Organosulfur compounds from alliaceae in the prevention of human pathologies

Abstract

A strong association between elevated plasma low-density-lipoprotein (LDL) and the development of cardiovascular diseases (CVD) has been established. Oxidation of LDL (Ox-LDL) promotes vascular dysfunction, enhances the production and release of inflammatory mediators such as reactive oxygen species and contribute to the initiation and progression of atherosclerosis. In addition, Ox-LDL enhances the production and release of tumor necrosis factor (TNF-α), interleukin (IL)-6, arachidonic acid metabolites and nitric oxide (NO) that are responsible for various human pathologies including cancer. Organosulfur compounds (OSC) from alliaceae modulate the glutathione (GSH) redox cycle and inhibits NFk-B activation in human T cells. Furthermore, OSC bioactivities include antioxidant, antibacterial, anticarcinogenic, antiatherogenic, immunostimulatory, and liver protection potential.

1. The antioxidant potential of organosulfur compounds

Oxidative modification of DNA, proteins and lipids by free radicals and non-radical oxidants plays a role in wide range of diseases including cardiovascular [1], neurodegenerative and inflammatory diseases [2] and cancer (Table 1) [3,4]. Free radicals include superoxide, nitric oxide (NO) and hydroxyl radical are the most toxic of the reactive oxygen species (ROS). The non-radical oxidants include hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂) and ozone (O₃) which form free radicals in some tissues through various chemical reactions (Table 2). Most of the ROS produced by cells are from:

1. normal aerobic respiration in mitochondria, which generates superoxide radical (O₂^·−) and the ensuing toxic products, and the highly reactive hydroxyl radical (OH^·),

2. stimulated macrophages and polymorphonuclear leukocytes, which release superoxide and the nitric oxide radical (NO^·), that can interact to form the non-radical destructive peroxynitrite,

3. peroxisomes, cell organelles that produce H₂O₂ as a by-product of degrading fatty acid and other molecules, and

4. oxidant by-products that occur during the induction of cytochrome P450 enzymes.

Table 1.

Mechanisms involved in the etiology of oxidant-mediated disorders

Disease	Pro-oxidative mechanism
Cancer	Oxidative damage to DNA Inactivation of DNA repair enzymes
Cardiovascular disease	Oxidative modification of LDL Adhesion of phagocytes to vascular endothe-lium
Pulmonary emphysema Tissue damage in autoimmune diseases (rheumatoid arthritis)	Potentiation of the proteolytic activity of phagocytes-derived proteases
Acquired immunosuppression	Oxidative inactivation of the protective activities of B- and T-lymphocytes and NK-cells

Table 2.

Reactive oxygen species (ROS). Quenching ability of specific antioxidants

Reactive species		Antioxidants
1O2	Singlet oxygen	Vitamins A, C, E, (β-carotene and other carotenoids
O2.−	Superoxide free radical	Superoxide dismutase, vitamins C, E, β-carotene
ROO.	Peroxyl-free radical	Vitamins C, E
H2O2	Hydrogen peroxide	Catalase, glutathione peroxidase
LOOH	Lipid peroxides	Glutathione peroxidase

Endogenous levels of ROS increase during chronic infection and inflammation, strenuous physical exercise, hyper-metabolic states, trauma and sepsis, and during exposure to exogenous sources. The most common exogenous sources of ROS are derived from tobacco smoke, (that generates free radicals in exposed tissues notably the highly reactive OH^· radical), UV light (which produces singlet oxygen (¹O₂) and OH^·, ozone (O₃), polluted air (which produces oxides of nitrogen), industrial toxins (such as carbon-tetrachloride), drugs (such as phenobarbital, a known tumor promoter in liver), and charcoal-broiled foods (which form a variety of carcinogens, notably benzo(a)pyrene). To protect molecules against toxic free radicals and other ROS, cells have developed antioxidant defences that include small molecules including glutathione, and the enzymes superoxide dismutase (SOD), which dismutates superoxide; catalase and glutathione peroxidase, which destroy toxic peroxides. External sources of antioxidant nutrients include antioxidant vitamins C and E, vitamin A/provitamin A and the mineral selenium, a component of selenium-dependent glutathione peroxidase (Table 2). Hence, dietary supplementation of these antioxidants may decrease the incidence of tissue damage that leads to a wide variety of diseases. One such source is phytochemicals.

Phytochemicals from plant-rich diets, including organo-sulfur compounds (OSC) from Allium sativum (garlic) such as alliin, diallylsulfides and allicin [5], provide most of its potent biological activity in the protection against oxidant damage (Fig. 1) [6]. Lipid-soluble allyl sulfur compounds are formed from the parent sulfur compound alliin by the action of alliinase, an enzyme released by crushing or chopping of garlic. The most commonly used lipid-soluble allyl sulfur compounds are ajoene, diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS). Water soluble compounds can also occur in garlic especially after alcoholic fermentation. The parent compound to alliin, γ-glutamyl-S-allylcysteine is converted to S-allylcysteine (SAC), S-allylmercaptocysteine (SAMC) and others (Fig. 2) [7].

Fig. 1.

Hydro- and lipo-soluble compounds from Allium sativum.

Fig. 2.

Thiosulfonate in age garlic extract (AGE).

Water-soluble allyl sulfur compounds include SAC and SAMC and the lipid-soluble sulfur compounds, DADS, DATS and diallylpolysulfides that are obtained from aged garlic extract (AGE, Kyolic), a naturally modified form of raw garlic produced by a unique aging process. These compounds are characterized by their high antioxidant content and health protective potential [8–11]. Other antioxidants in AGE include phenolic compounds, allixin, [12], selenium, and N-(1-deoxy-D-fructos-1-yl)-L-arginine (Fru-Arg) and N-fructosyl glutamate that could not be detected in raw nor heated garlic (Fig. 3) [13]. These data suggest that supple mentation with aged garlic extracts are therapeutically beneficial.

Fig. 3.

Chemical structure of Fru-Arg.

The oxidant injury caused by ROS is linked to various pathologies including the development of cardiovascular disease (Table 1). The potential of AGE to protect endothelial cells is by modifying cellular scavenging enzymes. [10,19]. Endothelial exposure to the oxidants hypoxanthine and xanthine oxidase or H₂O₂, in the presence of AGE generated increased levels of SOD, catalase, and glutathione peroxidase, and in a dose- and time-related fashion suppressed the production of O₂^·− and H₂O₂ [10]. In the blood circulatory system, AGE components improve peripheral circulation, [27], protect vascular endothelial cells from oxidant injury [9–11,19], reduce plasma lipids [28] and alter platelet function [29].

Decreased tissue-glutathione levels are associated with cell damage, depressed immunity, progression of aging, and increased risk of cancer. Reduced GSH, as a substrate for the antioxidant enzyme glutathione peroxidase, protects cellular constituents from the damaging effects of peroxides formed in metabolism and through ROS reactions. AGE increases cellular GSH in a variety of cells, including those in normal liver and mammary tissue [16]. AGE increases also glutathione peroxidase and other ROS scavenging enzymes [10] that are important in radioprotection and UV suppression of certain forms of immunity [17]. In addition, AGE supplementation has been shown to reduce or prevent the range of ROS-induced DNA, lipid and protein damage implicated in aging processes [17,18] as well as chemically induced cancers (Fig. 4) [4]. It has been shown also to act as immuno-modulator [30–33], antiallergic [34], liver protective agent [35] and has an antiaging effect [36].

Fig. 4.

Oxidative damage in carcinogenesis.

Pharmacologic studies of SAC and SAMC, were proposed to be responsible in part for scavenging of active oxygen species [20,21], inhibition of lipid peroxidation [22] and cancer prevention [23–26]. AGE and SAC protect vascular endothelial cells (EC) from H₂O₂-induced injury [11], inhibit Cu²⁺-induced LDL oxidation [14], modulates the glutathione (GSH) redox cycle, inhibits NFk-B activation in human T cells [15] and prevent oxidant-induced dense-body formation in sickle red blood cells [37].

2. Effect of organosulfur compounds in cardiovascular diseases

Cardiovascular diseases (CVD) are the major cause of death among people living a Western life style. The risk of developing CVD is greater for men than for premenopausal women. Moreover, as a person ages, there is a greater risk of CVD.A multitude of factors contribute to the development of CVD. Specifically, hypertension, diabetes and obesity are three clinical conditions that contribute to the increased incidence of CVD. Lifestyle, smoking, inactivity and stress are also important CVD risk factors [38–40]. Atherosclerosis is the principal contributor to the pathogenesis of myocardial and cerebral infarction. It is a complex disease that is associated with an excessive inflammatory, fibro-fatty, proliferative response to damage of the artery wall involving several cell types such as smooth muscle cells, monocyte-derived macrophages, T-lymphocytes and platelets [41].

The presence of a thrombus in a stenosed coronary artery can lead to acute syndromes such as myocardial infarction and angina [42]. Platelet aggregation plays a central role in coronary thrombosis and is related to a cascade of events which includes expression of adhesion molecules on the surface of the endothelium, the oxidation of lipoproteins, monocyte invasion of the vessel wall, foam cell formation, smooth muscle phenotypic change and proliferation and platelet deposition [43]. These events could be induced from dysfunction of the endothelial lining that occurs in hyperlipidemia, hypertension or cigarette smoke causing imbalance of angiotensin II and NO production in the artery wall [44,45].

Elevated plasma cholesterol particularly the LDL cholesterol but not the high-density-lipoprotein cholesterol (HDL cholesterol) and triglycerides are associated with an increased risk of CVD [46]. Oxidation of LDL cholesterol has been recognized as playing an important role in the initiation and progression of atherosclerosis [47–49]. LDL has been shown to be oxidized by cultured cells such as macrophages, endothelial and smooth muscle cells with transition metals [50–54]. The effect of Ox-LDL on vascular endothelial cells (EC), is evaluated by:

1. lactate dehydrogenase (LDH) release, used as index of membrane damage. LDH is an intracellular enzyme that leaks into the culture medium when cell membranes are damaged. Thus, as compared to untreated cells, exposure to Ox-LDL caused increase in LDH release;

2. cell viability, which is determined by the methylthiazol tetrazolium (MTT) ring cleaved by mitochondrial dehydrogenase, a reaction that occurs only in living cells;

3. lipid peroxidation evaluated by thiobarbituric acid reactive substances (TBARS). Products of lipid peroxidation increased greatly when cells were incubated with OX-LDL for 24 h.

A primary histologic feature of incipient atherosclerosis [55] is the presence of cholesterol-loaded foam cells and the fatty streak which occur when Ox-LDL is taken up by macrophage. The contribution of Ox-LDL to the initiation and progression of the atherosclerotic process includes events such as chemotaxis for monocytes, inhibition of macrophage motility, formation of foam cells, up-regulation of endothelial adhesion molecules, stimulation of growth factors and chemokines, and proliferation of smooth muscle cells [56]. Ox-LDL also appears to exert cytotoxicity and to initiate vascular dysfunction by altering the composition and permeability of the endothelial barrier [57–59]. Ox-LDL enhances also the production and release of inflammatory mediators such as ROS, tumor necrosis factor (TNF-alpha), interleukin (IL)-6, arachidonic acid metabolites and NO [60–62]. Acting as second messengers, these mediators stimulate cells to activate transcription factors regulated by the intracellular redox state and promote the development of inflammation leading to injury of surrounding cells and tissues. Among these factors, is nuclear factor NFk-B, a well-known transcription factor activated by oxidative stress. NFj-B is a heterodimeric transcription factor complex composed of two DNA-binding subunits, p50 and p65, and it is associated with the regulation of numerous genes encoding proteins in immune function, inflammation and cellular growth control [63]. Under stressed conditions in EC, activation of NFk-B leads to the expression of cell adhesion factors such as vascular cell adhesion molecule-1 (VCAM-1) and inter-cellular adhesion molecule-1 (ICAM-1) [64,65]. These events further accelerate the formation of atherogenic lesions and cell death. However, antioxidants can inhibit oxidant-induced NFk-B activation [66–68], protect EC [69,70] and normalize vascular functioning in hypercholesterolemia and atherosclerosis [71–73].

Relaxation mediated by endothelium derived NO is impaired in arteries from hypercholesterolemic and atherosclerotic animals [74]. The mechanisms suggested for the effect on vascular relaxation include increased diffusional barrier for NO due to the intima thickening [75], L-arginine depletion [76,77] altered endothelial cell receptor coupling mechanism and inactivation of NO by oxygen free radicals [78,79]. In addition, although blood lipids are in the normal or lower range, elevated blood homocysteine has also been associated with an increased incidence of cardiovascular disease [80–85].

2.1. The antiatheroslerotic activity of organosulfur compounds

OSC derived from garlic possess antiatherosclerotic properties by reducing serum cholesterol levels in humans [86,87], inhibit cholesterol biosynthesis [28], suppress LDL oxidation, lower plasma fibrinogen and increase fibrinolytic activity [88,89]. It has been also reported that in cell free homogenate and in vivo, garlic activates the NO synthase [90]. Amino acid analysis of garlic powder demonstrated that it is a rich source of arginine the precursor of NO. Since the hypertension induced by NO synthase inhibitor, L-NAME, and the decrease in the urinary levels of NO₂⁻/NO₃⁻ induced by L-NAME in rats were prevented by treatment with garlic, it was suggested that garlic increases NO synthase activity in vivo.

2.2. Inhibition of platelet aggregation

The major arachidonic acid metabolite formed by platelets is TXA2, a potent vasoconstrictor and stimulator of aggregation. In contrast, PGI₂, the major arachidonic acid metabolite formed by the vascular endothelial cells is a potent vasodilatator and inhibitor of platelet aggregation. It is the balance between these eicosanoids that is important in regulating hemostasis and platelet aggregation. These eicosanoids are extremely short-lived in plasma and are invariably measured as their stable metabolites, 6-keto-PGF₁ and TXB₂.

The production of NO by endothelial cells is another important regulator of platelet activity [91]. Aggregation of platelets is a consequence of exposure of fibrinogen receptors on the surface of the cells. These receptors bind fibrinogen in the presence of extracellular Ca²⁺ and cross-link the platelets to form aggregates. The fibrinogen receptor is a heterodimer of the membrane glycoproteins (GP)IIb and IIIa. Although unstimulated platelets express the GPIIb–IIIa complex at their surface, this complex is unable to bind fibrinogen until platelets are activated [92].

Although the mechanism of action of organosulfur is not clear, it was suggested that it may be due to a sulfhydryl group-mediated effect [100]. Studies in humans and animals have shown that certain constituents of fresh garlic or its extract have inhibitory activity on platelet aggregation [93–98]. The GPIIb–IIIa receptor has a high content of -SH groups, and binding of fibrinogen is inhibited by the organo-sulfur compound ajoene [99].

The inhibition of fibrinogen mediated platelet adhesion is due to an organosulfur compound in dried AGE reducing the functional competence of some GPIIb–IIIa receptors, whereas sufficient receptors remain to sustain full ADP-induced aggregation. In contrast, ADP-induced platelet aggregation was inhibited by the AGE supplementation. Thus, the most likely mechanism involves the ADP receptor. Platelet ADP receptors belong to the P2T subtype of purinoreceptors whose activation leads to a rise in intracellular Ca²⁺. Reduction of adhesion to collagen- and fibrinogen-coated surfaces and specificity of inhibition of platelet adhesion and individual receptors for these adhesive proteins may be affected differently by AGE [102]. Moreover, AGE contains a number of organosulfur components [101] and a large number of other substances including carbohydrates, proteins and saponins. Specific receptors, (epinephrine and collagen) could be also the mechanism by which AGE induced reduction of platelet aggregation rather than an inhibition of mediators of platelet aggregation such as observed with nonsteroidal antiinflammatory agents.

2.3. Cholesterol-lowering effect of organosulfur compounds

Elevated total and LDL cholesterol levels and blood pressure are the main cardiovascular risk factors [103].

Organosulfur compounds were shown to lower cholesterol level from cultured hepatocytes [104,105], blood cholesterol from animals [106–108] and humans [109–112]. Compounds such as S-allylcysteine (SAC), diallylsulfide (DADS) or allicin and its derivative (ajoene) were among compounds that contribute to reduce cholesterol level [113–115]. It has been suggested that the mechanism underlying the inhibitory action is related to the inhibition of cholesterol synthesis pathway and a decreased activity of several cholesterolgenic enzymes, including 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase and acetyl-CoA synthetase [114]. Several of the water soluble compounds present in AGE such as SAC, S-ethyl cysteine, and S-propyl cysteine have been shown to inhibit cholesterogenesis in hepatocytes [28]. Lipid-soluble organosulfurs such as diallylsulfide, diallyldisulfides and diallyltrisulfides as well as dipropylsulfide, dipropyldisulfides and methylallylsulfide, also decrease cholesterol synthesis of hepatocytes but to a less extent and by damaging the cells as evidenced by release of cellular lactate dehydrogenase. However, allicin one of the most effective organosulfurs of fresh garlic in vitro shows rapid reduction in the blood and appears to interact with the iron in hemoglobin, oxidizing it to the trivalent form to produce methemoglobin [116].

3. The antiproliferative actions on human cancers

Evidence for the anticancer effect of OSC comes from both epidemiologic and laboratory investigations. In 1958, Weisberger and Pensky [117] demonstrated in vitro and in vivo that thiosulfinate extracts of garlic inhibited the growth of malignant cells and prevented growth of sarcoma 180 as-cites tumor.

Moreover, it was found thatA 549 lung and BJA-B Burkitt lymphoma cells [118], human prostate cancer cells (LNCaP) [119], human breast cancer cells (MCF-7) [120] and animal mammary tumor cells in culture [121] were more sensitive to the antiproliferative effects of DATS and ajoene than the non-neoplastic cells. However, the antiproliferative effects of allyl sulfides are reversible [122] and both ajoene and SAMC have been found to be inversely proportional to cell density [118,123].

The transformation of normal to neoplastic cells in vivo involves at least three distinctive phases, initiation, promotion and progression. Much of the work focused on OSC to suppress tumor incidence in breast [23,25,124–127]. In rats DMBA-induced mammary tumor, treatment with Se-garlic inhibited both the initiation and post-initiation phases of chemical carcinogenesis [128]. Organosulfur compounds suppressed tumor incidence in skin [129,130], uterine [131], oesophagus [132], gastric [133,134], human prostate [135] and colon [26,136] cancer models. It was also reported that diallyl disulfide is as effective as 5-fluorouracil (5-FU) in inhibiting tumor growth. Although, combining the diallyl disulfide and 5-FU did not increase the effect, concurrent diallyl disulfide did significantly reduce the depression of leukocytes counts and splenic weight associated with chemo-therapy administration [137].

3.1. Mechanisms of action

While the allyl group appears to be responsible for the growth depression, not all allyl sulfides are equal in their ability to reduce tumor proliferation. The magnitude of the increase in the G2/M phase of the cell cycle reflects the antiproliferative potential of allyl sulfur compounds. DADS and diallyl trisulfide (DATS) were more effective in the cell cycle alteration and growth of neoplasms inhibition than soluble allyl sulfur compounds such as SAC. Increased DADS, DAS or SAMC exposure led to a proportional but not permanent increase in the percentage of cells arrested in the G2/M phase of the cell cycle [122,138,139]. G2/M phase arrest induced by the allyl sulfur compounds coincide with a suppression in p34cdc2 kinase activity. The p34cdc2 kinase complex which governs the progression of cells from the G2 into the M phase of the cell cycle, is controlled by the association of the p34cdc2 catalytic unit with the cyclin B1 regulatory unit [140]. Activation of this complex is governed by both cyclin B1 protein synthesis and degradation and by the phosphorylation and dephosphorylation of threonine and tyrosine residues on the p34cdc2 subunit [140,141]. It promotes chromosomal condensation and cytoskeletal organization through the phosphorylation of multiple substrates, including histone H1 [141,142]. Factors that inhibit p34cdc2 kinase activity lead to a block in the G2/M phase.

The effects of allium derivatives is mediated by various mechanisms, including blockage of N-nitroso compound (NOC) formation. A reduction in nitrosamines may occur as a result of enhanced formation of nitrosothiols after ingestion of allium foods [143–146] and the bioactivation and carcinogenicity of several non-nitrosamines [147]. It suppresses also the bioactivation of several carcinogens [148–150]. Depressed carcinogen bioactivation may be related to cyclooxygenase and lipoxygenase activity reduction [150–152].

Ajoene a major component of garlic induces apoptosis in a human promyeloleukemic cell line (HL-60) as well as in peripheral blood cells of a chronic leukemic patients suffering from a myeloid blast crisis. In contrast ajoene does not induce apoptosis in proliferating as well as non-proliferating PMBC of healthy human donors. [153]. Induction of apoptosis and peroxide production by ajoene may be linked through activation of NF-kB which is known to be induced by oxidative stress. NF-kB has been shown to be involved in signaling of apoptotic processes [154] regulation of the cell cycle [155,156], enhanced DNA repair [157], modifying the signal transduction pathway, and regulating nuclear factors involved in immune function and inflammation [65]. In animal studies, AGE is reported to induce release of IL-2, TNFα and INFγ [158].

The loss of cancer progression with allyl sulfur compounds can be also related to several epigenetic changes such as DNA methylation and histone acetylation that can be modified by enhanced intake of allyl sulfur compounds. Thus, after treatment with N-nitrosomethylbenzylamine, DAS inhibited the formation of O6-methyldeoxyguanosine in lung by 78% [162] and DADS caused a marked increase in the acetylation of H4 and H3 histones in DS19 and K562 human leukemic cells [163].

3.2. Inhibition of protooncogenes

DADS can also inhibits the growth of H-ras oncogene-transformed tumors in nude mice. This inhibition correlate with the inhibition of p21H-ras membrane association in the tumor tissue [164]. The ras family of protooncogenes encode 21-kDa proteins (p21ras) which play an important role in the transduction of extracellular signals to the cell nucleus [165,166]. Plasma membrane association of mutated p21ras encoded by oncogenic ras, is essential for its cell transformation activity. A lipid post-translational modification (farnesylation) is critical for the plasma membrane association of p21ras and this reaction is catalyzed by a specific cytosolic enzyme, farnesyltransferase [167–169]. The inhibition of p21ras farnesylation can be achieved either by inhibiting farnesyltransferase activity or by lowering the farnesylpyro-phosphate pool through inhibition of HMG-CoA reductase activity. Naturally occurring organosulfides from garlic were shown to inhibit chemically induced cancers, including those in which tumorigenesis is associated with the activation of ras oncogenes. The organosulfides-mediated suppression of tumor growth correlates with the inhibition of p21H-ras membrane association in tumor tissues. It seems that this occurs via depletion of the farnesyl pyrophosphate pool through inhibition of hepatic as well as tumoral HMG-Co A reductase activity [26,164,170].

3.3. Antibacterial activity

While the exact cause of gastric cancer is not understood, there is strong evidence that adenocarcinomas of the distal stomach are largely due to environmental exposures early in life, including a diet rich in highly salted foods and infection by the bacterium Helicobacter pylori. Bacterial activation of procarcinogens may lead to the development of metaplasia dysplasia and ultimately carcinoma [159]. Garlic extract exhibited a selective potency against H. pylori. Thiosulfinates and particularly allicin account for all the antibiotic activity. Upon reduction of allicin to diallyl disulfide, the antibacterial activity is greatly reduced [134,160]. Allicin exhibits its antimicrobial activity by a rapid and total inhibition of RNA synthesis [161]. The lipid content of the membranes and their electrical charge will have an effect on the permeability of allicin [160].

4. Conclusion

A plurality of factors likely lead to ROS-induced tissue damage that contribute to a wide variety of diseases. However, reducing endogenously or exogenous can block the toxic effects. Thus, garlic-derived allylsulfides may exert also their anticarcinogenic effects in experimental animals and in cell culture systems by blocking P450 enzymes included in carcinogen activation and/or by enhancing P450 enzymes that catabolize carcinogens to less reactive intermediates [171–173]. Allium constituents inhibit the covalent binding of carcinogens to DNA [124] and inhibit the activities of demethylating and hydroxylating cytochromes P450 2E1, 2B1, 1A1 and 1A2 in both hormone-responsive and hormone unresponsive cells [174,175]. In addition allium derivatives stimulate the GSH synthesis and enhance glutathione peroxidase activity [176]. Allium has been shown to induce phase II conjugation systems which inactivate most carcinogens. Ingestion of allium derivatives by rats increases the activity of gutathione-S-transferase (GST) in both liver and mammary tissue [24,177–180]. However, not all GST isozymes are influenced equally and induction of GST pi may be particularly important in the anticarcinogenic properties associated with allyl sulfur components [181].

It was also reported that S-methylcysteine a water soluble organosulfur compound prevented elevation of ornithine decarboxylase and spermidine/spermine N1-acetyltransferase (SAT) both rate limiting enzymes of polyamine metabolism which are increased by chemically induced carcinogenesis [182]. Ornithine decarboxylase, contains nucleophilic thiol moieties at cysteines 360 and 70 that are highly accessible to oxidants and sulfhydril agents [183]. Depletion of reduced GSH by oxidative stress produces a concomitant induction of ornithine decarboxylase, whereas treatment of cells with compounds that increase reduced GSH formation inhibits ornithine decarboxylase induction [184]. The balance of polyamines and reduced glutathione may be critical to the regulation of cell proliferation and differentiation. S-Allylmercaptocysteine inhibits ornithine decarboxylase activity either by elevating intracellular reduced GSH, which in turn, inhibits induction of ornithine decarboxylase, or by reacting directly with ornithine decarboxylase at the nucleophilic thiol moieties of cysteines or both [119]. Thus, garlic derivatives markedly influence the steady state concentration of reduced GSH as well as the activities of enzymes that control its metabolism.