein arginine, proline, or lysine oxidation by a radical mechanism formed semialdehydes, whereas alanine, valine, and serine oxidation formed HCHO (Headlam et al., 2002). However, acrolein and 2-hydroxypropanal were the major products formed via intermediate chloramines during the myeloperoxidase/H^sub 2^O^sub 2^/Cl- or HOCl-catalyzed oxidation of threonine, and glycolaldehyde was the major product formed from serine via intermediate chloramines (Anderson et al., 1997). Tyrosine formed p-hydroxyphenylacetaldehyde and 3-chlorotyrosine when oxidized by MPO/H^sub 2^O^sub 2^/C1 (Fu et al., 2000), whereas neutrophils/Cl incubated with glycine formed HCHO and alanine formed acetaldehyde (Figure 5) (Hazen et al., 1998). Such aldehyde formation in blood vessels may accelerate diabetic vascular disease. Itis also very likely that the aldehyde yield was much higher than was found, as aldehyde autoxidation is markedly catalyzed by peroxidases. This is toxicologically significant, as the electronically excited carbonyl intermediates (as indicated by chemiluminescence) and hydroperoxy-aldehyde intermediates formed by peroxidases from some aldehydes are reactive DNA oxidants (Adam et al., 1999; Oliveira et al., 1978).
c. Dietary Aldehydes
Methanol is present in fruit and vegetables, fruit juices, and fermented beverages (Kavet and Nauss, 1990), and ethanol constitutes a substantial portion of alcoholic beverages. They are enzymatically converted to formaldehyde and acetaldehyde, respectively, in vivo. However, formaldehyde is also present in fruit and vegetables, meat, cheese, and seafood, but its origin is not known (WHO, 1989). Aspartame, an artificial sweetener, is hydrolyzed in the gut to produce methanol, which is then converted to formaldehyde. Acetaldehyde is a natural component of many fluids including fruits, vegetables, and alcoholic products (Feron et al., 1991). It was also added to food and beverages as a flavoring substance. Thus, acetaldehyde concentrations in vegetables have been reported to be from 0.2 to 400 g/g, and in wine to be from 0.7 to 290 g/g. Acetaldehyde, formaldehyde, and acrolein are also produced during the cooking of fat-containing foods (Lane and Smathers, 1991 ; Svensson et al., 1999). Peas contain traces of acetaldehyde, whereas cinnamon contains high amounts of cinnamaldehyde. Almonds and cherries contain benzaldehyde, the characteristic impact substance, whereas hexanal and 2-hexenal cause the “unripe odors” in fruits and vegetables. Anise and vanilla extracts contain anisaldehyde and salicylaldehyde, respectively. Dietary aldehydes are much more varied and complex, unlike environmental aldehydes, which are dominated by formaldehyde and, to a lesser extent, acetaldehyde, acrolein, glyoxal, and methylglyoxal. These dietary carbonyls are described next under the following four classes:
FIG. 5. Myeloperoxidase-catalyzed metabolic activation.
1. Phospholipid and fatty acid hydroperoxlde decomposition aldehydic products. Over a wide range, alkenals, alkanals, hydroxyalkenals, and glyoxals are products of LPO (Lane and Smathers, 1991; Mlakar et al., 1996; Spiteller et al., 2001 ; Uchida et al., 1998). Recently the pathway for 4-HNE formation from 9- hydroperoxylinoleic acid was shown to involve a Hock cleavage to nonenal followed by oxygenation to form 4-hydroperoxynonenal (Schneider et al., 2001; Uchida et al., 1998). Fruits/vegetables have enzymic pathways involving lipoxygenase-hydroperoxide lyases, as their 2-alkenal and alkanal contents are markedly increased on physical disruption and subsequent processing as a result of these enzymes being brought into contact with their unsaturated fatty acid substrates (Gardner et al., 2004). For instance, the 13- hydroperoxides of linoleic/linolenic acid cleave into hexanal and cis-3-hexenal. The volatile aldehydes and alcohols formed contribute to the characteristic flavor of each fruit or vegetable but could be a primary defense mechanism against microbial infection. For example, hexenal up to 76 ppm can be found in bananas, with up to 25 ppm found in black tea. Hexenal has been approved by the Food and Drug Administration (FDA) to use as a food artificial flavoring additive to generate “green notes” because of its “green” and fruity flavor. Citrus peel can contain up to 500 ppm decadienal and 410 ppm nonadienal (Adam et al., 1999). Glycolaldehyde was the major lipid peroxide decomposition product formed during the ferrous-catalyzed autoxidation of arachidonic acid (Mlakar et al., 1996). Glyoxal is also a threefold more abundant LPO decomposition product than malondialdehyde (Mlakar et al., 1996). Acrolein formation from unsaturated lipids or glycerol dehydration is markedly increased at cooking temperatures. Furthermore, acrolein is likely also formed as a result of cooking other food components, as acrolein was formed when amino acids (or polyamines) and sugars were boiled together. A Strecker degradation (decarboxylation plus oxidative deamination) reaction was proposed (Alarcon, 1976).
2. Monoterpene aldehydes/ketones. These aldehydes/ketones are odoriferous compounds found in the essential oils obtained from plants that are also widely used as flavoring additives for food and beverages, as well as scenting agents in household products. Citral (3,7-dimethyl-2,6-octadienal) is a reactive alkenal found in all citrus fruits and forms up to 80% of the essential oil of Cymbopogon citratus, known as lemongrass (Cimanga et al., 2002), and 2.7% of lemon peel essential oil, where limonene is the major component (Umano et al., 2002). The approved herbal medicine lemon balm contains up to 30% citral and 40% citronellal. Because of its intense lemon aroma and flavor, citral has also been used extensively as an additive/agent in the cosmetic, detergent, and food industries. The FDA included citral on the list of “generally recognized as safe” (GRAS) substances and approved it for food use (GRAS 21, CFR 182.60). It is now a major additive/agent with a world consumption of 1200 tons in 1996. Citral has a low acute toxicity in rats, although short-term use at high doses caused hepatomegaly. However, 60 mg/kg citral given orally to pregnant rats from day 6 to 15 of pregnancy interfered with implantation and caused detectable embryofetal and maternal toxicity with overt toxicity at higher doses (Nogueira et al., 1995). Terpene ketones are the main constituents of the essential oil of the leaves of spearmint, caraway, and mint, which are also used widely, for example, as spearmint and peppermint in food, toothpastes, mouthwashes, chewing gum, aroma therapy, and herbal medicines. Toxic terpene ketones include thujone in wormwood oil, used in absinthe liqueur or herbal medicine but that can cause convulsions and other neurotoxic effects. The direct use of thujone in food is not allowed, but it is present in a number of approved flavorings and additives, as well as in perfumes and Vicks ointments (Hold et al., 2000). Another terpene ketone is pulegone, found in pennyroyal oil (a mint oil), which is still used as an abortifacient, even though pulegone causes life- threatening centrilobular hepatic necrosis, likely as a result of a P450-catalyzed metabolism of the menthofuran metabolite to form a highly reactive y-ketoenal (Madyastha et al., 1993).
3. Aromatic aldehydes, Most of the aromatic aldehydes are substituted benzaldehydes that are abundant in the most commonly used spices. Cinnamaldehyde is formed from cinnamic acid in plants and constitutes up to 90% of cinnamon oil extracted from cinnamon leaves and bark and in essential oils such as myrrh and patchouli. It is also widely used as a flavoring/aroma agent when added to food or beverages, including ice cream, confectionary, baked goods, chewing gums, condiments, and meat preparations, with up to 6400 ppm permitted as an additive to fruits and juices (Friedman et al., 2000). A cinnamaldehyde analysis showed that cinnamon sticks and powder (commonly added to tea or rice) contained 2470 mg/100 g, whereas cinnamon-containing bread, buns, cereals, and cookies contained up to 31 mg/100 g (Friedman et al., 2000). It would therefore be easy to exceed the acceptable daily intake permitted for cinnamaldehyde of 1.2 5 mg/kg. Another flavoring/aroma agent, vanilla, contains the aromatic aldehydes vanillin, 4- hydroxybenzaldehyde, and salicylaldehyde. Both additives act as food preservatives because of their antimicrobial properties. Benzaldehyde is the major constituent of the essential oils of the seeds and kernels of bitter almonds, apricots, peaches, plums, and cherries. Furfural (2-furaldehyde) is also used as a flavor ingredient (maximal use of 63 ppm in meat products) but is formed when polysaccaharides are heated and is therefore highest in coffee and cocoa (55-255 ppm), whereas furfuryl alcohol is highest in heated skim milk (230 ppm) and coffee (90-881 ppm). Intake levels of furfural and derivatives are approximately 0.3 mg/kg/day, whereas furfural intake as a flavor ingredient is 100 times lower (Adam et al., 1999).
4. α-Oxoaldehydes, dialdehydes, and advanced glycation end products formed by protein glycation and lipoxidation. Oxoaldehydes include glyoxal, methylglyoxal, and 3-deoxyglucosone, whereas the dialdehydes include malondialdehyde and diacetyl. As shown in Figure 6, the Maillard reaction, the reaction between reducing sugars and amino groups of proteins (glycation), results first in the conversion of reversible Schiff base adducts to covalently bound Amadori rearrangement products (particularly fructosamine) and is a reversible step. The Amadori products can then undergo multiple dehydration and rearrangements resulting in the release from the protein of 3-deoxyglucosone, and is the major pathway for 3- deoxyglucosone formation in vivo. The deoxyglucosone can then react with more protein amine groups, causing cross-linking, and browning of the proteins via the formation of AGEs in the late stage of the Maillard reaction. Another series of reactions accelerated by oxygen and transition metals in a process called glycoxidation can also cause the glycated protein Amadori product to form AGEs such as N^sub ε^-carboxymethyl lysine (CML), pentosidine, pyrraline, and other unidentified compounds. CML is also formed during metal- catalyzed oxidation of polyunsaturated fatty acids in the presence of proteins in a process called lipoxidation (Figure 7) (Baynes and Thorpe, 2000). Glyoxal and methylglyoxal can also be formed from the degradation of proteins glycated by glucose or the degradation of fructosyl-lysine (Thornalley et al., 1999). Possible intermediates involved in glyoxal formation are suggested in Figure 4. Cooking food at high temperatures mark\edly accelerates these Maillard or browning reactions. Although some food or cooked food has been analyzed for methylglyoxal, the glyoxal content has not been analyzed. The various structures of the AGEs formed from glyoxal, methylglyoxal, and deoxyglucosone are compared in Figure 8.
FIG. 6. Endogenous glyoxal formation by autoxidation of glycoladehyde formed by (left) sorbitol metabolism (right) ascorbate or xylitol oxidative degreadation.
Recently, it has been shown that two-thirds of orally absorbed food AGEs (known as glycotoxins) were retained in tissues. Furthermore, feeding these AGEs to diabetics also triggered changes in inflammation biomarkers that were linked to diabetes and vascular dysfunction and could thus be considered as a risk factor for diabetic angiopathy (Vlassara et al., 2002). Modification of plasma proteins by AGE precursors form ligands that bind to AGE receptors inducing changes in gene expression in various cells.
Hydroxymethylfarfural (HMF) and furaldehyde are common furan AGE hydrolysis products of the Maillard reaction for hexoses and pentoses, respectively, for example, 1% HMF is formed when sugar is heated under household cooking conditions. HMF sometimes exceeds 1 g/ kg in dried fruits and up to 9.5 g/kg in caramel products. The oral LD50 value for the rat is 3.1 g/kg. However, the high concentrations of HMF found in food are not considered to pose a health risk. Two HMF doses of 300 mg/kg po induced aberrant crypt foci, which suggests in rats it may act as a weak initiator and promoter of colon cancer (Zhang et al., 1993). Cellular glutathione (GSH) was depleted by high concentrations of HMF, but GSH conjugates or reactive metabolites have not been identified (Janzowski et al., 2000). Furaldehyde (Figure 1) is found in cocoa, coffee, alcoholic beverages, etc., and is used as a flavor ingredient. Our approximate daily consumption is 0.3 mg/kg/day. The oral LD50 value in the rat is 50-149 mg/kg, with hepatotoxicity occuring at doses above 50 mg/ kg (Adams et al., 1997), and is also a metabolic inhibitor (inhibits pyruvate dehydrogenase (Modig et al., 2002). It is readily metabolized by aldehyde dehydrogenase (ALDH) to furoic acid, but it is not known whether furaldehyde or reactive metabolites deplete cellular GSH (Adams et al., 1997). Furfural is also formed endogenously as an ROS breakdown product of deoxyribose in DNA (Barciszewski et al., 1997). Furan, another Maillard reaction product, is a rodent hepatocarcinogen and hepatotoxin found in many processed foods and beverages, with up to 3900 ppb in brewed coffee extracts (part of the aroma). It is also an environmental toxin and occupational hazard, as it is used as a resin and lacquer solvent and is volatile. The toxicity has been attributed to the major dialdehyde metabolite cis-2-butene-1,4 dial (formed by P450), which reacts with DNA to form mutagenic adducts and forms adducts with deoxycytidine, deoxyguanosine, and deoxyguanosine (Byrns et al., 2002). It also rapidly cross-links protein amino acids to form pyrrole/pyrrolin-2-one derivatives or reacts with glutathione to form adducts (Chen et al., 1997). 2-Methylfuran also found in coffee, cigarette smoke or air pollution (as an SOC) is metabolically activated to acetylacrolein and causes liver, lung, and kidney necrosis in rodents (Ravindranath et al., 1985). As described earlier, lethal hepatotoxicity induced by ingesting the herbal medicine pennyroyal oil for abortifaciant purposes has been attributed to the reactive γ-ketoenal metabolite of menthofuran, the major metabolite of pulegone (Madyastha et al., 1993).
FIG. 7. Mechanism of protein carbonylation and glycation by aldehydes.
FIG. 7. Mechanism of protein carbonylation and glycation by aldehydes.
Glyoxal and methylglyoxal (Figure 1) are major products formed from the reaction of carbohydrates amd amino acids during the nonenzymyatic browning that occurs during thermal processing of foods such as roasted coffee or bread crust. Glycolaldehyde was more effective than glyoxal in generating radicals for the browning reaction with alanine at 98C (Hofmann et al., 1999). Cooking food increases its glyoxal content because, as described earlier, glyoxal (but not methylglyoxal) was formed by glucose autoxidation during heating (Wells-Knecht et al., 1995). Glyoxal/methylglyoxal formation by lipid autoxidation or DNA oxidation discussed earlier would also be expected to increase during cooking. The methylglyoxal content of some foods have been determined but not their glyoxal content. The highest methylglyoxal content of food found was in soy sauce (498 g/ ml) and soybean paste (939 g/ml) (Sugimura et al., 1983). By far the highest methylglyoxal containing beverage is brewed coffee (75 g/ gm). The methylglyoxal content of coffee has been attributed to the roasting process. An estimated 150 g methylglyoxal and 750 g hydrogen peroxide was found per cup of coffee. It was also found that hydrogen peroxide increases methylglyoxal mutagenicity (Fujita et al., 1985). Coffee also contains more furfural than methylglyoxal, but less glyoxal and diacetyl (an aroma component). Because of their reactivity associated with pathophysiological consequences, it is clear that the content of glyoxals as well as AGEs in our food, particularly cooked food, needs to be analyzed.
FIG. 8. Advanced glycation end-products formed by reactive carbonyl products.
Acrylamide, a probable human carcinogen (IARC, 1994), has recently caused some concern, as it was found in fried and oven- cooked food. It also could be formed when glucose was heated with asparagine, which forms 40% of the amino acids of potato and 18% of wheat flour. A Strecker degradation of the amino acid by glyoxal products of a Maillard reaction was proposed (Mottram et al., 2002).
d. Aldehyde Drugs or Reactive Drug Metabolites
Drugs that are aldehydes or form reactive aldehyde metabolites which cause side effect toxicity are summarized in Table 1. The anticancer drugs cyclophosphamide and ifosfamide undergo a P450- catalyzed metabolism to acrolein and chloroacetaldehyde, which cause hemorrhagic cystitis and neurotoxicity, respectively (Maki and Sladek, 1993; Sladek et al., 1985; Sood and O’Brien, 1996). The anticancer drug misonidazole is reduced to DNA-adduct-forming glyoxal, catalyzed by P450 reductase (Heimbrook et al., 1986). The nonsteroidal antiinflammatory drug sudoxicam was also metabolized to glyoxal and was withdrawn because of hepatotoxicity. Felbamate antiepileptic drug therapy has limited use because of hepatotoxicity and aplastic anemia, which has been attributed to atropaldehyde, a major metabolite (Kapetanovic et al., 2002). Abacavir, a reverse transcriptase inhibitor, marketed for the treatment of HIV-I infection, forms a reactive aldehyde intermediate that plays a role in covalently binding proteins (Walsh et al., 2002). Sorbinil, an aldose reductase inhibitor, formed an erythenatous rash in humans and formed a reactive aldehyde intermediate from 2-hydroxysorbinil, its major metabolite (Maggs and Park, 1988).
TABLE 1
Examples of therapeutic drugs and toxic aldehyde metabolites
2. ALDEHYDE METABOLISM AND METABOLIZING ENZYMES
The body carbonyl burden is of concern because aldehydes are reactive electrophiles that form adducts with cellular protein thiols and amines and can therefore cause toxicity. Lipoxidation is the description of the process for forming carbonylated proteins with LPO aldehydes, whereas glycoxidation is used to describe the process for forming cabonylated proteins with carbohydrate-derived dicarbonyls. The protein adducts formed are often reversible, but can undergo degradation to form reactive advanced lipoxidation (ALE) or glycoxidation (AGE) end-products that covalently cross-link proteins that accumulate with aging and increasingly disrupt protein and cellular function. ALEs probably contribute to the pathogenesis of atherosclerosis, whereas AGEs are generally regarded as a cause of the increased risk for microvascular disease in diabetes.
The ability of cells to metabolize and detoxify aldehydes largely depends on the aldehyde and the cellular content of aldehyde- metabolizing enzymes. Thus 4-HNE and methylglyoxal were very rapidly metabolized (within 3 min) by cells, whereas formaldehyde metabolism was slower but more rapid than acetaldehyde metabolism. The rapid metabolism of 3 mM 4-HNE (t^sub 1/2^ of 2 min) by rat hepatocytes (106/ml) was attributed to glutathione S-transferase (GST) and ALDH2 (aldehyde dehydrogenase) activities and to a minor extent alcohol dehydrogenase (ADH), with only 1% binding to proteins (Grune et al., 1991,1994; Hartley et al., 1995; Siemsetal., 1997). Ratenterocytes also rapidly metabolized 4-HNE. The rapid metabolism of 3 mM methylglyoxal (t^sub 1/2^ of 2 min) by hepatocytes was likely due to metabolism catalyzed by glyoxalase (GLOX) and reduction catalysed by aldo-keto reductase (AKR) 1A2. Because of this rapid metabolism, both of these aldehydes were much less toxic than would be expected from their reactivity towards macromolecules. Furthermore, 4-HNE was metabolized by mouse Ehrlich Ascites cells at one-third of the hepatocyte rate and was also more toxic to ascites cells than to hepatocytes (Grune et al., 1991; Niknahad et al., 2003a). The slower metabolism of 3 mM formaldehyde (t^sub 1/2^ of 30 min) by hepatocytes was likely due to metabolism by ADH5 and ALDH2. Furthermore, formaldehyde cytotoxicity was markedly increased by inhibiting the activity of these enzymes or by depleting hepatocyte glutathione levels (Teng et al., 2001).Chloroacetaldehyde cytotoxicity was markedly increased by ADH1 and ALDH2 inhibitors or by depleting glutathione levels (Sood and O’Brien, 1993). While the major in vivo metabolism route for eliminating alkanals and aromatic aldehydes is via oxidation catalyzed by dehydrogenases, the major urinary metabolite for the dicarbonyl 3-deoxyglucosone in \humans or when administered to rats is by reduction catalyzed by aldehyde reductases to 3-deoxyfructose (Kato et al., 1990). Ketones and 2- hydroxy- or 2-carboxybenzaldehyde are also mostly reduced. Conjugation with glutathione catalyzed by glutathione transferase is a major in vivo elimination route for alkenals, while a glutathione- catalyzed isomerization catalyzed by glyoxalase is the major route for the dicarbonyl phenylglyoxal. The following 19 classes of aldehyde-metabolizing enzymes described include 9 aldehyde- oxidizing enzymes, 7 aldehyde-reducing enzymes, and 3 glutathione- dependent enzymes, which function to help the cell detoxify the various aldehydes that the cell is exposed to.
a. Aldehyde-Oxidizing Enzymes
Aldehydes are somewhat unique toxicants in that they are metabolically detoxified by oxidation in vivo (to acids) and/or reduction to alcohols. Alcohol reoxidation can then be prevented by glucuronidation or sulfation of the alcohol, whereas acids often form glycine conjugates (hippuric acids). In general, aldehydes are primarily oxidized usually by NAD^sup +^ and ALDH, whereas some ketones are reduced by NADH and ADH to form secondary alcohols. Alcohol dehydrogenase (described later under Aldehyde-Reducing Enzymes) can also oxidize aldehydes; for example; ADH5 utilizes NAD^sup +^ to oxidize formaldehyde (via its glutathione conjugate) to form formate; ADH1 (particulary γ^sub 2^γ^sub 2^) and to a lesser extent ADH2 but not ADH3 (yeast) catalyzes the dismutation of aldehyde hydrates to form alcohol and carboxylic acid. However, the K^sub m^ for acetaldehyde was 100-fold higher than that for acetaldehyde reduction. Other K^sub m^ values are 1.2mM for butanal and 0.15 mM for 5-hydroxindole 3-acetaldehyde (the major serotonin metabolite) (Svensson et al., 1999). The alcohol dehydrogenase ADH5 also utilizes NAD^sup +^ to oxidize formaldehyde (via its glutathione conjugate) to form formate. ADH catalyzes the oxidation of aldehydes via gem diols. Sixteen aldehyde dehydrogenase (ALDH) genes and three pseudogenes have been identified and their chromosome locations determined in the human genome. Their role in endogenous and xenobiotic metabolism has recently been reviewed (Vasiliou and Pappa, 2000). The toxicologically more significant hepatic ALDHs, other dehydrogenases, and P450s describe by their gene name are as follows.
Aldehyde dehydrogenase superfamily
1. ALDHlA (chromosome location 9q21) is cytosolic in location, which is constitutively expressed in various tissues , with highest levels in the liver. It is the major ALDH that catalyzes the oxidation of retinal to retinoic acid (Ambroziak et al., 1999; Klyosov, 1996) and detoxifies aldophosphamide (the active metabolite of the anticancer agent cyclophosphamide). It is not efficient at oxidizing acetaldehyde but is efficient at oxidizing longer chain alkanals. The aldehydes of naphthalene, phenanthrene, and coumarin are also oxidized (Keung, 1991; Vidal et al., 1998).
2. ALDH2 (chromosome 15 location) is mitochondrial in location, which is constitutively expressed in various tissues, with the highest levels in the liver. The enzyme has two cysteine residues immediately adjacent to the active site thiol and can form an inactive intramolecular disulfide bond with NO or nitroglycerin. Indeed, this process results in the bioactivation of nitroglycerin with ALDH2 acting as a nitrate reductase (Chen et al., 2002). A solubilized purified preparation of mitochondrial ALDH catalyzed aldehyde oxidation at pH 9.5 with the following order of effectiveness as determined by k^sub cat^: formaldehyde > acetaldehyde, heptanal > octanal > 4-nitrobenzaldehyde > benzaldehyde, whereas retinal or alkenals were found to be inhibitors (Klyosov, 1996). However, the order found for mitochondrial ALDH activity using a mitochondrial fraction at pH 8.5 and much lower aldehyde concentrations (10 M) was heptanal, octanal > phenylacetaldehyde > acetaldehyde > retinal [much greater than] formaldehyde, benzaldehyde (Wang et al., 2002). The order found for rat liver mitochondrial ALDH2 activity based on K^sub m^ and V^sub m^ was chloroacetaldehyde [much greater than] propionaldehyde, > dichloroacetaldehyde with no activity for chloral hydrate presumably because it is a strong ALDH2 inhibitor with a K^sub i^ of 150 M (Sharpe and Carter, 1993). The order found for human ALDH was methoxyacetaldehyde > propionaldehyde > acetaldehyde > glycolaldehyde > methylglyoxal > glyoxal > benzaldehyde with no activity for chloral hydrate or glyoxylic acid (Kraemer et al., 1968). Perhaps the best substrate is 4-HNE, as addition of 1 mM 4- HNE to hepatocytes resulted in a very rapid metabolism (half-life of 1 min) that was attributed to metabolism by ALDH2 and GST (Siems et al., 1997; Hartly et al., 1995). Methylglyoxal was a poor substrate (K^sub m^ 8.6 mM) and was also an ALDH2 inhibitor (Izaguirre et al., 1998). Because of the high efficiency of ALDH2 in catalyzing acetaldehyde oxidation, it is likely that ALDH2 plays the major role in acetaldehyde detoxification in humans. It is highly polymorphic in Asians, and individuals with the ALDH2.2 deficient allele exhibit the “alcohol flushing” syndrome as a result of the elevated blood acetaldehyde (Vasiliou et al., 2000). As a result of this discomfort, it is possible that this mutation is strongly protective against alcoholism. However, individuals with this mutation who drink have an increased risk of esophageal and upper aerodigestive tract cancers. Interestingly, although the mutant allele has a marked decrease in activity toward acetaldehyde, formaldehyde, propionaldehyde, or hexanal, its activity toward longer alkanals or aromatic aldehydes was the same as the wild allele (Wang et al., 2002).
3. ALDHlBl, formerly known as ALDH5 (chromosome location 9pl3), is mitochondrial in location and found in the liver, heart, brain, testis, skeletal muscle and fetal tissues. It is active with short- chain aldehydes (acetaldehyde and propionaldehyde) and NAD^sup +^ (not NADP^sup +^) but is insensitive to disulfiram unlike ALDH 1 and 2 (Stewart et al., 1995). It is polymorphic and three variants have been identified (Vasiliou et al., 2000).
4. ALDH3A1 (chromosome location 17pll.2) belongs to a group of homodimeric enzymes tthat are cytosolic in location and utilize NAD^sup +^ or NADP^sup +^ as a cofactor, depending on the isozyme. It is constitutive in the stomach, heart, skin, and corneal epithelium. This enzyme is highly polymorphic in the general population. Although it is not found in the liver, colon, spleen, bladder, lung, and thymus of rodents but can be induced with AhR ligands, it is expressed in hepatoma cells of rats and can be induced along with CYPlAl more than 100-fold in rat liver by AhR ligands, such as the carcinogenic polycyclic aromatic hydrocarbons benzo[a]pyrene and 3-methylcholanthrene. The ALDH3A1 induction is in response to acute-phase proteins induced by an atypical hepatocyte inflammation and can be prevented by NSAIDs (Pappas et al., 2003). It is also overexpressed in the hepatocellular carcinoma tissues of 50% of cancer patients and is associated with ALDH2 downregulation (Park et al., 2002). These patients are also resistant to oxazaphosphorine chemotherapy, for example, cyclophosphamide, ifosfamide, 4-hydroperoxycyclophosphamide, and mafosfamide. Genotyping cancer patients for ALDH3A1 could therefore be useful for selecting the best type of chemotherapy. The chemotherapy resistance is due ostensibly to the enzyme-catalyzed oxidative detoxication of aldophosphamide, the pivotal aldehyde metabolite of these prodrugs. The disulfiram and chloral hydrate ALDH inhibitors do not inhibit this enzyme. However, chlorproamide analogues are inhibitors for this enzyme and may prove useful for overcoming tumor resistance (Rekha et al., 1998). Substrates include LPO aldehydes, such as 4- HNE. The order of effectiveness of other substrates was phenylacetaldehyde > benzaldehyde [much greater than] propionaldehyde. Dicarbonyls have not been reported to be substrates.
5. ALDH3A2 (chromosome location 17pl 1) is microsomal in location utilizes NAD^sup +^ as a cofactor and is considered to perform a housekeeping function in all tissues. A solubilized preparation of microsomal ALDH catalyzed the oxidation of fatty aldehydes to fatty acids, as well as the oxidation of medium- and long-chain alkenals, and alkanals, whereas acetaldehyde, benzaldehyde, retinaldehyde, and crotonaldehyde were poor substrates (Kelson et al., 1997). A genetic deficiency of ALDH3A2 is associated with the SjogrenLarson syndrome, an inherited neurocutaneous disorder, whose symptoms include mental retardation, spasticity, and ichthyosis. ALDH3A2 is also inducible by peroxisome proliferators. The enzyme is polymorphic and seven variants have been identified (Agarwal, 2001).
6. ALDH9 (betaine aldehyde dehydrogenase) is found as cytosolic and mitochondria! isozymes in the liver, kidney, adrenal glands, brain, and plants and utilizes NAD^sup +^ as a cofactor (Pietruszko et al., 2001). ALDH9 catalyzes the oxidation of betaine aldehyde (formed from choline by choline dehydrogenase located in the inner mitochondrila membrane) to betaine (an intracellular osmolyte and methyl donor) and the final step in the conversion of choline to betaine. Betaine aldehyde is the preferred substrate, but aminoaldehydes (e.g., trimethylaminobutyraldehyde, a carnitine biosynthesis intermediate, as well as aldehydes of polyamine metabolism), aliphatic aldehydes (e.g., acetaldehyde [formerly E3]), aromatic aldehydes, and methylglyoxal (and glycolaldehyde) are also substrates (vander Jagt and Hunsaker, 2003). Cimetidine is an inhibitor of this isoform.
7. α-Oxoaldehydedehydrogenase (ODH), NADP^sup +^ dependent, is located in the liver cytosolic fraction and erythrocytes and utilizes NADP^sup +^ for catalyzing the oxidation of methylglyoxal (K^sub m^ 0.\26 mM) to pyruvate but did not catalyse the oxidation of glyoxal or 3-deoxyglucosone. Amines, such as aminopropanol, markedly activate this dehydrogenase (vander Jagt and Hunsaker, 2003). The α-oxoaldehyde substrates are essentially fully hydrated and are not good substrates for ALDHl and -2. It is therefore believed that the role of 2-ODH is to prevent protein glycation (Fujii et al., 1995). The enzymes are monomeric with a 42,000-MW cysteine at the active center and are inhibited by nucleotides and thiol reagents.
8. NAD^sup +^-dependent a-ketoaldehyde-dehydrogenase or pyruvate dehydrogenase. A goat liver cytosol enzyme was isolated that catalyzed the oxidation of methylglyoxal to pyruvate (Ray et al., 1982), whereas methylglyoxal oxidative decarboxylation to acetyl coenzyme A (CoA) and formate was catalyzed by the thiamine pyrophosphatedependent decarboxylase part of the mitochondrial pyruvate dehydrogenase multienzyme complex (Argiles, 1989).
9. P450 or aldehyde oxidase. P450 is located in the endoplasmic reticulum and uses NADPH, oxygen, and NADPH:P450 reductase to catalyze the oxidation of alcohols and aldehydes. Alcohol oxidation is catalyzed by CYP2E1 (alcohol-inducible P450). Alcohols oxidized include ethanol and cyclohexenol. We have also shown that P450, not ADH, is the major metabolizing enzyme in hepatocytes for propargyl alcohol and 2-bromoethanol but not allyl alcohol (Khan et al., 1996; Moridani et al., 2001 ). The ethanol oxidation mechanism involves two one-electron oxidations, with the first forming 1-hydroxyethyl radicals and the second forming acetaldehyde. The mechanism of cyclohexenol oxidation to cyclohex-2-en-l-one, however, involves a radical-mediated formation of a gem-diol intermediate (Bellucci et al., 1996). P450 also catalyzes the oxidation of most aldehydes and includes alkanals (e.g., nonanal), alkenals (e.g., citronellal), benzaldehydes (e.g., 4-nitrobenzaldehyde), terpene-aldehydes (e.g., perillaldehyde), and unsaturated ketones (e.g., cyclohexenone) to products that inactivate P450. The examples given are the most effective aldehyde in that class (Raner et al., 1997). Many of these aldehydes were oxidized by specific P450 isozymes to the corresponding acid, for example, retinal (CYPlAl, 1A2, IBl, 3A4), acetaldehyde, trifluoroacetaldehyde (CYP2E1 ), tolualdehyde (CYP2C), and 9-anthraldehyde (CYP3A4) (Marini et al., 2003). However, the aldehyde oxidation mechanism found when 3phenylpropionaldehyde or cyclohexanal oxidation was catalyzed by P450 involved the formation of peroxyhemiacetal intermediates, which underwent scission (deformylation) to form formic acid and carbonyl carbon radicals. The latter then formed olefins and alkyl P450 heme adducts, which irreversibly inactivated P450 (Kuo et al., 1999). 4-HNE was also metabolically activated by P450 to carbonyl carbon radicals, which were oxidized to 4-hydroxy-2-nonenoic acid or inactivated P450 (Kuo et al., 1997). These reactive radicals also caused mitochondrial toxicity and formed LPO/ROS, which contributed to the aldehyde- cytotoxicity mechanism. Acrolein or allyl alcohol oxidation to glycidaldehyde (a reactive epoxide) and glycidol, respectively, was also catalized by the P450 mixed, function oxidase system (Patel et al., 1980). Furthermore-P450 activation by caffeine has been suggested as an explanation for the caffeine potentiation of allyl alcohol-induced hepatotoxicity (Karas and Chakrabarti, 2001).
10. Molybdenum hydroxylases: aldehyde oxidase (AOX) and xanthine oxidase (XO). Aldehyde oxidase (AOX) is mostly cytosolic with some microsomal activity, primarily in the liver (then kidney and intestine), that utilizes oxygen (or ferricyanide) to oxidize aldehydes to carboxylic acids. AOX has a broad specificity for aldehydes, including all aromatic aldehydes. Aldehyde substrates commonly used include benzaldehydes (e.g., vanillin, 4-hydroxy-3- methoxybenzaldehyde, although steric hindrance occurred with ortho substituted benzaldehydes) or 1- or 2-naphthaldehyde, while butyraldehyde was the best alkanal substrate. Endogenous or biogenic aldehyde substrates included indole-aldehydes, retinal, pyridoxal, and TV-methylnicotinamide. AOX also utilized aldehydes to reduce nitroarene drugs such as nitrofurazone (Wolpert et al., 1973). AOX is a dimeric protein of molecular mass 300 with two identical independent subunits containing molybdopterin, FAD, and two iron- sulfur centers. AOX had a higher affinity for aldehydes than xanthine oxidase (XO), but did not oxidize xanthine as did XO. Superoxide radicals were also formed when acetaldehyde was oxidized by AOX catalysis and seemed to contribute to ethanol hepatotoxicity in vivo or ischemia-reperfusion injury (Mira et al., 1995; Shaw and Jayatilleke, 1990). AOX also oxidized and detoxified lipophilic aromatic compounds, including pteridines, phthalazines, phenanthridine, pyrimidines, and purines. Its activity was higher in guinea pigs and rabbits than in pigs, mice, hamsters, or humans, but is absent in dogs, birds, and some Sprague-Dawley (SD) rat strains, including the one used by most researchers. It was inhibited by isovanillin, menadione, chlorpromazine, promazine, hydralazine, and estrogen receptor modulators (with raloxifene the most effective), but not allopurinol. Other drugs also effectively inhibit AO; therefore, these drugs have the potential to cause drug/drug interactions with other drugs metabolized by AO (Obach, 2004).
XO normally exists in cells as xanthine dehydrogenase (XDH), which differs from XO in having an accessible NAD^sup +^ binding site so that XDH can oxidize xanthine or hypoxanthine to form uric acid, the terminal part of the purine catabolism pathway in humans. Other purines and pteridines were also oxidized. XDH also oxidized ethanol to hydroxyethyl radicals and acetaldehyde (Castro et al., 2001). X-ray crystallography showed that XO was formed from XDH by a reversible oxidation of Cys535 and Cys992 or if the peptide chain was knicked by a Ca^sup 2+^-ac
