Antigenotoxic Potential of Some Dietary Non-phenolic Phytochemicals

Chapter 7

Antigenotoxic Potential of Some Dietary Non-phenolic Phytochemicals Ana Clara Aprotosoaie1, Vlad Simon Luca, Adriana Trifan and Anca Miron Department of Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy Grigore T. Popa Iasi, Iasi, Romania 1 Corresponding author: e-mail: [email protected]; [email protected]

Chapter Outline Introduction Main Genotoxic Agents and Their DNA-Damaging Effects Physical Genotoxicants Chemical Genotoxicants Biological Genotoxicants Antigenotoxic Properties of Organosulfur Compounds Glucosinolates and Isothiocyanates

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224 224 225 228

228 229

Allium Organosulfur Compounds Antigenotoxic Properties of Terpenoids and Volatile Phenylpropanoids Monoterpenoids Volatile Phenylpropanoids Triterpenoids Antigenotoxic Properties of Polysaccharides Conclusion References

238

253 253 261 263 268 287 291

INTRODUCTION In recent decades human exposure to various genotoxicants has dramatically increased. Both endogenous (oxidative stress from pathological processes) and exogenous agents (ultraviolet light, ionizing radiation, air, water and food pollutants, smoking) can alter the structure and functions of DNA and other biomacromolecules through a series of molecular events, mainly triggered by major production of reactive oxygen species (ROS). The structural integrity of a DNA macromolecule can be adversely affected by the degradation of bases and sugars, breakage of the hydrogen and sugar-phosphate bonds, and cross-linkage induced by ROS. The predominant features of DNA injury Studies in Natural Products Chemistry, Vol. 60. https://doi.org/10.1016/B978-0-444-64181-6.00007-3 Copyright © 2018 Elsevier B.V. All rights reserved.

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include nucleotide base damage, single-strand breaks, DNA adducts, DNA– protein cross-linkages and double-strand breaks. If the capacity of cells to detect and repair DNA lesions is affected, continuous and cumulative DNA damage leads to genomic instability, cell dysfunction, mutation events, and cancer. Moreover, DNA damage can be associated with initiation and promotion of aging, infertility, and some human diseases (neurodegenerative and cardiovascular disorders, metabolic syndrome) [1]. There is increased interest in the compounds that might protect humans against genomic damage and its consequences [2,3]. These compounds are often included in the class of chemopreventive agents [4]. Numerous epidemiological and experimental studies have revealed the chemopreventive potential of many medicinal and dietary plants and mushrooms and their components. Various phenolic and nonphenolic phytochemicals have been reported to exert genoprotective and antimutagenic effects primarily related to their antioxidant properties and abilities to modulate DNA metabolism and repair carcinogen metabolism. This chapter focuses on the antigenotoxic and chemopreventive potential of some important dietary nonphenolic phytochemicals, such as organosulfur compounds, terpenoids, and polysaccharides. The antigenotoxic properties of these phytochemicals are discussed in terms of study type, mechanisms of activity, structure–activity relationships, and their clinical significance. In addition, the chapter presents the main genotoxic agents whose effects have been prevented or attenuated by nonphenolic phytochemicals.

MAIN GENOTOXIC AGENTS AND THEIR DNA-DAMAGING EFFECTS Genotoxic agents are classified into three main categories: physical (ultraviolet light, ionizing radiation, temperature), chemical (hydrogen peroxide, aromatic hydrocarbons, anticancer agents, heavy metals, asbestos, organic solvents, food additives, pesticides), and biological (mycotoxins, parasites, bacteria, viruses) [3]. Genotoxic events are triggered directly or indirectly and involve oxidative damage and structural modifications of DNA. Most of these agents require metabolic activation to exert their genotoxic properties. Several genotoxicants from each group are presented below. They have been used predominantly in studies related to the assessment of the genoprotective properties of phytochemicals discussed in this chapter.

Physical Genotoxicants Ultraviolet B (UVB) radiation is known to cause direct molecular lesions by generating dimeric DNA photoproducts (cyclobutane pyrimidine dimers and pyrimidine-6,4-pyrimidone photoproducts) and indirect cellular injuries by

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producing ROS; both effects lead to base alterations and oxidative DNA damage [5]. Gamma radiation is used for both diagnostic and therapeutic reasons. Gamma rays induce DNA damage by increasing the production of ROS and lipid peroxidation; ROS interact with deoxyribose and DNA bases leading to single- and double-strand breaks [1].

Chemical Genotoxicants Hydrogen peroxide (H2O2) is produced endogenously by several physiological and pathological processes [6]. It is responsible for more than 200 genotoxic effects [7]. H2O2 damages DNA both directly and indirectly (by generating hydroxyl radicals via a Fenton-type reaction) causing oxidative DNA damage that leads to DNA single-strand breaks, chromosomal aberrations, and gene mutations [1]. Polycyclic aromatic hydrocarbons (PAHs) like benzo[a]pyrene (B[a]P), which is one of the main PAHs used in genotoxic assays, are found in grilled foods, cigarette smoke, and automobile and industrial emissions [1]. It is a potent human carcinogen, an immunosuppressive, and a proinflammatory agent. Most of its effects have been linked to activation of the aryl hydrocarbon receptor (AhR). Thus, B[a]P becomes a substrate for induced phase I enzymes of the CYP450 1 superfamily (CYP1A1, CYP1B1). CYP1A1 metabolizes B[a]P to epoxide derivatives, such as benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), responsible for attacking DNA structures by forming BPDE–DNA adducts. Furthermore, BPDE metabolite is subjected to subsequent hydrolytic reactions catalyzed by phase II enzymes, such as UGT and GST, leading to its detoxification [8,9]. In addition, the generation of B[a]P intermediates by AhRdependent CYP1A1 upregulation may lead directly to the formation of ROS [10]. Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are also widely distributed environmental contaminants present in smaller quantities than PAHs, but still mutagenic and carcinogenic [11]. Styrene, a monocyclic aromatic hydrocarbon, is one of the most widely used industrial chemicals. It has recently been listed by IARC as a group 2B carcinogen. Under the influence of CYP450 isoenzymes, mostly 2E1 and 2F, it is converted to the genotoxic metabolite styrene-7,8-oxide (SO). The highly electrophilic molecule of SO can react with the nucleophilic residues of DNA, leading to DNA strand breaks and mutations [12]. N-Nitrosamines have been observed in a wide variety of foods and smoked tobacco. Their carcinogenic potential is significantly augmented after CYP450mediated generation of DNA metabolites responsible for DNA adduct formation; concomitantly, ROS are generated with subsequent oxidative DNA damage [1].

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Heterocyclic aromatic amines (HAAs), such as 2-amino-3-methylimidazo [4,5-f]quinolone (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), are produced when protein-rich products are heated. They are converted to highly reactive metabolites by the CYP450 1A superfamily (1A1, 1A2), N-acetyltransferases, and sulfotransferases. HAA intermediates can covalently bind to DNA-generating adducts and DNA strand breaks, chromosomal aberrations, and sister chromatid exchanges [1]. Heterocyclic aromatic nitroderivatives (HANs) can be exemplified by a well-known compound from this class, 4-nitroquinoline-1-oxide (4-NQO), which produces CYP-mediated metabolites that can bind covalently to DNA and produce ROS that can induce single DNA strand breaks, alkali-labile sites, and chromosomal aberrations [13,14]. Mercuric chloride (HgCl2) is an environmental pollutant that has been widely used in the past in agriculture as a fungicide and in medicine as an antiseptic and disinfectant. Inorganic mercury is mutagenic and genotoxic, inducing DNA single-strand breaks, chromosomal aberrations, and sister chromatid exchanges [15]. Asbestos is an occupational pollutant known to cause bronchial carcinoma and mesothelioma of the pleural and peritoneal cavity. Asbestos fibers can induce genotoxicity by generating ROS either via Fenton’s reaction catalyzed by the ferrous ion present in the composition of the fibers, or via oxidative bursts during phagocytosis of the fibers by pulmonary macrophages and neutrophils [16]. Anticancer agents, such as doxorubicin, vincristine, mitomycin C, and bleomycin, are chemotherapic agents currently used in cancer treatment, whereas urethane and methyl methanesulfonate are used mainly in experimental research. Doxorubicin (Adriamycin, DXR) is an anthracycline antibiotic originally isolated from Streptomyces peucetius, a soil bacterium. As a topoisomerase II inhibitor and a DNA intercalating agent, DXR induces double-strand DNA breaks and DXR–DNA adduct generation. The quinone structure of DXR can be easily oxidized via a process catalyzed by various NAD(P)H-oxidoreductases to a semiquinone radical that quickly reacts with molecular oxygen, leading to superoxide anion radicals and H2O2. Moreover, DXR is an iron chelator. DXR–iron complexes mediate the conversion of hydrogen peroxide to highly reactive hydroxyl radicals. Thus, DXR generation of free radicals may cause oxidative stress and impair DNA functionality [17]. Vincristine (VCR) is a dimeric alkaloid isolated from the periwinkle plant Catharanthus roseus. It is listed by the IARC as one of the 10 anticancer agents classified as group 1 carcinogens. The in vitro and in vivo genotoxicity of VCR has been widely reported, showing aneugenic and clastogenic properties [18,19].

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Mitomycin C (MMC) is a quinone–carbamate–aziridine antibiotic that becomes active after an enzymatic reduction process catalyzed by xanthine oxidase, xanthine dehydrogenase, DT-diaphorase, and other NADPH-CYP450 oxidoreductases [20]. After bioreductive transformation, MMC generates ROS (hydroxyl and superoxide anion radicals) and forms highly reactive electrophile intermediates (2,7-diaminomitosene, 1,2-cis-, and 1,2-trans-1-hydroxy-2,7diaminomitosene), which act as DNA alkylating and cross-linkage agents [14,21]. Thus, MMC has been recognized as a classical DNA-damaging agent, as a result of it inhibiting DNA synthesis, mutagenesis, and clastogenesis [22]. Bleomycin (BLM) is a glycopeptide-derived antitumor antibiotic that generates single- and double-strand DNA breaks and clustered DNA lesions. BLMinduced DNA damage involves free radical attack on DNA nucleotides and deoxyribose residues, a process that is ferrous ion and oxygen-dependent [23]. Methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) are direct alkylating agents that do not require biotransformation to produce potentially mutagenic and carcinogenic DNA adducts. MMS has been classified by the IARC as group 2A, whereas EMS are group 2B [24]. They both readily react with DNA-generating methylated and ethylated nucleotides; alkylation can occur at various locations on the nucleotide base, depending on the physicochemical properties of the agent [25]. In addition, they can indirectly produce ROS [13]. Cyclophosphamide (CPA) is an oxazaphosphorine bifunctional alkylating agent that belongs to the nitrogen mustard class. CPA is widely used at high doses in the chemotherapy of various forms of cancer (pulmonary, mammary, ovarian, lymphoma, leukemia) and in low concentrations for the treatment of autoimmune diseases and bone marrow transplantation. CPA is activated by CYP450, generating various metabolites, such as phosphoramide mustard and acrolein, which are linked to its antineoplastic and toxic side effects. CPA can generate free radicals and ROS and produce lethal mutations, micronuclei, and DNA damage [26,27]. Procarbazine (PCB) is an alkylating chemotherapeutic agent that undergoes bioactivation to azoxy derivatives via the CYP450 system; its toxicity is mostly related to DNA strand breakage and inhibition of DNA, RNA, and protein synthesis [28]. Urethane (ethyl carbamate, URE), a group of 2B human carcinogens, is a natural constituent of tobacco leaves and tobacco smoke. It is also generated during fermentation processes in bread, yogurt, and cheese. It undergoes bioactivation to vinyl carbamate, an intermediate capable of forming DNA adducts [29].

Other Chemical Genotoxic Agents Estradiol, a natural estrogenic hormone, and diethylstilbestrol (DES), a synthetic estrogen that mimics the metabolism of endogenous estrogens, are reported to induce structural chromosomal aberrations and sister chromatid exchanges.

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Estrogens can also lead to endogenous DNA adduct formation in both humans and animals by attacking the N3 position of adenine and N7 of guanidine. They can suffer CYP-mediated bioactivation to catechol estrogens that can be further oxidized in the presence or absence of metal ions to O-(semi)quinones, which can give rise to ROS responsible for cell damage [30,31].

Biological Genotoxicants Mycotoxins are secondary metabolites produced by fungi or molds. They contaminate different human and animal foodstuffs and present a considerable risk for human and animal health due to their hepatotoxic, nephrotoxic, immunosuppressive, carcinogenic, and mutagenic characteristics [32]. The main groups of mycotoxins of greatest interest are aflatoxins, ochratoxins, zearalenone, trichothecenes, and fumonisins [33]. Of these, the most commonly used in studies on the genoprotective potential of plant compounds are aflatoxin B1 and zearalenone. Aflatoxin B1 (AFB1) is a mycotoxin with a difuranocoumarin chemical structure produced by Aspergillus flavus and Aspergillus parasiticus spp. According to the IARC, it is included in group 1 human carcinogens, known to cause liver, lung, and colon cancer [1]. Their genotoxicity comes about as a result of both direct and indirect effects. In humans AFB1 undergoes biotransformation to AFB1-8,9-epoxide (AFBO), which irreversibly damages DNA by binding to N7-guanine residue. Metabolic activation is catalyzed by several CYP450 isoenzymes (3A4, 1A1, 1A2, 1B1, 2A13). In rats CYP2C11 and 3A2 have been reported to enhance this process. Nevertheless, this electrophilic metabolite can increase ROS with subsequent oxidative DNA damage. However, AFB1 CYP-mediated oxidation (1A, 2B, 3A) can also generate some nontoxic hydroxylated metabolites, such as aflatoxins M1 (AFM1), P1 (AFP1), and Q1 (AFQ1) [34]. It has been shown that GST-mediated conjugation with glutathione (GSH) is the main pathway [1,35] for AFBO detoxification. In addition, it has been suggested that AFB1 aldehyde reductase (AFAR) might also play an important role in decreasing AFB1 toxicity by preventing the binding of the dialdehydic form of the mycotoxin to intracellular proteins [34]. Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin produced by numerous Fusarium fungi species, such as F. culmorum, F. roseum, and F. graminearum [36,37]. Its genotoxicity is related to production of DNA adducts, sister chromatid exchanges, chromosome aberrations, and polyploidy [38].

ANTIGENOTOXIC PROPERTIES OF ORGANOSULFUR COMPOUNDS Organosulfur compounds (OSCs) are widely distributed in the natural environment and in living organisms. The main plant OSCs include glucosinolates, isothiocyanates, and sulfur compounds from Allium spp.

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Glucosinolates and Isothiocyanates Glucosinolates (GLSs) comprise a large group of nonvolatile secondary metabolites that are widely spread in numerous vegetables of the Brassicaceae family, such as broccoli, cauliflower, cabbage, Brussels sprouts, mustard, cress, and radish [39,40]. They have a common core structure based on the b-D-thioglucoside group linked to a sulfonated aldoxime moiety and a variable-length sidechain derived from different aliphatic or aromatic amino acids [40,41]. After plant tissue damage during harvesting, processing, or chewing, GLSs are converted by endogenous myrosinase enzymes to biologically active compounds named isothiocyanates, which contain a –N]C]S functional group [42,43]. Alongside GLSs, isothiocyanates (ITCs) are components of plant defense response to different agents, such as insects, herbivores, or microbial pathogens. They are largely responsible for the characteristic taste and flavor of cruciferous vegetables and various plants belongs to the Capparidaceae, Resedaceae, and Tropaeolaceae families [40,41]. ITCs have been associated with multiple potential health benefits in several chronic diseases, including cancer, cardiovascular pathologies, neurodegenerative diseases, and diabetes [43]. In the next few sections we review the antigenotoxic potential of some natural ITCs classified as thioalkyl ITCs (sulforaphane, erucin, erysolin, 3-methylthiopropyl-1-isothiocyanate, 5-methylthiopentyl-1-isothiocyanate), thioalkenyl ITCs (4-methylthio-3butenyl-1-isothiocyanate), alkenyl ITCs (allyl isothiocyanate), and aromatic ITCs (benzyl isothiocyanate, phenethyl isothiocyanate) (Table 7.1).

Thioalkyl ITCs Sulforaphane (4-methylsulfinylbutyl-1-isothiocyanate, SFN) (Fig. 7.1) is a thioalkyl ITC generated from the GLS glucoraphanin, which is present in high amounts in broccoli. As a dietary phytochemical with low toxicity, SFN is widely consumed and has been qualified as a food, dietary supplement, or drug, depending on its intended use [54]. SFN is known to have a variety of beneficial effects, including antioxidant [39], antiinflammatory [55], and anticancer [56]. In addition, recent reports have suggested that SFN might also possess cardioprotective and vasculoprotective [39,57], neuroprotective [58], antidepressant and anxiolytic [54], antiobesity [59], and antithrombotic [60] properties. The antigenotoxic potential of SFN has also been investigated. SFN reduced the DNA damage induced by several genotoxic agents, such as H2O2, gamma radiation, PAHs (B[a]P), nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) (1,6-DNP), N-nitrosamines (NDMA), HAAs (IQ, PhIP, MeIQx), HANs (4-NQO), and cancer chemotherapeutic agents (BLM, DXR, EMS, VCR, MMC, MMS, URE) in different experimental models and cell-based assays: human peripheral blood lymphocyte cells, human colon adenocarcinoma cells, human liver epithelial T5 cells, mouse hepatocytes, Drosophila melanogaster, or Salmonella typhimurium (Table 7.1). SFN exerted its antigenotoxic effects predominantly in pretreatment and/or cotreatment protocols [7,44,45].

TABLE 7.1 Antigenotoxic Potential of Isothiocyanates

Compound

Genotoxic Agent

Antigenotoxic Assay

Cell Lines/ Experimental Animals

Experimental Protocol

Outcome

References

SFN

H2O2

CBMN

HPBLC

H2O2 (3.4 mg mL
1, 30 min); SFN (1–10 mg , 30 min) pretreatment and cotreatment

Reduction of MN frequency

[44]

SMART

DM

H2O2 (0.12 M); SFN (1.575–12.6 mM) cotreatment

Reduction of total spot frequency

[6]

SCGE

LS-174

H2O2 (0.1 M, 5 min); SFN (5 mM, 24 h) pretreatment

Reduction of % DNA in tail

[45]

Gamma radiation

CBMN

HWBLC

0.25–2 Gy gamma radiation and PHA stimulation (2 h or 20 h); SFN (400 nM, 42 or 22 h) posttreatment

Reduction of MN frequency

[46]

B[a]P

SCGE

LS-174

B[a]P (25 mM, 24 h); SFN (5 mM, 24 h) pretreatment followed by cotreatment

Reduction of % DNA in tail

[45]

32

MCF-10F

B[a]P (0.3 mM, 12 h); SFN (0.1–2 mM, 7 days) pretreatment

Reduction of B[a]P-DNA adduct level

[11]

P-postlabeling

1,6-DNP

32

MCF-10F

1,6-DNP (10 mM, 22 h); SFN (0.1–2 mM, 7 days) pretreatment

Reduction of 1,6-DNP– DNA adduct level

[11]

NDMA

SCGE

T5-2E1

NDMA (0.1 mg mL
1, 1 h); SFN (0.1–10 mM, 1 h) cotreatment

Dose-dependent reduction of % DNA in tail and TM

[47]

Ames

ST (TA100)

NDMA (4.4 mg/plate); SFN (0.8–200 mM)

Dose-dependent reduction of revertant frequency

[48]

DNA repair assay

Mouse hepatocytes

NDMA (33.5 mM, 20 h); SFN (0.032–20 mM, 20 h) cotreatment

Dose-dependent reduction of nuclear grain counts

[48]

IQ

SCGE

T5-1A2

IQ (1 mg mL
1, 1 h); SFN (0.1–10 mM, 1 h) cotreatment

Reduction of % DNA in tail and TM

[37]

PhIP

14

Cpostlabeling

HepG2

PhIP (10 nM, 24 h); SFN (1–10 mM, 24 h) cotreatment

Dose-dependent reduction of PhIP–DNA adduct level

[49]

MeIQx

Ames

ST (YG1024 and TA98)

MeIQx (0.0002–0.002 nM); SFN (0.02 nM) in ST YG1024; MeIQx (0.02–0.2 nM); SFN (2 nM) in ST TA98

Reduction of revertant frequency

[50]

4-NQO

SMART

DM

4-NQO (2 mM); SFN (0.14–0.56 mM); STD

Reduction of small and total spot frequency at all tested concentrations and of twin spot frequency at 0.28 mM

[13]

P-postlabeling

Continued

TABLE 7.1 Antigenotoxic Potential of Isothiocyanates—Cont’d

Compound

Genotoxic Agent

Antigenotoxic Assay

Cell Lines/ Experimental Animals

Experimental Protocol
1

Outcome

References

BLM, DXR

CBMN

HWBLC

BLM (15 mg mL , 1 h) or DXR (25 mg mL
1, 1 h); SFN (400 nM; 22 or 42 h) posttreatment

Reduction of MN frequency

[46]

EMS, VCR

CBMN

HPBLC

EMS (120 mg mL
1, 24 h) or VCR (0.05 mg mL
1, 24 h); SFN (1–10 mg mL
1, 24 h) pretreatment, cotreatment, and posttreatment

Reduction of MN frequency

[44]

MMC

CBMN

HPBLC

MMC (0.1 mg mL
1, 24 h); SFN (1–10 mg mL
1, 24 h) cotreatment and posttreatment

Reduction of MN frequency

[44]

MMS

SMART

DM

MMS (0.5 mM); SFN (0.14–0.56 mM); STD and HB

Reduction of small, large, and total spot frequency in STD at all tested concentrations and of large and total spot frequency in HB at 0.56 mM

[13]

URE

SMART

DM

URE (20 mM); SFN (0.14 mM); HB

Reduction of small spot frequency

[13]

Erysolin

B[a]P

SCGE

HepG2

B[a]P (50 mM, 24 h); erysolin (1.25–2.5 mM, 24 h) pretreatment

Reduction of OTM

[10]

Erucin

B[a]P

SCGE, MN

HepG2

B[a]P (50 mM, 24 h); erucin (0.01–3 mM, 24 h) pretreatment

Reduction of OTM at 1 mM and of MN frequency at 0.3–3 mM

[10,51]

MTPITC, MTPnITC

B[a]P

SCGE, MN

HepG2

B[a]P (50 mM, 24 h); MTPITC (0.01–3 mM, 24 h) or MTPnITC (0.01–3 mM, 24 h) pretreatment

Reduction of OTM at 0.1–3 mM for MTPITC and 0.1–0.3 mM for MTPnITC; reduction of MN frequency at 0.01–1 mM for MTPITC and 0.1–1 mM for MTPnITC

[51]

MTBITC

ZEN

CBMN, CA

Balb/c mice spleen lymphocytes

ZEN (15 mM, 24 h); MTBITC (25 mM, 24 h) cotreatment

Reduction of MN and CA frequency

[38]

Balb/c mice

ZEN (40 mg kg
1; 10 days); MTBITC (5 mg kg
1; 10 days) cotreatment

Reduction of MN and CA frequency in bone marrow cells

ST (YG1024 and TA98)

MeIQx (0.0002–0.02 nM) and PEITC (0.02 nM) in ST YG1024; MeIQx (0.02–0.2 nM) and PEITC (2 nM) in ST TA98

Reduction of revertant frequency

AITC

MeIQx

Ames

[50]

Continued

TABLE 7.1 Antigenotoxic Potential of Isothiocyanates—Cont’d

Compound PEITC

BITC

Genotoxic Agent

Antigenotoxic Assay

Cell Lines/ Experimental Animals

Experimental Protocol
1

Outcome

References

NDMA

CBMN

HepG2

NDMA (2.2 mg mL ; 26 h); PETIC (0.25–1 mg mL
1, 26 h) cotreatment

Reduction of MN frequency

[52]

MeIQx

Ames

ST (YG1024 and TA98)

MeIQx (0.0002–0.002 nM) and PEITC (0.02 nM) in ST YG1024; MeIQx (0.02–0.2 nM) and PEITC (2 nM) in ST TA98

Reduction of revertant frequency

[50]

B[a]P

SCGE

HepG2

B[a]P (4 mg mL
1), BITC (0.8 mg mL
1)

Reduction of TM

[53]

IQ

SCGE

SD rats

IQ (90 mg kg
1); BITC (70 mg kg
1, 3 days)

Reduction of TM in liver and colon cells

[53]

PhIP

32

SD rats

PhIP (50 mg kg (70 mg kg
1)

Reduction of PhIP–DNA adduct level

[53]

P-postlabeling


1

); BITC

B[a]P, benzo[a]pyrene; BLM, bleomycin; CA, chromosomal aberrations; CBMN, cytokinesis-block micronucleus; DM, Drosophila melanogaster; 1,6-DNP, 1,6-dinitropyrene; DXR, doxorubicin; EMS, ethyl methanesulfonate; H2O2, hydrogen peroxide; HB, high bioactivation; HepG2, human hepatocellular liver carcinoma cells; HPBLC, human peripheral blood lymphocyte cells; HWBC, human whole blood lymphocytes; IQ, 2-amino-3-methylimidazo[4,5-f]quinolone; LS-174, human colon adenocarcinoma cells; MCF-10F, human normal breast epithelial cells; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; MMC, mitomycin C; MMS, methyl methanesulfonate; MN, micronuclei; MTBITC, 4-methylthio-3-butenyl-1-isothiocyanate; MTPITC, 3-methylthiopropyl-1-isothiocyanate; MTPnITC, 5-methylthiopentyl-1-isothiocyanate; NDMA, N-nitrosodimethylamine; 4-NQO, 4-nitroquinoline-1-oxide; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; SCGE, single-cell gel electrophoresis; SD, Sprague–Dawley; SFN, sulforaphane; SMART, somatic mutation and recombination test; ST, Salmonella typhimurium; STD, standard bioactivation; T5-1A2/2E1, T-antigen immortalized human liver epithelial cells expressing human CYP1A2/2E1; TM, tail moment; URE, urethane; VCR, vincristine; ZEN, zearalenone.

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FIG. 7.1 Chemical structure of main natural ITCs.

When gamma radiation was used as a genotoxic agent the protective effects of SFN were observed in posttreatment protocols, suggesting a possible beneficial intervention on DNA repair mechanisms [46]. The antigenotoxic activity of SFN seems to be based on its ability to: l

l

l

Indirectly protect against oxidative stress by inhibiting the phase I enzymes CYP450 1A1, 2B1, 2B2, 3A4, and 2E1 responsible for bioactivation of some chemical genotoxicants to highly reactive metabolites [47,48,61], and inducing the phase II enzymes glutathione transferase (GST), NAD(P)H quinone oxido-reductase 1 (NQO1), aldo-keto reductase (AKR), UDPglucuronyl transferase (UGT), glutathione reductase (GR), glutathione peroxidase (GPx), gamma-glutamylcysteine synthetase (GCS), and heme oxygenase 1 (HO1) [49,61]. Inhibit histone deacetylase (HDAC). It has been shown that SFN prolonged the H4 histone acetylation status that decreased after gamma irradiation of human peripheral blood lymphocyte cells; an increased amount of acetylated histones facilitates DNA double-strand break repair [46]. Enhance the apoptotic response. In cells exposed to genotoxicants the induction of apoptosis can be regarded as a selection process for surviving cells that may contain sublethal levels of DNA damage, which might proliferate and facilitate the acquisition of mutations in the genome [44].

Several less studied SFN derivatives (erysolin, erucin, 3-methylthiopropyl-1isothiocyanate, and 5-methylthiopentyl-1-isothiocyanate) have been reported to protect against the genotoxicity induced by B[a]P in HepG2 cells.

236 Studies in Natural Products Chemistry

Erysolin (4-methylsulfonylbutyl-1-isothiocyanate) (Fig. 7.1) is an ITC found in Eruca sativa (rocket), Erysimum allionii (Siberian wallflower), and other Brassicaceae plants. It is derived from the GLS glucoerysolin through the same enzymatic pathway as SFN [10,62]. Erucin (4-methylthiobutyl-1-isothiocyanate) (Fig. 7.1) is an ITC obtained enzymatically not only through hydrolysis of the GLS glucoerucin, but also through in vivo reduction of SFN, its structurally oxidized analog. Erucin was isolated for the first time from the seeds of E. sativa, which is found in high levels in rocket salad species [63]. Erysolin and erucin protected against B[a]P-induced DNA damage in HepG2 cells, but erysolin was active at higher concentrations (1.25–2.5 mM) than erucin (1 mM) [10]. The protective effects of erysolin might be correlated with the fact that it strongly decreased B[a]P-induced CYP1A1 protein expression. In contrast, although erucin did not alter the CYP1A1 level, it augmented the concentration of GST, a phase II enzyme involved in the detoxification of B[a]P. When the antigenotoxic activities of 3-methylthiopropyl-1-isothiocyanate (MTPITC) and 5-methylthiopentyl-1-isothiocyanate (MTPnITC) (Fig. 7.1) were investigated together with erucin, MTPITC offered the greatest protection as assessed by comet assay, followed by erucin, and then by MTPnITC. This order was not confirmed in a cytokinesis-blocked micronucleus (CBMN) assay, although the three compounds showed a significant reduction in MN frequency [51].

Thioalkenyl ITCs 4-Methylthio-3-butenyl-1-isothiocyanate (MTBITC) (Fig. 7.1) can be isolated from Raphanus sativus (radish) and possibly from other Brassicaceae vegetables. It is known to possess antibacterial and anticancer properties [64]. SalahAbbe`s et al. [38] showed that MTBITC has a strong protective effect against zearalenone-induced genotoxicity both in vivo (Balb/c mice) and in vitro (mice spleen lymphocyte cells). Suggested mechanisms for the antigenotoxic effects of MTBITC include direct and indirect antioxidant activity, modulation of phase I and phase II detoxification enzymes, induction of apoptosis, control of cell cycle and cell signaling, and antioxidant activity [38]. Alkenyl ITCs Allyl isothiocyanate (allyl senevol, allyl mustard oil, AITC) (Fig. 7.1), an ITC derived from the GLS sinigrin, is an oily colorless compound responsible for the pungent taste of mustard, radish, horseradish, and wasabi. It has been reported that AITC possesses antioxidant, antiinflammatory, hypoglycemic, antimicrobial, and anticancer effects [65,66]. Similar to SFN, AITC protected against MeIQx-induced mutagenicity in S. typhimurium strains (TA98 and YG1024). The antigenotoxic effects were observed when ITC was applied in excess (at a 1:10 or 1:100 molar ratio). Unlike SFN, AITC also manifested activity for a molar reagent ratio of 1:1, but only in the YG1024 strain [50].

Antigenotoxic Potential of Phytochemicals Chapter

7 237

Aromatic ITCs Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) (Fig. 7.1) are two naturally occurring ITCs, derived from gluconasturtin present in watercress and wasabi and glucotropaeolin present in red cabbage, respectively [45]. Several studies have managed to show the protective effects of these compounds against the genotoxicity induced by some genotoxic agents, such as B[a]P, IQ, and PhIP in the case of BITC [53] and NMDA [52] and MeIQx [50] in that of PEITC. Based on the aforementioned literature reports, several features affecting the antigenotoxic potential of ITCs can tentatively be highlighted: l

l

l

increase in the length of the alkyl chain results in a decrease in activity: MTPITC was more active than erucin, which was more active than MTPnITC [51]; oxidation of the sulfur atom from the alkyl chain might reduce antigenotoxic potency: erucin was more active than erysolin [10]; disposal of the sulfur atom from the alkyl chain can still maintain protective effects: AITC, BITC, and PEITC were still active in multiple assays; AITC was even more effective than SFN against the DNA damage induced by MeIQx [50].

Since DNA damage leads to gene mutations and chromosomal aberrations, genotoxic agents are often associated with cancer development, especially at the initiation phase when the normal (epi)genetic machinery is irreversibly altered. Moreover, it has also been pointed out that most of the previously described genotoxic agents are included by the IARC in group 2 (2A, 2B) of human carcinogens, based on their possible or probable involvement in carcinogenesis. With ITCs proving to be able to protect DNA against injury induced by various genotoxicants, they should be considered for use in cancer chemoprevention or chemotherapy. Discovering new antitumor molecules with improved toxicological profiles is a primary objective in anticancer drug development in the context that even modern agents, such as imatinib, can induce chromosomal aberrations and gene mutations [42]. However, it is hard to transpose these remarks into a clinical context, due to the fact that there are few available consistent data regarding the relationship between the intake of specific ITCs and outcomes in cancer management. In a phase II single-arm trial, SFNrich broccoli sprout extract (50 mmol/capsule, 4 capsules/day) was administered for 20 weeks to prostate adenocarcinoma patients with increased prostate specific antigen (PSA) values after prostatectomy or radiation. At the end of the study period only 3 patients had lower values of PSA than the baseline, while the other 17 subjects had higher PSA values than the baseline [67]. Despite this low efficacy of SFN, another trial conducted for a longer period of time (6 months) on a similar group (patients with biochemically recurrent prostate cancer after prostatectomy)

238 Studies in Natural Products Chemistry

but with a more vigorous design (a double-blind, randomized, placebo-controlled multicenter trial) showed that the administration of stabilized SFN from broccoli seeds (10 mg/tablet, 2 tablets  3/day) produced a significant reduction in mean PSA values from the baseline in the SFN-treated group vs the placebo group [68]. Nevertheless, all becomes clearer when taking into consideration the consumption of vegetables from the Brassicaceae family, which are rich in ITCs precursors. Many epidemiological studies have shown that dietary intake is correlated with a decreased risk of lung, pancreatic, colorectal, bladder, and prostate cancer. The explanation for such observations might reside in the well-known fact that plant extracts contain a high variety of phytochemicals that can act synergistically, potentiating a desired biological effect [50,61]. It is known that people consume daily about 100mg of ITCs containing around 77.2% (435mmol) SFN or even higher amounts of GLSs from Brassicaceae vegetables [44]. One hour after the intake of 200mmol (35.46 mg) SFN the peak concentrations in the plasma, serum, and erythrocytes of healthy volunteers were found to range between 0.94 and 2.27mM [42]. As described above, at a concentration of 0.4 mM, SFN protected human blood cells against the DNA damage induced by various genotoxic agents [46]. Thus we might conclude that a normal diet containing ITCs might confer significant protection against the genotoxicity of environmental, food, or drug xenobiotics. Nevertheless, clinical studies are needed to support these assumptions.

Allium Organosulfur Compounds The best known Allium spp., Allium sativum (garlic) and Allium cepa (onion), have been used since ancient times as spices and remedies to treat earache, deafness, diarrhea, constipation, respiratory disorders (chronic bronchitis, recurrent infections of the upper respiratory tract, influenza), parasitic infections, and tumors [69–71]. A number of significant bioactivities have been reported for these plants, such as antioxidant, antimicrobial, cardioprotective, vasculoprotective, and anticancer. Organosulfur compounds are the main bioactive components of Allium spp. They have a distinct flavor that is responsible for the pungency of Allium plants and their lachrymatory effect. Intact plants contain S-alk(en)yl-1-cysteine sulfoxides (alliin) and g-glutamylcysteine. The disruption of plant tissues (cutting, crushing, chopping, chewing) causes enzymatic hydrolysis of cysteine sulfoxides to volatile unstable thiosulfinates, such as allicin. Depending on the Allium sp. and the conditions, subsequent decomposition of thiosulfinates yields additional active organosulfur compounds, such as monoallylsulfides, diallylsulfides, and triallylsulfides, vinyldithiins, and ajoene [72–74]. Allicin (diallyl thiosulfinate) (Fig. 7.2) was isolated and identified for the first time in 1944 as the molecule responsible for the antibacterial activity of garlic oil [71]. Besides its antimicrobial properties, allicin is known to possess antioxidant, cardiovascular (antithrombotic, lipid-lowering, angiogenesisinhibiting, hypoglycemiant), and anticancer effects [75]. Further biotransformation of allicin yields diallyl sulfide (DAS) (Fig. 7.2), diallyl disulfide

Antigenotoxic Potential of Phytochemicals Chapter

7 239

FIG. 7.2 Chemical structure of Allium organosulfur compounds.

(DADS) (Fig. 7.2), diallyl trisulfide (DATS) (Fig. 7.2), dithiins, vinildithiins, and ajoene [71]. Allyl mercaptan (AM) (Fig. 7.2) has been identified as an in vivo metabolite of allicin and DADS, whereas S-allyl cysteine (SAC) (Fig. 7.2) is a hydrophilic compound found in high amounts in aged garlic extracts [35]. Dipropyl sulfide (DPS) (Fig. 7.2) and dipropyl disulfide (DPDS) (Fig. 7.2) are similar OSCs that are mainly found in onions. Conversely, we might also consider propyl mercaptan (PM) (Fig. 7.2) as a metabolite of DPS and DPDS [76]. The antigenotoxic potential of various Allium OSCs has been investigated against the DNA damage induced by H2O2, PAHs, mycotoxins, N-nitrosamines, HAAs, aromatic amines, alkylating compounds, and other agents (Table 7.2). DAS, DADS, AM, and SAC have shown protective effects against the genotoxicity induced by the direct-acting agent H2O2 in HepG2 cells, whereas allicin did not confer any protection. DAS and AM produced a significant reduction at lower concentrations (5 mM), as did SAC and DADS at higher doses (25 and 50 mM, respectively). The mechanisms underlying the antigenotoxic activity of these OSCs could be due to their antioxidant properties. OSCs have the ability to scavenge hydroxyl radicals and other free radicals, chelate ferrous ion thus reducing the conversion of H2O2 to highly reactive hydroxyl radicals via Fenton’s reaction, and increase the level of some antioxidant enzymes (namely, NQO1 and superoxide dismutase, SOD) [35]. Belloir et al. [35] showed that DAS, DADS, SAC, and AM, but not allicin, significantly decreased B[a]P-induced DNA damage in HepG2 cells, with DADS being the most efficient genoprotectant. The protective effects of DAS have been confirmed in animal-based studies, where it was found to reduce the frequency of chromosomal aberrations, micronuclei, and sister chromatid exchanges in mice bone marrow cells [78]. Moreover, DAS, DADS, DPS, DPDS, and SAC alleviated the mutagenicity altered by BPDE, the main active

TABLE 7.2 Antigenotoxic Potential of Allium Organosulfur Compounds

Compound

Genotoxic Agent

Antigenotoxic Assay

Cell Lines/ Experimental Animals

Allicin

AFB1

SCGE

MMS

DAS

Experimental Protocol

Outcome

References

HepG2

AFB1 (25 mM, 20 h); allicin (5–100 mM, 20 h) pretreatment

Reduction of OTM

[35]

SCE

HPBLC

MMS (60 mM, 48 h); allicin (1–20 mM, 48 h) cotreatment

Reduction of SCE frequency

[77]

SCGE

HepG2

MMS (75 mM, 4 h); allicin (5–100 mM, 4 h) cotreatment (4 h)

Reduction of OTM

[35]

17bEstradiol

CA, SCE

HPBLC

17b-Estradiol (20–40 mM, 48 h); allicin (5–15 mM, 48 h) cotreatment (48 h)

Reduction of CA and SCE frequency

[78]

H2O2

SCGE

HepG2

H2O2 (75 mM, 4 h); DAS (5–25 mM, 4 h) cotreatment

Reduction of OTM

[35]

B[a]P

CA, SCE, CBMN

Swiss albino mice

B[a]P (40 mg kg
1); DAS (40 and 60 mg kg
1, 5 days) pretreatment

Reduction of CA, MN, and SCE frequency

[78]

SCGE

HepG2

B[a]P (25 mM, 20 h); DAS (100 mM, 20 h) pretreatment

Reduction of OTM

[35]

Ames

ST TA100

B[a]P (20 mg/plate); DAS (40–80 mg/plate) cotreatment

Reduction of revertant frequency

[78]

BPDE

Ames

Wistar rats, ST TA100

BPDE (75–100 ng/plate); DAS (1 mmol kg
1 in Wistar rats, 4 days) pretreatment

Reduction of revertant frequency

[79]

DMBA

DAUA

Swiss albino mice

DMBA (5 mg kg
1, 23–95 h); DAS (5–10 mg kg
1, 1 h) pretreatment and posttreatment

Reduction of DNA strand breaks in mouse skin

[80]

SO

Ames

Wistar rats, ST TA100

SO (250–400 mg/plate); DAS (1 mmol kg
1 in Wistar rats, 4 days) pretreatment

Reduction of revertant frequency

[79]

AFB1

SCGE

HepG2

AFB1 (25 mM, 20 h); DAS (5–25 mM, 20 h) pretreatment

Reduction of OTM

[35]

SCGE

Wistar rats

AFB1 (2 mg kg
1); DAS (0.2%, 2 weeks) pretreatment

Reduction of elution constant

[81]

Ames

Wistar rats, ST TA100

AFB1 (25–100 ng/plate); DAS (0.2%, 2 weeks) pretreatment

Reduction of revertant frequency

[81]

Ames

Wistar rats, ST TA100

AFB1 (5–50 ng/plate); DAS (1 mmol kg
1, 4 days) pretreatment

Reduction of revertant frequency

[34]

Ames

Wistar rats, ST TA100

AFBO (5–30 ng/plate); DAS (1 mmol kg
1, 4 days) pretreatment

Reduction of revertants frequency

[34]

AFBO

Continued

TABLE 7.2 Antigenotoxic Potential of Allium Organosulfur Compounds—Cont’d

Compound

Genotoxic Agent

Antigenotoxic Assay

Cell Lines/ Experimental Animals

NDMA

SCGE

Experimental Protocol

Outcome

References

HepG2

NDMA (75 mM, 20 h); DAS (25 mM, 20 h) pretreatment

Reduction of OTM

[35]

SCGE

HepG2

NDMA (27 mM, 24 h); DAS (1–50 mM, 24 h) cotreatment

Dose-dependent reduction of OTM

[76]

SCGE

Wistar rats

NDMA (10 mg kg
1); DAS (0.2%, 2 weeks) pretreatment

Reduction of elution constant

[81]

Ames

Wistar rats, ST TA100

NDMA (1–5 mg/plate); DAS (0.2%, 2 weeks) pretreatment

Reduction of revertant frequency

[81]

NPYR

SCGE

HepG2

NPYR (5 mM, 24 h); DADS (1–5 mM, 24 h) cotreatment

Dose-dependent reduction of OTM

[76]

NPIP

SCGE

HepG2

NPIP (44 mM, 24 h); DAS (1–50 mM, 24 h) cotreatment

Reduction of OTM

[82]

NDBA

SCGE

HepG2

NDBA (3 mM, 24 h); DAS (50 mM, 24 h) cotreatment

Reduction of OTM

[82]

PhIP

SCGE

MCF-10A

PhIP (100 mM, 24 h) or N-OH PhIP (5 mM, 24–72 h); DAS (100 mM, 6 h) pretreatment

Reduction of OTM

[83]

DADS

2-AF

32

T24

2-AF (30–60 mM, 18 h); DAS (50 mM) cotreatment

Reduction of AF–DNA adduct level

[84]

MMS

SCGE

HepG2

MMS (75 mM, 4 h); DAS (5–25, 100 mM, 4 h) cotreatment

Reduction of OTM

[35]

CPA

CA, SCE, CBMN

Swiss albino mice

CPA (25 mg kg
1); DAS (40 and 60 mg kg
1, 5 days) pretreatment

Reduction of CA, MN, and SCE frequency

[78]

Ames

ST TA100

CPA (10 mg/plate); DAS (40–80 mg/plate) cotreatment

Reduction of revertant frequency

[78]

DES

SCGE

MCF-10A

DES (100 mM, 3 h); DAS (1–10 mM; 3 and 24 h) cotreatment

Reduction of OTM

[16]

Asbestos

CMBN

HMC

Asbestos (1 mg cm
2, 72 h), DAS (5–10 mM, 72 h) cotreatment

Reduction of MN frequency

[16]

H2O2

SCGE

HepG2

H2O2 (75 mM, 4 h); DADS (50–100 mM, 4 h) cotreatment

Reduction of OTM

[35]

B[a]P

SCGE

HepG2

B[a]P (25 mM, 20 h); DADS (25–100 mM, 20 h) pretreatment

Reduction of OTM

[35]

Ames

ST (TA98 and TA100)

B[a]P (5 mg/plate); DADS (2–10 mg/plate)

Dose-dependent reduction of revertant frequency

[85]

P-postlabeling

Continued

TABLE 7.2 Antigenotoxic Potential of Allium Organosulfur Compounds—Cont’d

Compound

Genotoxic Agent

Antigenotoxic Assay

BPDE

Ames

SO

AFB1

AFBO

Cell Lines/ Experimental Animals

Experimental Protocol

Outcome

References

Wistar rats, ST TA100

BPDE (25–100 ng/plate); DADS (1 mmoL kg
1 in Wistar rats, 4 days) pretreatment

Reduction of revertant frequency

[79]

Ames

Wistar rats ST TA100

SO (100–400 mg/plate); DADS (1 mmoL kg
1 in Wistar rats, 4 days) pretreatment

Reduction of revertant frequency

[79]

SCGE

HepG2

AFB1 (25 mM, 20 h); DADS (5–100 mM, 20 h) pretreatment

Reduction of OTM

[35]

SCGE

Wistar rats

AFB1 (2 mg kg
1); DADS (0.2%, 2 weeks) pretreatment

Reduction of elution constant

[81]

Ames

Wistar rats, ST TA100

AFB1 (25–100 ng/plate); DADS (0.2%, 2 weeks) pretreatment

Reduction of revertant frequency

[81]

Ames

Wistar rats, ST TA100

AFBO (5–30 ng/plate); DADS (1 mmol kg
1, 4 days) pretreatment

Reduction of revertant frequency

[34]

NDMA

SCGE

HepG2

NDMA (75 mM, 20 h); DADS (5 mM, 20 h) pretreatment

Reduction of OTM

[35]

SCGE

HepG2

NDMA (27 mM, 24 h); DADS (1–50 mM, 24 h) cotreatment

Dose dependent reduction of OTM

[76]

SCGE

Wistar rats

NDMA (10 mg kg
1); DADS (0.2%, 2 weeks) pretreatment

Reduction of elution constant

[81]

Ames

Wistar rats, ST TA100

NDMA (1–5 mg/plate); DADS (0.2%, 2 weeks) pretreatment

Reduction of revertant frequency

[81]

NPYR

SCGE

HepG2

NPYR (5 mM, 24 h); DADS (1–5 mM, 24 h) cotreatment

Dose-dependent reduction of OTM

[76]

NPIP

SCGE

HepG2

NPIP (44 mM, 24 h); DADS (0.1–2.5 mM, 24 h) cotreatment

Dose-dependent reduction of OTM

[82]

NDBA

SCGE

HepG2

NDBA (3 mM, 24 h); DADS (0.1–2.5 mM, 24 h) cotreatment

Reduction of OTM

[82]

2-AF

32

T24

2-AF (30–60 mM, 18 h); DADS (50 mM) cotreatment

Reduction of AF–DNA adduct level

[84]

4-NQO

Ames

ST (TA98 and TA100)

4-NQO (0.5 mg/plate); DADS (2–10 mg/plate)

Dose-dependent reduction of revertant frequency

[85]

P-postlabeling

Continued

TABLE 7.2 Antigenotoxic Potential of Allium Organosulfur Compounds—Cont’d

Compound

Genotoxic Agent

Antigenotoxic Assay

Cell Lines/ Experimental Animals

Experimental Protocol

Outcome

References

Ames

Wistar rats, ST TA100

4-NQO (100–500 ng/plate); DADS (1 mmoL kg
1 in Wistar rats, 4 days) pretreatment

Reduction of revertant frequency

[79]

MMS

SCGE

HepG2

MMS (75 mM, 4 h); DADS (25–100 mM, 4 h) cotreatment

Reduction of OTM

[35]

HgCl2

CA

Albino rats

HgCl2 (20 mg kg
1, 3 weeks); DADS (80 mg kg
1, 3  week, 3 weeks) pretreatment and posttreatment

Reduction of CA frequency in mice bone marrow cells

[15]

DATS

B[a]P

SCGE

MCF-10A

B[a]P (1 mM, 3–24 h); DATS (6 and 60 mM, 4 h) pretreatment and cotreatment

Reduction of OTM

[86]

DPS, DPDS

BPDE, SO, 4-NQO

Ames

Wistar rats, ST TA100

BPDE (75–100 ng/plate) or SO (100–400 mg/plate) or 4-NQO (100–500 ng/plate); DPS or DPDS (1 mmol kg
1 in Wistar rats, 4 days) pretreatment

Reduction of revertant frequency

[79]

NDMA

SCGE

HepG2

NDMA (27 mM, 24 h); DPS or DPDS (1–50 mM, 24 h) cotreatment

Dose-dependent reduction of OTM

[76]

NPYR

SCGE

HepG2

NPYR (5 mM, 24 h); DPS or DPDS (1–5 mM, 24 h) cotreatment

Dose-dependent reduction of OTM

[76]

NPIP

SCGE

HepG2

NPIP (44 mM, 24 h); DPS (1–10 mM, 24 h) or DPDS (0.1–2.5 mM, 24 h) cotreatment

Reduction of OTM

[82]

NDBA

SCGE

HepG2

NDBA (3 mM, 24 h); DPS (1–10 mM, 24 h) or DPDS (0.1–2.5 mM, 24 h) cotreatment

Reduction of OTM

[82]

DPDS, DMDS

B[a]P, 4-NQO

Ames

ST (TA98 and TA100)

B[a]P (5 mg/plate) or 4-NQO (0.5 mg/plate); DMDS or DPDS (2–10 mg/plate)

Dose-dependent reduction of revertant frequency

[85]

AM

H2O2

SCGE

HepG2

H2O2 (75 mM, 4 h); AM (5–100 mM, 4 h) cotreatment

Reduction of OTM

[35]

B[a]P

SCGE

HepG2

B[a]P (25 mM, 20 h); AM (5–100 mM, 20 h) pretreatment

Reduction of OTM

[35]

Ames

ST (TA98 and TA100)

B[a]P (5 mg/plate); DADS (2–10 mg/plate)

Dose-dependent reduction of revertant frequency

[85]

Continued

TABLE 7.2 Antigenotoxic Potential of Allium Organosulfur Compounds—Cont’d

Compound

Genotoxic Agent

Antigenotoxic Assay

Cell Lines/ Experimental Animals

AFB1

SCGE

NDMA

Experimental Protocol

Outcome

References

HepG2

AFB1 (25 mM, 20 h); AM (100 mM, 20 h) pretreatment

Reduction of OTM

[35]

SCGE

Wistar rats

AFB1 (2 mg kg
1); AM (0.2%, 2 weeks) pretreatment

Reduction of elution constant

[81]

Ames

Wistar rats, ST TA100

AFB1 (25–100 ng/plate); AM (0.2%, 2 weeks) pretreatment

Reduction of revertant frequency

[81]

SCGE

HepG2

AFB1 (25 mM, 20 h) or B[a]P (25 mM, 20 h) or NDMA (75 mM, 20 h); AM (5–100 mM, 20 h) pretreatment

Reduction of OTM at 100 mM for AFB1, 5 and 50–100 mM for BaP, and 25–100 mM for NDMA

[35]

SCGE

Wistar rats

NDMA (10 mg kg
1); AM (0.2%, 2 weeks) pretreatment

Reduction of elution constant

[81]

Ames

Wistar rats, ST TA100

NDMA (1–5 mg/plate); AM (0.2%, 2 weeks) pretreatment

Reduction of revertant frequency

[81]

MMS

SCGE

HepG2

MMS (75 mM, 4 h); AM (100 mM, 4 h) cotreatment

Reduction of OTM

[35]

PM

B[a]P, 4-NQO

Ames

ST (TA98 and TA100)

B[a]P (5 mg/plate) or 4-NQO (0.5 mg/plate); PM (2–10 mg/plate)

Dose-dependent reduction of revertant frequency

[85]

SAC

MMS, H2O2

SCGE

HepG2

MMS (75 mM, 4 h) or H2O2 (75 mM, 4 h); SAC (5–100 mM, 4 h) cotreatment

Reduction of OTM at 5–100 mM for MMS and 25–100 mM for H2O2

[35]

AFB1, B[a] P, NDMA

SCGE

HepG2

AFB1 (25 mM, 20 h) or B[a]P (25 mM, 20 h) or NDMA (75 mM, 20 h); SAC (5–100 mM, 20 h) pretreatment

Reduction of OTM at 5–100 mM for AFB1, 50–100 mM for B[a]P, and 25 and 100 mM for NDMA

[35]

BPDE

32

DNA from HPBLC

BPDE (0.1 nM, 2 h); SAC (1 nM, 2 h) cotreatment

Reduction of BPDE–DNA adduct level

[87]

DMBA

32

Rats

DMBA (25 mg kg
1, 24 h); SAC (0.1–1 mg kg
1, 2 weeks) pretreatment

Reduction of DMBA–DNA adduct level in mammary tissue

[88]

MNNG

CBMN, SCE

Swiss albino mice

MNNG (40 mg kg
1); SAC (100 mg kg
1, 5 days) pretreatment

Reduction of MN and SCE frequency

[89]

P-postlabeling P-postlabeling

2-AF, 2-aminofluorene; AFB1, aflatoxin B1; AFBO, AFB1-8,9-epoxide; AM, allyl mercaptan; B[a]P, benzo[a]pyrene; BPDE, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide; CBMN, cytokinesis-block micronucleus; CPA, cyclophosphamide; DADS, diallyl disulfide; DAS, diallyl sulfide; DATS, diallyl trisulfide; DES, diethylstilbestrol; DMBA, 7,12-dimethylbenz[a] anthracene; DMDS, dimethyl disulfide; DPDS, dipropyl disulfide; DPS, dipropyl sulfide; H2O2, hydrogen peroxide; HepG2, human hepatocellular liver carcinoma cells; HgCl2, mercuric chloride; HMC, human mesothelial cells; HPBLC, human peripheral blood lymphocytes; MCF-10A, human normal breast epithelial cells; MMS, methyl methanesulfonate; MN, micronuclei; MNNG, N-methyl-N0 -nitro-N-nitrosoguanidine; NDBA, N-nitrosodibutylamine; NDMA, N-nitrosodimethylamine; NPIP, N-nitrosopiperidine; NPYR, N-nitrosopyrrolidine; 4-NQO, 4-nitroquinoline-1-oxide; OTM, olive tail moment; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; PM, propyl mercaptan; SAC, S-allyl cysteine; SCE, sister chromatid exchange; SCGE, single-cell gel electrophoresis; SO, styrene oxide; T24, human bladder carcinoma cells.

250 Studies in Natural Products Chemistry

metabolite of B[a]P [79,87]. It was suggested that modulation of phase I and II enzymes is likely to be the main mechanism by which these OSCs exert their protective effects against the genotoxicity induced by B[a]P. It has been shown that various OSCs inhibited CYP1A1 activity in HepG2 cells, thus reducing the bioactivation of B[a]P, and induced the activity of GST, NQO1, and UGT, enzymes responsible for the detoxification of BPDE [35,79,85]. DAS and DADS have been shown to possess antimutagenic properties against the genotoxic metabolite of styrene (SO) in rat hepatic cytosols and microsomes, while DPS and DPDS only protected rat hepatic microsomes [79]. A plausible mechanism explaining the antigenotoxic capacity of Allium OSCs against SO-induced DNA damage has been correlated with the fact that they can induce the activity of epoxide hydrolase involved in the inactivation of SO. Nevertheless, all the other actions of OSCs (modulation of phase I and phase II enzymes) might also be responsible for these effects. Different authors have investigated the antigenotoxic potential of allicin, DAS, DADS, AM, and SAC against the DNA damage induced by AFB1 mycotoxin and its main metabolite AFBO in bacterial-, cell-, and animalbased assays. In the study of Belloir et al. [35], allicin, DAS, DADS, and SAC significantly reduced AFB1-induced OTM in HepG2 cells from 5 mM, whereas AM was only able to show protective effects at 100 mM. When DAS, DADS, and AM were administered orally to rats for 2 weeks the mutagenicity of AFB1 was decreased when assayed in the Ames test [81]. DAS and DADS also showed protection against the genotoxicity induced by AFBO metabolite when the OSCs were given for 4 days to rats [34]. Despite the fact that the mechanisms involved in AFB1 inhibition of genotoxicity were intensively investigated, they are still not fully elucidated. The data are contradictory regarding CYP activity: on the one hand, OSCs can increase 3A2 and 2B1/2 levels, thus enhancing the production of genotoxic AFBO metabolite but, on the other hand, they can reduce the expression of 2E1 or 3A4. It was also shown that DAS and DADS augmented the production not only of AFBO, but also of the nongenotoxic metabolites AFM1 and AFQ1. Generation of the latter metabolites might counterbalance increase in the AFBO level by competing for the same substrate (AFB1). Nevertheless, it has been suggested that the simultaneous induction of phase II detoxifying enzymes might be the key pathway to reducing the toxicity of AFB1 and AFBO. Indeed, DAS and DADS have been shown to be potent inducers of GST and AFAR1 in rats [34,35,81]. Allium OSCs have also been studied for their potential to protect against the genotoxic effects of various N-nitrosamines, such as NDMA, N-nitrosopyrrolidine (NPYR), N-nitrosopiperidine (NPIP), N-nitrosodibutylamine (NDBA), and N-methyl-N0 -nitro-N-nitrosoguanidine (MNNG) [35,76,81,82,89]. AM and SAC significantly inhibited NDMA-induced DNA damage, whereas DAS and DADS were found to have lower protective effects in HepG2 cells. Allicin showed no efficacy against NDMA toxicity [35]. In the study of Le Bon et al. [81],

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AM showed better antigenotoxic activity than DAS and DADS when the OSCs were orally administered in rats. DAS, DADS, DPS, and DPDS inhibited to a variable extent NDMA-, NPYR-, NPIP-, and NDBA-induced DNA damage in HepG2 cells [76,82]. In all experiments DADS and DPDS were more efficacious at lower concentrations than DAS and DPS. SAC, administered in mice, also showed protection against MNNG by reducing the frequency of micronuclei and sister chromatid exchanges [89]. One feasible mechanism by which Allium OSCs exert their protective effects against DNA damage induced by N-nitrosamines is that these compounds may interact with the CYP isoenzymes required for N-nitrosamine bioactivation. It has been shown that various OSCs inhibited CYP2E1, 2A6, and 1A1 that are deeply involved in N-nitrosamine metabolism. Moreover, stimulation of GSH level and of phase II enzymes (UGT, GST, NQO1, GPx, GR) might also contribute to the antigenotoxic potential of OSCs [76,82,89]. DAS protected MCF-10A cells against PhIP-induced DNA strand breaks [83]. As previously described, PhIP is oxidized by CYP1A2 to form N-hydroxy PhIP (NPhIP), which subsequently undergoes acetylation or sulfation to a nitrenium ion free radical that covalently binds to C8-guanine residue. The same group of researchers also showed that DAS has the potential to inhibit the DNA damage produced by NPhIP, the genotoxic metabolite of PhIP. To the best of our knowledge the mechanisms underlying DAS protection against the action of this HAA have not been investigated, but it is assumed that the modulation of phase I and phase II enzymes might be an important factor. Various authors studied the efficacy of OSCs against the genotoxicity induced by HAN 4-NQO in mixed bacterial- and animal-based models [79,85]. Therefore DADS, DPDS, DMDS, DPS, and PM decreased the mutagenicity of 4-NQO when assessed by the Ames test. The possible mechanisms explaining the protective effects might be correlated with the ability of these OSCs to reduce ROS level, increase intracellular GSH level, and inhibit phase I enzymes, such as CYP1A1, and induce phase II enzymes, such as GST. For instance, PM was the most efficient CYP1A1 inhibitor among DADS, DPDS, and DMDS. Allicin, DAS, DADS, AM, and SAC protected HepG2 cells against the DNA-damaging effects induced by alkylating agents, such as MMS. At 5 mM, allicin, DAS, and SAC prevented the genotoxic effects of MMS, whereas DADS and AM showed significant protective activity at 25 and 100 mM, respectively [35]. The preventive properties of allicin against the mutagenicity of MMS were confirmed in human peripheral blood lymphocytes [77]. It is assumed that OSCs may act as molecular interceptors of the electrophilic species (methyl groups) generated by MMS, thus blocking their attack on the nucleophilic sites of DNA [35,77]. In addition, DAS inhibited the genotoxicity of another alkylating agent, CPA. The proposed mechanisms for the protective effects involve scavenging of toxic and mutagenic electrophiles and free radicals and modulation of phase I and phase II enzymes [78].

252 Studies in Natural Products Chemistry

Allicin showed efficacy against DNA damage induced by 17b-estradiol, whereas DAS protected against DES toxicity. The protective effects of OSCs against estrogen-induced genotoxicity can be explained by their ability to prevent free radicals from reaching critical target sites. Indeed, it has been shown that DAS can reduce the intensity of lipid peroxidation induced by DES in MCF-10A cells [30,31]. When DADS was administered for 3 weeks in rats, it showed protection against the DNA damage induced by administration of HgCl2 both in pretreatment and posttreatment protocols. The inhibitory effects of DADS against HgCl2 genotoxicity may be related to the presence of the sulfhydryl group that may chelate HgCl2 before DNA attack. Moreover, the antioxidant potential of DADS may provide protection against free radical formation induced by HgCl2 [15]. The cotreatment of HMC cells with asbestos fibers and DAS yielded a significant reduction in micronuclei frequency. To the best of our knowledge the mechanisms underlying the protective effects of DAS against asbestos-induced genotoxicity have not been investigated, but it has been suggested that they could be attributed to the ability of OSCs to increase the level of GSH and GST activity [16]. To sum up, the antigenotoxic activity of Allium OSCs is based on: l

l l

l

direct antioxidant activity, such as free radical scavenging activity (hydroxyl radical) and ferrous ion chelating activity; indirect antioxidant activity, such as increase in GSH level and SOD activity; modulation of xenobiotic metabolism, such as inhibition of phase I enzymes (CYP 1A1, 2A6, 2E1, 3A4) and phase II enzymes (GST, UGT, NQO1, EH, AFAR, GPx, GR); direct scavenging activity of the electrophilic metabolites of genotoxic agents.

Based on the aforementioned literature reports, we can tentatively point up some structure–activity relationships regarding the antigenotoxic potential of Allium OSCs: l l

l

l

the presence of the sulfhydryl group is essential for DNA-protective effects; the number of sulfur atoms in the OSC structure is an important feature since it was shown that disulfides (DADS, DPDS) were more active than monosulfides (DAS, DPS). There are no literature data to support the belief that trisulfides would be more or less active than the previous ones [76]; oxidation of one of the sulfur atoms maintains the activity, but diminishes the efficacy: allicin was several times less active or even inactive in different antigenotoxic assays compared with other OSCs [35]; the nature of alk(en)yl side chains can also affect the activity, but the data are inconsistent. In some studies, allyl derivatives (DAS, DADS) were more active than propyl derivatives (DPS, DPDS) [79], whereas in others the situation looked totally different [82].

Cancer chemoprevention and chemotherapy are among the leading potential clinical applications of the previously described Allium OSCs in relation to their DNA-protective efficacy. Numerous epidemiological studies have shown

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an inverse association between increased garlic consumption and risk of stomach and colorectal cancer [90]. Moreover, many garlic constituents have been proven to act as promising anticancer agents in preclinical experiments, but they seem to be unsuitable for clinical application due to chemical stability issues [91]. Indeed, to the best of our knowledge, no clinical trials have been conducted on the efficacy of specific Allium OSCs in cancer management. Until further human investigations have taken place, their application as antigenotoxic drugs in cancer chemoprevention or chemotherapy thus remains uncertain.

ANTIGENOTOXIC PROPERTIES OF TERPENOIDS AND VOLATILE PHENYLPROPANOIDS Terpenoids are the largest group of natural substances biosynthetically derived from isoprene units (C5H8). They are secondary metabolites produced by plants for protection against herbivores, infections, and parasites and for pollination purposes; they are mostly found in fruit, vegetables, and aromatic and medicinal plants [92]. Terpenoids are endowed with various biological activities, such as antimicrobial, antiinflammatory, antinociceptive, antifoaming and carminative, and hepatoprotective. They are therefore used to treat different health disorders [93–95]. It has been shown that terpenes possess significant chemopreventive and antitumor effects, with numerous studies indicating both the protective capacity of terpenes against endogenous/ environmental sources of genomic stress and their putative use in the prevention of mutation-related diseases [94,96]. In this section we will give an overview of the antimutagenic properties of two major classes of terpenoids: monoterpenoids and triterpenoids. Monoterpenoids are compounds formed by two isoprene units and are generally known as the main constituents of essential oils [97]. Such compounds as carvacrol, limonene, eucalyptol, citral, and borneol, as well as volatile phenylpropanoids like anethole and eugenol will be discussed in this chapter regarding their antigenotoxic potential. Triterpenoids are derived biosynthetically from squalene, which consists of a 30-carbon backbone (6 isoprene units) [95]; this chapter will focus mainly on the antimutagenic properties of oleanolic and ursolic acids. The selection of these particular terpenoids was made on the basis of antigenotoxic effects shown in in vitro and in vivo experimental models and the existence of complementary studies aiming to elucidate their underlying mechanisms.

Monoterpenoids Numerous studies have reported on the antigenotoxic effects of essential oils [94]. Frequently, these properties have been ascribed to their major constituents, which exhibit their own antimutagenic activities [98–100]. Keeping this in mind, we now summarize the most relevant monoterpenoids that are known for their protective abilities against DNA damage induced by various genotoxicants (Table 7.3).

TABLE 7.3 Antigenotoxic Potential of Monoterpenoids and Volatile Phenylpropanoids Compound Carvacrol

Genotoxic Agent

Antigenotoxic Assay

Cell Lines/ Animals

Experimental Protocol
1

Outcome

References

Malathion

SCE

HPBLC

Malathion (30 mg mL , 72 h); carvacrol (2.5–5 mg mL
1, 72 h) cotreatment

Reduction of SCE frequency

[101]

H2O2

SCGE

HepG2

H2O2 (50–200 mM, 5 min); carvacrol (200 mM, 24 h) pretreatment

Reduction of % DNA in tail

[102]

D-

SCGE

Wistar rats

D-Galactosamine

(400 mg kg
1); carvacrol (20 mg kg
1, 6 days) posttreatment

Reduction of % DNA in tail, TL, TM, and OTM in blood lymphocytes and liver cells

[103]

SCGE

HepG2

H2O2 (250 mM, 5 min); carvacrol (25–200 mM, 24 h) pretreatment

Reduction of % DNA in tail

[104]

Caco-2

H2O2 (250 mM, 5 min); carvacrol (25–300 mM, 24 h) pretreatment

Reduction of % DNA in tail

[104]

K562

H2O2 (50–250 mM, 5 min); carvacrol (150 and 200 mM, 24 h) pretreatment

Reduction of % DNA in tail

[105]

HPBLC

MMC (1 mg mL
1, 24 h); carvacrol (0.1–5 mg mL
1, 24 h) cotreatment

Reduction of SCE frequency

[106]

Galactosamine H2O2

MMC

SCE

Anethole

Eugenol

Citral

Bupropion

CA

Swiss albino mice

Bupropion (0.4 mg, 14 days); anethole (0.2 mg/30 g, 14 days) cotreatment

Reduction of CA frequency in bone marrow cells and spermatocytes

[107]

PCB

MN assay

Swiss albino mice

PCB (50 mg kg
1 body weight); anethole (100–400 mg kg
1 body weight, 2 and 24 h) pretreatment

Reduction of MN frequency

[98]

URE

MN assay

Swiss albino mice

URE (400 mg kg
1 body weight); anethole (100–400 mg kg
1 body weight, 2 and 24 h) pretreatment

Reduction of MN frequency

[98]

DXR

SCGE

Murine peritoneal macrophages

DXR (34 mg mL
1, 6 h); eugenol (0.31–1.24 mg mL
1, 6 h) pretreatment, cotreatment, and posttreatment

Reduction of TI

[108]

TAA

SCGE

Wistar rats

TAA (300 mg kg
1, 2 days); eugenol (10.7 mg kg
1, 15 days) pretreatment

Reduction of % DNA in tail TL, TM, and OTM in blood cells

[109]

PCB

MN assay

Swiss albino mice

PCB (50 mg kg
1 body weight); eugenol (125–500 mg kg
1 body weight, 2 and 24 h) pretreatment

Reduction of MN frequency

[98]

URE

MN assay

Swiss albino mice

URE (400 mg kg
1 body weight); eugenol (125–500 mg kg
1 body weight, 2 and 24 h) pretreatment

Reduction of MN frequency

[98]

DXR

SCGE

Murine peritoneal macrophages

DXR (34 mg mL
1, 6 h); citral (50–100 mg mL
1, 6 h) pretreatment, cotreatment, and posttreatment

Reduction of TI

[108]

Continued

TABLE 7.3 Antigenotoxic Potential of Monoterpenoids and Volatile Phenylpropanoids—Cont’d Compound

Genotoxic Agent

Antigenotoxic Assay

Cell Lines/ Animals

Limonene

H2O2

SCGE

CBMN

Borneol

Experimental Protocol

Outcome

References

HPBLC

H2O2 (50 mM, 5 min); limonene (0.5–10 mM, 30 min) pretreatment

Reduction of TL, TM, and TI

[110]

V79

H2O2 (50 mM, 5 min); limonene (0.5–10 mM, 1 h) pretreatment

Reduction of TL, TM, and TI

[110]

HPBLC

H2O2 (50 mM, 24 h); limonene (0.5–10 mM, 24 h) cotreatment

Reduction of MN frequency

[110]

V79

H2O2 (50 mM, 18 h); limonene (0.5–10 mM, 18 h) cotreatment

Reduction of MN frequency

[110]

H2O2

SCGE

HepG2, VH10

H2O2 (50 mM, 5 min); borneol (0.5–10 mM, 24 h) pretreatment

Reduction of % DNA in tail

[111]

t-BHP

SCGE

HepG2

t-BHP (1 mM, 20 min); eucalyptol (0.01–1 mg mL
1, 20 h) pretreatment and cotreatment

Reduction of DNA damage

[112]

NC–NC

t-BHP (1 mM, 20 min); (0.01–1 mg mL
1, 20 min) cotreatment

Reduction of DNA damage

[112]

Vero

4-NQO (5 mM, 1 h); eucalyptol (0.05–10 mM, 20 h) pretreatment and posttreatment

Reduction of TM

[100,113]

4-NQO

SCGE

CA, chromosomal aberrations; CBMN, cytokinesis-block micronucleus; DXR, doxorubicin; H2O2, hydrogen peroxide; HepG2, human hepatocellular liver carcinoma cells; HPBLC, human peripheral blood lymphocytes; MN, micronuclei; NC–NC, human B lymphoblast cells; 4-NQO, 4-nitroquinoline-1-oxide; OTM, olive tail moment; PCB, procarbazine; SCE, sister chromatid exchange; SCGE, single-cell gel electrophoresis; TAA, thioacetamide; t-BHP, tert-butylhydroperoxide; TI, tail intensity; TL, tail length; TM, tail moment; URE, urethane; V79, Chinese hamster lung fibroblast cells; Vero, African green monkey kidney cells; VH10, human fibroblasts.

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7 257

FIG. 7.3 Chemical structure of genoprotective monoterpenoids and volatile phenylpropanoides.

Carvacrol Carvacrol (2-methyl-5-(1-methyl ethyl) phenol) (CVC) (Fig. 7.3) is a monoterpene phenol that occurs in various essential oils of Lamiaceae species, including Origanum, Thymus, Satureja, Thymbra, and Coridothymus [114]; the highest naturally occurring concentration of carvacrol (ca. 80%) has been found in Origanum vulgare. CVC has been shown to possess a wide range of biological activities, such as antibacterial, antifungal, insecticidal, antioxidant, and antitumor [94,114]. The antigenotoxic potential of CVC has also been investigated. In vitro studies revealed the ability of CVC to reduce the DNA damage induced by several genotoxic agents. Kumar et al. [101] studied the antigenotoxic potential of CVC against malathion-induced DNA damage, using sister chromatid exchange (SCE) as a biomarker of genotoxicity. Malathion (diethyl 2-[(dimethoxyphosphorothioyl) sulfanyl]butanedioate) is an organophosphate insecticide, extensively used in gardens and orchards to control various pests. Its mutagenic effects are related to increased SCEs and chromosome aberrations (CAs). CVC has significantly reduced SCE frequency induced by malathion in human peripheral blood lymphocytes [101]. The antioxidant properties of CVC might explain its antimutagenic activity against malathion, alongside the interaction of its own hydroxyl group with the cytoplasmic membrane. CVC thus exerts its protective activities through changes in membrane lipids and the permeability of ion channels,

258 Studies in Natural Products Chemistry

which inhibit mutagen uptake into cells [101,115]. In addition, CVC proved its protective effects by decreasing the SCE frequency induced by the cross-linking agent MMC in human peripheral blood lymphocytes [106]. The ability of CVC to protect lymphocytes against DNA damage might be due to its antioxidant effects; CVC acts as a strong free radical scavenger, which may explain its antimutagenic activity [116,117]. It has been shown that pretreatment with CVC mitigates DNA damage induced by H2O2 in several human cell-based models (K562, HepG2, and Caco-2 cells) [102,104,105]. This effect could be explained by an increase in the metabolic activity of mammalian cells during incubation with CVC, which is known as a bifunctional inducer of phase I and phase II enzymes. Moreover, the significant protection that CVC affords cells against H2O2-induced DNA strand breaks might also be down to its antioxidant activity [117,118]. The in vivo antimutagenic effects of CVC have also been demonstrated [103]. In male albino Wistar rats with D-galactosamine-induced hepatotoxicity, orally administered CVC counteracted mutagenic effects, as assayed by the comet test (with consequent reduction of % DNA in the tail, TL, TM, and OTM in both lymphocytes and liver cells). D-Galactosamine-induced toxicity is mainly due to the formation of hydroxyl radicals, which in turn determine lipid peroxidation and damage to DNA and the cell membrane. CVC posttreatment returned the values of enzymatic (SOD and detoxifying phase II enzymes like GPx) and nonenzymatic antioxidants (vitamin C, vitamin E, and GSH) toward normality. The study authors concluded that the in vivo antigenotoxic activity of CVC is based on its ability to induce detoxification mechanisms, suppress metabolic bioactivation, and protect against oxidative stress [103].

Limonene Limonene (p-mentha-1,8-diene) (LMN) (Fig. 7.3) is a component found in nearly every essential oil isolated from a number of Citrus species; the highest amount (up to 97%) is found in sweet orange (Citrus sinensis syn. Citrus aurantium var. sinensis) essential oil [97]. It is commonly used as a flavoring agent in the perfume, food, and pharmaceutical industries, being generally recognized as safe (GRAS) by the joint FAO/WHO Committee on Food Additives and the FDA. LMN is endowed with antibacterial, antioxidant, and antiproliferative properties [94,97]. Bacanli et al. [110] have shown that LMN exhibited significant antigenotoxic properties when used in an in vitro experimental design (human peripheral blood lymphocyte cells and Chinese hamster fibroblast V79 cells). Two assays were used to determine the protective effects of LMN against H2O2induced oxidative damage in cultured cells. A MN assay detects clastogenicity (chromosome breakage) and aneugenicity (chromosome lagging due to dysfunction of mitotic apparatus), whereas comet assay detects primary

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DNA damage. Even though these methods differ in terms of sensitivity, endpoints, and type of data, their combination significantly improves the capacity to detect genotoxicity. H2O2 induced chromosome breakage and loss and DNA damage in human lymphocytes and in mammalian fibroblasts. LMN not only exhibited antioxidant properties and protective effects against DNA damage (with a reduction in TL, TM, and tail intensity as determined by comet assay), but also increased DNA repair capacity (with a reduction in micronuclei frequency as shown by a MN assay). According to the study authors these protective effects have been more efficient in human lymphocytes than in Chinese hamster fibroblast (V79) cells; this outcome could be due to differences in the monoterpene absorption rates in the two cell lines. Ferna´ndez-Bedmar et al. [119] have shown that LMN exhibited genotoxic effects in the Drosophila wing spot test at concentrations of 0.73 mM, while low concentrations of LMN (0.011 mM) protected against H2O2-induced genotoxicity. The activity of LMN seems to be closely related to the concentrations; at relatively high concentrations LMN may act as a prooxidant agent [119].

Eucalyptol Eucalyptol (1,8-cineole) (EUC) (Fig. 7.3) is a monoterpene cyclic ether found abundantly in nature. It is the major component of an essential oil isolated from Eucalyptus globulus leaves (up to 80%), but it can be also found in other species such as rosemary (Rosmarinus officinalis), sage (Salvia officinalis), and mint (Mentha ssp.) [120]. The pharmacological effects of EUC have been well documented; it is used in the treatment of respiratory tract diseases due to its antimicrobial, mucolytic, broncholytic, and antiinflammatory properties [97]. The antigenotoxic potential of EUC has also been investigated, mostly in cell-based assays against DNA damage induced by several agents. For example, Miti
c-Culafi
c et al. [112] reported that EUC is able to reduce the percentage of DNA damage when hepatoma HepG2 and lymphoma NC–NC cell lines are pretreated and cotreated with tert-butylhydroperoxide (t-BHP). t-BHP is an organic hydroperoxide that exerts mutagenic effects through iron-dependent decomposition to more reactive species, such as hydroxyl, alkoxyl, and peroxyl radicals, generating in this way alkaline-labile sites and single-strand breaks [1,121]. According to the study authors, EUC acted through a number of different mechanisms related mostly to the treatment protocol. In the case of cotreatment the protective activity was therefore mostly due to the capacity of EUC to scavenge ROS generated by t-BHP, while pretreatment with EUC induced enzymatic and nonenzymatic cellular antioxidants and metabolic phase II detoxifying enzymes [112]. At low concentrations (0.05–10 mM), EUC reduced tail intensity and TM via comet assay of Vero cells incubated for 1 h with 4-NQO [100,113]. Pretreatment and posttreatment with EUC stimulated DNA repair processes, probably mediated by

260 Studies in Natural Products Chemistry

NER via the cytoprotective transcription factors Nrf1 and/or Nrf2, both essential for the expression of phase II detoxifying and antioxidant genes [122]. EUC thus induced the repair of 4-NQO–DNA adducts. Some essential oils that contain EUC as the main compound, such as Ravensara aromatica essential oil (56.45% EUC), also showed antigenotoxic activity against urethane in the Drosophila wing spot test. The protective mechanism probably involves reducing urethane activation via CYP450 enzymes [123]. In addition, no genotoxic effects were observed with other EUC-containing essential oils, such as Laurus nobilis (38.73% EUC) or R. officinalis (10.71% EUC), by SMART assay in D. melanogaster [124].

Citral Citral (3,7-dimethyl-2,6-octadienal) (CTR) (Fig. 7.3) is a monoterpene aldehyde that represents a mixture of cis-isomers and trans-isomers called geranial and neral, respectively. The major source of CTR (65%–85%) is an essential oil isolated from lemongrass (Cymbopogon citratus) leaves, but it can also be found in Citrus spp. fruits. CTR displays spasmolytic, antimicrobial, antiinflammatory, analgesic, and chemopreventive activities [94,120]. As far as antimutagenic activity is concerned, Porto et al. [108] have shown that CTR was able to reduce DXR-induced genotoxicity in murine peritoneal macrophages. CTR pretreatment, simultaneous treatment, and posttreatment has shown DNA protective effects and a reduction in tail intensity (as assayed by the comet method). Independently of the protocol, CTR presented antigenotoxic potential at concentrations in the range 25–100 mg mL
1 [108]. To the best of our knowledge the mechanisms by which CTR protects against the genotoxic effects of DXR have still not been fully elucidated. It seems that CTR antimutagenic effects might be explained by its antioxidant activity. The CTR mechanism could thus be similar to other structurally related monoterpenes (e.g., linalool, myrcene). The protective effects might be ascribed to the ability of CTR to act as a direct antioxidant agent by scavenging ROS generated by DXR; indirect antioxidant effects (induction of the enzymatic and nonenzymatic cellular antioxidants—GSH and SOD) and inducing phase II detoxifying enzymes might also be involved [112]. Borneol Borneol (1,7,7-trimethylbiciyclo[2.2.1]heptan-2-ol) (BRN) (Fig. 7.3) is a bicyclic monoterpene found in several Artemisia species and members of the Dipterocarpaceae family. BRN is used in Europe as a food additive (flavoring agent) [125], but it is also used as a traditional medicine in Japan and China due to its antibacterial, antiinflammatory, analgesic, and antithrombotic activities [94]. The antimutagenic effects of BRN have been investigated in vitro by Slamenova´ et al. [111]. The ability of borneol to modulate the resistance of two human cell lines (hepatoma cell HepG2 and fibroblast V10) against

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7 261

the DNA-damaging effects of H2O2 was assessed by comet assay. The level of H2O2-induced DNA strand breaks was significantly reduced by BRN in both cell lines. As BRN did not exhibit in vitro antioxidant activity the study authors concluded that its protective properties were not due to direct antioxidant effects, but might be related to stimulation of DNA repair mechanisms, since the HepG2 cell line possesses a significant number of metabolic enzymes (phase I and II and antioxidant enzymes) [111].

Volatile Phenylpropanoids Anethole Anethole (1-methoxy-4-propenyl-benzene, isoestragole) (ANT) (Fig. 7.3) is an alkoxypropenylbenzene derivative and an important bioactive component of the essential oils of more than 20 plant species. Essential oils from seeds of anise (Pimpinella anisum), star anise (Illicium verum), and fennel (Foeniculum vulgare) are the major sources used for the isolation of anethole [126]. ANT is an agent that is GRAS by the US FDA and the Flavors and Extracts Manufacturers Association [127]. ANT is considered nongenotoxic and noncarcinogenic. It is widely used as a flavor agent in the food, cosmetics, perfume, and pharmaceutical industries. Many studies in recent years have shown the beneficial effects of ANT for human health, such as antiinflammatory, anticarcinogenic and chemopreventive, antidiabetic, neuroprotective, and immunomodulatory [127,128]. To date, several in vivo studies have described the protective effects of ANT against DNA damage (Table 7.3). Abraham et al. reported the antigenotoxic potential of ANT against PCB and URE agents [98]. Pretreatment with low doses of ANT (2 and 24 h before administration of genotoxicants) translated into a reduction in MN frequency induced by both PCB and URE in mouse bone marrow cells. The chemoprevention mechanism of ANT seems to be able to enhance the metabolic detoxication process by inducing phase II enzymes, such as glutathione S-transferase, UDP-glucuronyl transferase, and DT-diaphorase [98,129]. Some mechanisms like ROS inactivation could be involved in alleviating DNA damage due to oxidative stress induced by mutagenic compounds [98]. ANT had significant repair influence on different mutagenic symptoms caused by bupropion hydrochloride in Swiss albino mice [107]. Bupropion hydrochloride is primarily used as an antidepressant and smoking cessation medication; it is bioactivated by the CYP450 isoenzyme CYP2B6 and its biological activity can be attributed to its active metabolites. It is reported to be a carcinogenic agent inducing such symptoms as undesirable cell division and DNA damage [130]. Simultaneous treatment with ANT (14 days) significantly inhibited the frequency of aberrant metaphases and CAs in mouse bone marrow cells induced by bupropion hydrochloride. Moreover, ANT brought about a significant reduction in abnormal sperm by reducing the

262 Studies in Natural Products Chemistry

amount of CAs in mouse spermatocytes. The mechanisms involved in DNA protection by ANT seem to be mostly related to the upregulation of enzymes involved in the detoxification of bupropion hydrochloride metabolites, and less related to mutagen bioactivation. It has been established that ANT preferentially induces hepatic phase II but not phase I biotransformation enzymes in the liver [129]. In addition, ANT might act as a chain-breaking antioxidant, inhibiting the binding of mutagenic products to DNA. As a result of its phenolic structure ANT may inactivate the metabolites of bupropion, donate electrons to reactive metabolites of the toxin, and render them inactive or may bind to DNA and prevent the reactive intermediates from interacting directly with DNA [107].

Eugenol Eugenol (4-allyl-2-methoxyphenol) (EUG) (Fig. 7.3) is the major biologically active component of an essential oil isolated from cloves (Syzygium aromaticum) (up to 90%). EUG is widely used as a flavoring agent in cosmetic, food, and pharmaceutical products [120]. The joint FAO/WHO Committee on Food Additives and the FDA have recognized EUG as a safe dietary agent, with no carcinogenic or mutagenic effects. EUG is endowed with a number of biological properties (such as antimicrobial, antioxidant, antiinflammatory, and analgesic) [131]. Recently, the antigenotoxic potential of EUG has been extensively investigated by in vitro and in vivo experimental models (Table 7.3). EUG has been shown to be able to reduce DXR-induced genotoxicity in mouse macrophages [108]. EUG pretreatment, simultaneous treatment, and posttreatment (6 h) has shown DNA-protective effects in murine peritoneal macrophages, with a reduction in tail intensity (as assayed by the comet method). Independently of the protocol, EUG showed antigenotoxic potential at low doses (0.31–1.24 mg mL
1) [108]. This activity might be explained by the phenoxyl radical mechanism: for example, as a result of its o-methoxyphenolic structure, EUG is transformed into phenoxyl radicals after interaction with ROS via donation of a phenolic hydrogen atom [132]. Yogalakshmi et al. [109] demonstrated the DNA-protective ability of eugenol in thioacetamide-administered rats. Biotransformation of thioacetamide (TAA) into its toxic metabolites sulfoxide and sulfone is mediated by CYP450 (namely, CYP2E1). Further, TAA metabolites exert hepatotoxic effects by binding to macromolecules and generating ROS, with subsequent damage to proteins, lipids, and DNA [133]. Pretreatment for 15 days with EUG attenuated genetic damage, with a reduction of TL, TM, OTM, and DNA in the tail (%) in rat blood cells. Inhibition of CYP2E1 isoenzymes (TAA genotoxicity is strongly dependent on this enzyme-mediated biotransformation) was the mechanism by which EUG protected against genotoxic effects induced by the mutagenic agent. Moreover, EUG induced phase II detoxification enzymes (UDP-glucuronyl transferase, DT-diaphorase, and glutathione-S-transferase)

Antigenotoxic Potential of Phytochemicals Chapter

7 263

[109]. Abraham et al. [98] reported the antigenotoxic potential of EUG against PCB- and URE-induced genotoxicity in mice. Pretreatment with low doses of EUG (125–500 mg kg
1 body weight) reduced MN frequency in mouse bone marrow cells [98]. This might be due to the antioxidant effects of EUG and its ability to induce enzymatic and nonenzymatic cellular antioxidants and phase II detoxifying enzymes [134,135]. Overall, the antimutagenic effects of monoterpenoids and phenylpropanoids may be due to: l l l

l

l

l

inhibition of mutagen penetration into cells; activation of cell enzymatic and nonenzymatic antioxidants; direct scavenging activity of mutagenic agents or inactivation of radicals produced by mutagens; inhibition of phase I metabolic enzymes involved in the bioactivation of genotoxic agents; activation of phase II metabolic enzymes responsible for the detoxification of mutagens; and interference with DNA repair systems.

Having reviewed the antimutagenic effects of several monoterpenoids, it is reasonable to conclude that such compounds are interesting candidates for the development of chemopreventive and anticancer agents. One of the aforementioned monoterpenes, limonene, was included in clinical trials to evaluate its antitumor potential. Limonene demonstrated partial response in a phase I clinical trial in patients with breast cancer that was followed up by a limited phase II evaluation [136]. Monoterpenoids are lipophilic compounds that have poor water solubility. This translates into low therapeutic potential in cancer therapy and restricts their use in other clinical applications. The low water solubility of monoterpenes could be overcome by including them in vectorized liposomes to ensure their efficacy [137]. Moreover, the putative clinical use of monoterpenes and corresponding essential oils should be kept in mind when considering the possible synergy that may occur between volatile compounds. In this section we pointed up several advantages associated with the use of essential oils, such as reduced genotoxicity, ability to act on multiple cellular targets, abundance of raw materials necessary for their extraction, and the low cost of production.

Triterpenoids Ursolic acid (3b-hydroxy-urs-12-en-28-oic acid) (Fig. 7.4) and its isomer oleanolic acid (3b-hydroxy-olea-12-en-28-oic acid) (Fig. 7.4) are two pentacyclic triterpenoids widely distributed in the plant kingdom in free form or as aglycons of triterpenoid saponins linked to one or more sugar residues [138]. Ursolic acid is found in apple peel, cranberries, bilberries, basil, rosemary, lavender,

264 Studies in Natural Products Chemistry

FIG. 7.4 Chemical structure of main antigenotoxic triterpenoids.

peppermint, and thyme, whereas oleanolic acid is especially prevalent in olive trees (leaves, fruit, oil), mistletoe sprouts, and clove flowers [139]. Having similar molecular structures (a change in position of one methyl group being the only difference), ursolic and oleanolic acids share many of their pharmacological properties, including hepatoprotective, gastroprotective, cardioprotective, vasculoprotective, neuroprotective, antiinflammatory, anti-HIV, antiobesity, immunomodulatory, and anticancer [140,141]. Their antigenotoxic potential was also investigated in cell- and animal-based assays against the DNA damage brought on by various agents (H2O2, t-BHP, zidovudine, DXR, carbon tetrachloride, UVB radiation) (Table 7.4). Ursolic acid and oleanolic acid (2.5–10 mM) reduced the percentage of DNA in the tail of rats when leukemic L1210, K562, and HL60 cell lines postincubated for 5 min with H2O2. Ursolic acid’s isomer demonstrated a significantly higher efficiency in the first two types of cells than ursolic acid itself, with no relevant difference between their potency in HL60 cells. Although the influence of different positions in the methyl group on the chemical structures of these triterpenoids has been investigated by different groups of researchers, there is still not enough information available to differentiate them from a concise pharmacological point of view [143]. Additionally, ursolic acid proved to possess antigenotoxic properties by reducing the oxidative DNA damage induced by H2O2 in Caco-2 cells or t-BHP in HepG2 cells and enhancing DNA repair. The DNArepairing activity of ursolic acid has been illustrated by its ability to diminish the level of 8-oxo-7,8-dihydroguanine induced by Ro 19-8022 photosensitizer together with visible light and alleviate DNA rejoining of strand breaks induced by the oxidative agents [121,142]. Zidovudine (azidothymidine, AZT) was the first nucleoside analogue to be approved for HIV-1/AIDS treatment. Despite its high clinical efficiency, it possesses a low safety profile, being currently included by IARC in group 2B carcinogens. AZT induces mutagenicity by getting incorporated into cellular DNA or generating DNA double-strand breaks. It has been shown that pretreatment of Caco-2 and HepG2 cells with ursolic acid decreased the percentage of DNA in the tail induced by AZT [144].

TABLE 7.4 Antigenotoxic Potential of Triterpenoids

Compound

Genotoxic Agent

Antigenotoxic Assay

Cell Lines/ Animals

Ursolic acid

H2O2

SCGE

Experimental Protocol

Outcome

References

Caco-2

H2O2 (75 mM, 5 min); ursolic acid (5–10 mM, 2 or 24 h) pretreatment or ursolic acid (5 mM, 24 h) pretreatment and 30 min recovery period

Reduction of % DNA in tail

[142]

L1210, K562, HL-60

H2O2 (100 mM, 5 min); ursolic acid (2.5–10 mM, 24 h) pretreatment

Reduction of % DNA in tail

[143]

t-BHP

SCGE

HepG2

t-BHP (200 mM, 1 h); ursolic acid (25 mM, 24 h) pretreatment or ursolic acid (12.5 mM, 24 h) pretreatment and 2 h recovery period

Reduction of DNA damage

[121]

AZT

SCGE

Caco-2, HepG2

AZT (3 mg mL
1, 3 h); ursolic acid (1 mM, 1 h) pretreatment

Reduction of % DNA in tail

[144]

UVB

SCGE

HPBLC

UVB (19.8 mJ cm
2); ursolic acid (1–10 mg mL
1, 30 min) pretreatment

Reduction of TL, TM, and % DNA in tail

[145]

Continued

TABLE 7.4 Antigenotoxic Potential of Triterpenoids—Cont’d

Compound

Oleanolic acid

Genotoxic Agent

Antigenotoxic Assay

Cell Lines/ Animals

Experimental Protocol
1

Outcome

References

DXR

CBMN

Balb/c mice

DXR (90 mg kg ); ursolic acid (80 mg kg
1) cotreatment

Reduction of MN frequency in peripheral blood and bone marrow cells

[146]

CCl4

8-OHdG

ICR mice

CCl4 (0.5 mL kg
1, 2  week, 6 weeks); UA (25–50 mg kg
1, 1  day, 6 weeks) cotreatment

Reduction of 8-OHdG level in kidney cells

[147]

H2O2

SCGE

L1210, K562, HL-60

H2O2 (100 mM, 5 min); oleanolic acid (2.5–10 mM, 24 h) pretreatment

Reduction of % DNA in tail

[143]

DXR

CBMN

Balb/c mice

DXR (90 mg kg
1); oleanolic acid (80 mg kg
1 cotreatment)

Reduction of MN frequency in peripheral blood and bone marrow cells

[146]

AZT, zidovudine; Caco-2, human intestinal cells; CBMN, cytokinesis-block micronucleus; CCl4, carbon tetrachloride; DXR, doxorubicin; H2O2, hydrogen peroxide; HepG2, human hepatocellular liver carcinoma cells; HL60, human promyelocytic leukemia cells; HPBLC, human peripheral blood lymphocytes; K562, human myelogenous leukemia cells; L1210, murine leukemia cells; MN, micronuclei; 8-OHdG, 8-hydroxy-2-deoxyguanosine; SCGE, single-cell gel electrophoresis; t-BHP, tertbutylhydroperoxide; TL, tail length; TM, tail moment; UVB, ultraviolet B.

Antigenotoxic Potential of Phytochemicals Chapter

7 267

Ursolic acid has also been shown to have protective effects against DNA damage induced by UVB radiation by significantly reducing the TL, TM, and percentage of DNA in the tail after pretreating human peripheral blood lymphocytes with doses of ursolic acid ranging from 1 to 10mg mL
1 [145]. In addition, ursolic acid and oleanolic acid have been found to exert in vivo antimutagenic effects. The two triterpenoids were able to mitigate the clastogenic effects of DXR by significantly decreasing the frequency of micronuclei in Balb/c mice peripheral blood and bone marrow cells as compared with the group treated with DXR alone [146]. Moreover, ursolic acid has been shown to mitigate the 8-hydroxy-2deoxyguanosine (8-OHdG) level in kidney cells brought about by treating mice with carbon tetrachloride. 8-OHdG is a commonly used marker of oxidative DNA damage resulting from the action of hydroxyl radicals on 20 -deoxyguanosine [147]. Carbon tetrachloride (CCl4) is a colorless, volatile, heavy, and nonflammable alkyl halide widely encountered in grain fumigants, rodenticides, fire extinguishers, refrigerants, and cleaning agents. In animal experimental research CCl4 is employed as a liver, kidney, heart, lung, testis, brain, and blood toxicity-inducing agent. Hepatic CYP450 converts it into trichloromethyl free radicals that can generate ROS, leading to DNA strand breaks, crosslinks, sister chromatid exchanges, and chromosomal aberrations [148,149]. The aforementioned DNA-protective effects of oleanolic acid and ursolic acid have been attributed to their direct and indirect antioxidant activity. They have been found to inhibit vitamin C/iron(II)-, DXR-, curcumin hydroperoxide-, and CCl4/NADPH-induced lipid peroxidation and scavenge DPPH, ABTS, nitric oxide, hydroxyl, and superoxide anion radicals, with outcomes similar to well-known antioxidants (butylated hydroxytoluene, vitamins C and E) [143,145,150,151]. Moreover, ursolic acid and oleanolic acid possess the ability to enhance the biosynthesis of glutathione (GSH) and increase the expression of Nrf2-mediated intracellular antioxidant enzymes, such as CAT, GPx, and SOD [141,152]. Since genotoxic substances may cause mutations that can potentially lead to cancer the protection conferred by ursolic acid and oleanolic acid against oxidative DNA damage would represent an important mechanism in their potential use as anticancer agents. Indeed, it has been shown that these triterpenoids can act at any stage of carcinogenesis, including not only the initiation, promotion, and progression phases, but also the late events of angiogenesis and metastasis [143]. Despite the fact that oleanolic acid is registered on the pharmaceutical market in China for liver disease, there are no clinical studies of either of these two triterpenoids concerning their chemopreventive or chemotherapeutic potential [153]. Moreover, since current cancer or HIV/AIDS agents pose a lot of limitations as a result of the occurrence of severe toxic effects at the cellular and molecular level (such as the DNA strand breaks induced by DXR and AZT), integration in the therapeutic scheme of substances able to prevent their genotoxicity might be another suitable clinical direction for oleanolic acid

268 Studies in Natural Products Chemistry

and ursolic acid, molecules known to possess intrinsic anticancer and antiHIV properties too. Therefore, cancer or HIV/AIDS management should not focus only on novel drug discovery, but also on reinventing well-known agents by including them in proper combinations with natural antioxidants that have been proven to mitigate the DNA damage induced by DXR and AZT. Clearly, consistent clinical investigations should be conducted before using them for such purposes.

ANTIGENOTOXIC PROPERTIES OF POLYSACCHARIDES Polysaccharides are major biomacromolecules in living organisms that play multiple roles in the structure, energy storage, cell recognition, and differentiation or regulation of cell signaling. They are extremely diverse in chemical structure and have a wide range of applications in many domains (food, cosmetics, medical devices, pharmaceuticals). The most important biomedical uses include tissue engineering, biosensors, enzyme immobilization, and vehicles for the controlled release of drugs [154,155]. Moreover, in recent decades there has been increased interest in the biological activity of polysaccharides, including their antigenotoxic and chemopreventive effects. In Japan, China, and Korea some glucans, such as lentinan from Shiitake mushroom, krestin from Coriolus spp. or Polyporus versicolor, and schizophyllan from the fungus Schizophyllum commune, are already clinically used as immunotherapeutic agents in cancer treatment mainly in combination with chemotherapy or radiotherapy [156]. The most studied polysaccharides are the b-D-glucans, mannans, and their water-soluble derivatives. b-D-Glucans consist of a linear central backbone of D-glucopyranosyl units linked in the b-(1 ! 3) position to D-glucose side chains via b-(1 ! 6) or b-(1 ! 4) linkages. The side chains are of various lengths and occur at different intervals along the main chain, generating a tertiary structure stabilized by interchain hydrogen bonds [2,157,158]. b-D-Glucans are generally found in the cell walls of plants (mainly cereals like oats and barley), fungi, bacteria, yeasts, algae, and lichens, where they ensure the rigidity and define the morphological features of cells [155,157]. The macrostructure of glucans depends on both the source and methods of extraction or purification [159]. Microbial b-glucans are composed of D-glucose molecules joined by glycosidic bonds mainly via b-(1 ! 3) and b-(1 ! 6) linkages to a main skeleton branched with regular (bacterial) or irregular (fungal) sidechains of glucose units and different linkages, such as b-(1 ! 3) or b-(1 ! 6). b-Glucans from cereals consist of b-Dglucopyranosyl units linked by (1 ! 3) and (1 ! 4) glycosidic bonds (Fig. 7.5) [155]. Yeast b-glucans have a structure comprising a b-(1 ! 3)-D-glucose-linked backbone with side chains of glucosyl units attached by b-(1 ! 6) linkages (Fig. 7.5) [160]. b-D-Glucans, primarily (1 ! 3) (1 ! 6)-b-D-polymers, are known as biological response modifier agents that have the ability to nonspecifically activate the

FIG. 7.5 Chemical structure of b-D-glucans from yeasts and barley.

270 Studies in Natural Products Chemistry

cellular and humoral components of the host immune system (macrophages, neutrophils, mononuclear cells) [161]. They exert various and significant activities that bring about immunomodulatory, antitumor, chemopreventive, antiviral, antiinfective, antioxidant, antimutagenic, wound-healing, and hematopoetic effects [4,162]. b-Glucans from cereals are classified as soluble dietary fibers and have been shown to demonstrate cholesterol-lowering properties and beneficial effects in the regulation of postprandial glucose levels [155]. Moreover, valuable biological properties have been shown for chemically modified b-glucans. Chemical derivatization improves the water solubility, bioactivity, and application of parent glucans. It is mainly achieved by carboxymethylation, sulfonylation, phosphorylation, and acetylation. Carboxymethylation is achieved using chloroacetic acid under alkaline conditions. Carboxymethylated polysaccharides possess antioxidant, antitumor, and immunomodulatory properties with potential applications in the pharmaceutical, food, and cosmetic industries. Sulfonylation implies the use of pyridine and chlorosulfonic acid, or pyridine and sulfur trioxide reagents. Sulfonated polysaccharides have important antiinflammatory, anticoagulant, and antithrombotic properties. The phosphorylation of glucans is performed using phosphorus acid in molten urea or a mixture of sodium trimetaphosphate and sodium tripolyphosphate reagents. It has been shown to improve antiinflammatory, antiviral, and antiproliferative effects compared with unmodified glucans. The introduction of acetyl groups in glucan molecules is achieved by using acetic anhydride reagents. Acetylated glucans have been shown to have great antioxidant potential. In addition, other kinds of derivatization, such as linkages with amino acids, peptides, proteins, lipids, phenolic substituents, or other sugar molecules, can trigger improvements in the absorption, bioavailability, and bioactivity of glucans and broadens the spectrum of biological activities and the potential for clinical application [155]. Many studies have highlighted the genoprotective and anticlastogenic effects of natural b-glucans, which are mainly found in Saccharomyces cerevisiae yeast, the mushroom Agaricus brasiliensis S. Wasser (syn. Agaricus blazei Murrill ss. Heinemann), and barley (Hordeum vulgare), and their derivatives obtained mainly by carboxymethylation or sulfonylation. Genoprotective potential has been demonstrated against various genotoxicants (gamma radiation, mycotoxins, H2O2, PHAs, heterocyclic aromatic derivatives, N-nitroso compounds, aromatic amines, and cancer chemotherapeutic agents), using a number of experimental animal and cell models, and protocols of administration (preincubation, postincubation, simultaneous, and simultaneous with preincubation) (Table 7.5). Yeast and mushroom b-glucans protect against radiation-induced DNA breakage. b-Glucan from S. cerevisiae and its carboxymethyl derivative (CM-G) enhance the survival of irradiated mice. b-Glucan and CM-G do so due to their hematopoietic activity and free radical scavenging properties. Moreover, the administration of CM-G accelerates hematopoietic recovery in CPA-treated mice, and b-glucan and lentinan protect mice against total-body

TABLE 7.5 Antigenotoxic Potential of Polysaccharides

Compound

Genotoxic Agent

Antigenotoxic Assay

Cell Lines/ Experimental Models

Experimental Protocol
1

Outcome

References

Barley b-glucan

MMS, 2-AmA

MN

CHO (k1), HTC

MMS (37.6 mg mL ; 3 h), 2-AmA (1 mg mL
1, HTC, 3 h); glucan (5, 10, 20 mg mL
1, 17 h); pretreatment, posttreatment, simple simultaneous treatment, and simultaneous treatment with preincubation (1 h)

Reduction of DNA damage; high efficiency for highest doses and simultaneous treatment

[163]

Barley b-glucan

B[a]P

SCGE

HepG2

B[a]P (10 mg mL
1); glucan (1, 5, 25 mg mL
1); pretreatment, posttreatment, simple simultaneous treatment (24 h), and simultaneous treatment with preincubation (1 h)

Reduction of DNA migration

[164]

Barley b-glucan

MMS, 2-AmA

CA

CHO-k1, HTC

MMS (37.6 mg mL
1); 2-AmA (1 mg mL
1); glucan (2.5, 5, 10 mg mL
1)

Reduction of metaphases with chromosomal aberration frequency

[165]

Continued

TABLE 7.5 Antigenotoxic Potential of Polysaccharides—Cont’d

Compound

Genotoxic Agent

Antigenotoxic Assay

Cell Lines/ Experimental Models

Experimental Protocol
1

Outcome

References

BOT

DXR, H2O2, B [a]P

SCGE, MN

V79, HTC

DXR (0.75 mg mL , 3 h), H2O2 (1.7 mg mL
1, 20 min), B[a]P (20 mg mL
1, 24 h); BOT (7.5, 30, 100 mg mL
1, 3 h V79; 24 h HTC); simultaneous treatment and posttreatment

Reduction of DNA damage

[166]

BOT

MMS

(a) SCGE

(a) Normal human lymphocytes, Jurkat;

(a) MMS (10 mM); BOT (7.5, 30, 100 mg mL
1); simultaneous treatment (4 h), posttreatment (20 h);

[161]

(b) ST (TA98, TA100, TA102, TA97)

(b) BOT (75, 150, 300, 450, 600 mg/plate)

Reduction in DNA damage in normal (posttreatment) and tumor lymphocytes (simultaneous and posttreatments);

V79

H2O2 (200 mM); visible light (60-W bulb, 60 s); MB (1 mL); glucan derivatives (150, 750, 1500 mg mL
1, pretreatment 24 h)

(b) Ames

CM-G, SE-G, CM-Ch-G

H2O2, visible light-excited MB

SCGE

absence of mutagenic effect Reduction of oxidative DNA damage

[162]

CM-Ch-G

H2O2, MMS, MNNG

SCGE

HepG2, HeLa

H2O2 (50, 75, 100, 200 mM, 5 min); MMS (0.4, 0.8, 1.6, 3.2, 6.4 mM, 30 min); MNNG (0.08, 0.16, 0.3, 1, 2 mg mL
1, 30 min); CM-CG (50, 150, 300 mg mL
1, pretreatment 24 h)

Reduction of H2O2induced oxidative DNA lesions;

[157]

DNA repair stimulating effect

Curdlane

CPA

MN

Mice

CPA (50 mg kg
1 body weight, intraperitoneal); curdlane (70, 150, 300 mg kg
1, gavage), simultaneous treatment

Reduction of DNA damage

[167]

EPS

4-NQO

SCGE

Intestine 407 cells

4-NQO (0.03 mg mL
1), EPS (10, 50 mg mL
1), simultaneous treatment (12 h)

Decrease of DNA migration

[168]

GL-b-glucan

Gamma radiation

CA

Mice

Radiation (4 Gy); glucan (500 mg kg
1 body weight), 5 min postirradiation

Decrease in the percentage of aberrant cells

[169]

AB-b-glucan

B[a]P

SCGE, CBMN

HepG2

B[a]P (20 mM); glucan (7, 21, 63 mg mL
1); pretreatment 24 h, simultaneous treatment 24 h, presimultaneous treatment 24 h

Reduction of DNA fragmentation and MN frequency

[170]

Continued

TABLE 7.5 Antigenotoxic Potential of Polysaccharides—Cont’d

Antigenotoxic Assay

Cell Lines/ Experimental Models

H2O2, BLM, DXR

SCGE

AB-b-glucan

Trp-P-2, BPDE, H2O2

SC-b-glucan

SC-b-glucan

Compound

Genotoxic Agent

AB-TP, AB-b-glucan

Experimental Protocol

Outcome

References

HepG2

H2O2 (100 mM), BLM (0.5 mg mL
1), DXR (1 mg mL
1); TP/glucans (5, 15, 45 mg mL
1); simultaneous treatment (3 h BLM, DXR; 5 min H2O2)

Reduction of DNA migration; ineffective protection of TP against DXR

[2]

SCGE

Human peripheral lymphocytes

Trp-P-2 (50 mmol L
1, 40 min), BPDE (0.4 mmol L
1, 10 min), H2O2 (50 mmol L
1, 10 min); glucan (20–80 mg L
1), simultaneous treatment

Decrease of H2O2-and TrpP-2-induced DNA migration

[170]

CPA

SCGE, MN (peripheral blood)

Mice

CPA (50 mg kg
1 body weight/week, intraperitoneally, 6 weeks); glucan (100, 150, 200 mg kg
1 body weight, intraperitoneally, 3 consecutive days/week, 6 weeks)

Effective reduction in mutagenic and genotoxic damage rate in the first week of treatment

[171]

CPA

MN (peripheral blood)

Mice (pregnant and nonpregnant females)

CPA (20 mg kg
1, intraperitoneally); glucan (150 mg kg
1 body weight, intraperitoneally)

Reduction in MN frequency

[172]

SC-b-glucan

MMS

SCGE, CBMN

CHO (k1, xrs5)

MMS (18.8, 37.6 mg mL
1– CBMN; 4.23 mg mL
1— SCGE; 3 h); glucan (5, 10, 20, 40 mg mL
1); pretreatment, posttreatment (17 h, CBMN), simple simultaneous, simultaneous after preincubation (1 h) (CBMN, SCGE)

Reduction in DNA damage; lowest efficiency in posttreatment protocol

[158]

SC-b-glucan

AFB1

SCGE

Broiler chicken lymphocytes

AFB1 (20 mg mL
1, 72 h); glucan (0.1%, 1%, 10%, 72 h)

Reduction in DNA damage for 1% glucan treatment

[173]

SE-G

MMS, ofloxacin, MH

(a) CR;

(a) CR (WT, uvs10);

(a) MMS (0.1%, 0.2%, 0.3%), SEG (0.1 mM), simultaneous treatment, 30 min;

(a) Increase of uvs10 survival;

[4]

(b) ofloxacin (250 mg mL
1), SEG (1, 10 mM), cotreatment 22 h;

(c, d) reduction in the frequency of chromosomal aberrations

(b) SC; (c) cytogenetic test; (d) anticlastogenity assay

(b) SC (D7); (c) Vicia faba seeds; (d) Vicia sativa seeds

(b) decrease of revertant and convertant number;

(c) MH (25, 50, 100 mM), SEG (0.1 mM), cotreatment, 2 h; (d) MH (25, 50, 100 mM), SEG (0.1 mM), treatment, 24 h

SE-Ch-G

Fe2+, H2O2

AGE

Plasmid pBR322 DNA

Fe2+ (10 mmol, 5 min), H2O2 (100 mmol, 30 min); SE-Ch-G (1 mg mL
1), simultaneous treatment

Inhibition of Fe2+-induced DNA breakage; increase of H2O2-induced DNA breakage

[174]

Continued

TABLE 7.5 Antigenotoxic Potential of Polysaccharides—Cont’d

Compound Glucomannan

Genotoxic Agent 9-AA, MMS, MNNG, 4-NQO, NMU, NaN3

Antigenotoxic Assay (a) Ames; (b) SC; (c) CR; (d) anticlastogenity assay

Cell Lines/ Experimental Models (a) ST (TA97, TA98, TA100, TA102); (b) SC (D7); (c) CR (WT; uvs10, uvs12, uvs14); (d) Vicia sativa seeds

Experimental Protocol

Outcome

References

(a) 9-AA (50 mg/plate), 4-NQO (0.2 mg/plate), MNNG (10 mg/plate), NaN3 (5 mg/plate), 72 h; glucomannan (250, 500, 750 mg/plate); 1 h pretreatment;

(a) Reduction of base substitution and frameshift mutation in TA97 and TA100 strains;

[175]

(b) NQO (4  10
6 mol L
1); glucomannan (2  10
6, 2  10
5 mol L
1), cotreatment, 22 h; (c) MMS (0.2%, 0.3%, 0.4%); glucomannan (10
4 mol L
1), simultaneous treatment, 30 min; (d) NMU (1  10
3, 1.5  10
3, 2  10
3 mol L
1); glucomannan (2  10
7 mol L
1), treatment 24 h

(b) weak antimutagenic effect, statistically insignificant; (c) increase of survival (uvs10, uvs14); (d) reduction in the frequency of chromosome aberrations

CU glucomannan

Ofloxacin, MH, BLM, Fe2 + , H2O2

(a) SC bioprotectivity assay;

(a) SC (W3031B; JDY-1);

(b) cytogenetic test;

(b) Vicia faba;

(c) DNA topology assay

(c) pBR322 DNA

(a) ofloxacin (700 mM); glucomannan (0.1, 0.2, 2 mM); cotreatment 22 h; (b) MH (25, 50, 100 mM); glucomannan (0.1 mM); cotreatment 2 h; (c) BLM (300 mM, 15 min), Fe2+ (10 mM, 15 min), H2O2 (0.03%, 30 min); glucomannan (1 mg mL
1, cotreatment)

(a) Increase of cell survival;

[176]

(b) reduction in frequency of CA; (c) suppression of Fe2+induced DNA nicking and enhancement of BLM- and H2O2-induced DNA damage

a-Mannan

AFB1

SCGE

Mouse hepatocytes

AFB1 (1 mg kg
1); mannan (100, 400, 700 mg kg
1, pretreatment)

Reduction in DNA damage at high doses

[177]

Mannan

AFB1

MN, SCE

Mice

AFB1 (0.25 mg kg
1 of diet); mannan (50, 250, 500 mg kg
1 of diet), 4 weeks treatment and 4 weeks as recovery period

Decrease of MN and SCE rates

[178]

Mannan– protein conjugates

Ofloxacin, AO

EG

EG Z (12245/90)

Ofloxacin (15 mg mL
1), AO (5 mg mL
1); mannan conjugates (150, 300, 1500, 3000 mg mL
1); cotreatment 24 h

Reduction of mutant white colony proportion

[179]

Yeast mannans

Ofloxacin, AO

EG

EG Z

Ofloxacin (15 mg mL
1), AO (5 mg mL
1); mannans (150, 300, 1500, 3000, 4500 mg mL
1); cotreatment 24 h

Reduction of white colony number

[180]

Continued

TABLE 7.5 Antigenotoxic Potential of Polysaccharides—Cont’d

Compound

Genotoxic Agent

Antigenotoxic Assay

Cell Lines/ Experimental Models

Experimental Protocol
1

Outcome

References

Lasiodiplodan

DXR

MN, SCGE

Wistar rats

DXR (15 mg kg body weight, intraperitoneally, on day 14), lasiodiplodan (5, 10, 20 mg kg
1 body weight, gavage, 15 days), cotreatment

Reduction in comet and MN formation

[181]

APS

B[a]P

DAFA

NCTC clone1469

B[a]P (4 nmol L
1, 6 h); APS (6, 20, 60, 180 mg mL
1), incubation 12 h

Inhibition of B[a]P-DNA adduct formation; reduction of 8-OH-dG formation

[182]

ELP

Sulfur dioxide, UV

MN

Allium sativum root cells

Sulfur dioxide solution (0.01–10 mM, 4 h), UV (1540 W, 5 min); ELP (50, 100, 200 g mL
1, 4 h); pretreatment, posttreatment, and simultaneous treatment

Decrease in MN frequency

[183]

9-AA, 9-aminoacridine; AB, Agaricus blazei mushroom; AGE, agarose gel-electrophoresis; 2-AmA, 2-aminoanthracene; AO, acridine orange; APS, Aloe polysaccharides; B[a]P, benzo [a]pyrene; BLM, bleomycin; BOT, botryosphaeran; BPDE, B[a]P-7,8-dihydrodiol-9,10-epoxide; CA, chromosomal aberrations; CBMN, cytokinesis-block micronucleus; CHO-k1, Chinese hamster ovarian cell line (wild type); CM-Ch-G, carboxymethylchitin-glucan; CM-G, carboxymethyl glucan; CPA, cyclophosphamide; CR, Chlamydomonas reinhardtii; CU, Candida utilis; DAFA, DNA adduct formation assay; DXR, doxorubicin; EG, Euglena gracilis; ELP, Enteromorpha linza polysaccharide; EMS, ethyl methanesulfonate; EPS, exopolysaccharides; HepG2, human hepatocellular liver carcinoma cells; HTC, rat hepatoma cells; GL, Ganoderma lucidum; Jurkat, leukemic human lymphocytes; MB, methylene blue; MH, maleic hydrazide; MMS, methyl-methane-sulfonate; MN, micronuclei; MNNG, N-methyl-N0 -nitro-N-nitrosoguanidine; NaN3, sodium azide; NCTC, mouse normal liver cells; NMU, N-nitroso-N0 -methylurea; 4-NQO, 4-nitroquinoline 1-oxide; 8-OH-dG, 8-hydroxydeoxyguanosine; SC, Saccharomyces cerevisiae; SCE, sister chromatid exchange; SE-ChG, sulfoethyl chitin-glucan; SE-G, sulfoethyl glucan; SCGE, single-cell gel electrophoresis; TP, total polysaccharides; Trp-P-2, 3-amino-1-methyl-5H-pyrido[4,3-b]indole; UV, ultraviolet; uv10, recombination repair-deficient CR mutans; uv12, excision repair-deficient CR mutans; uv14, mismatch repair-deficient CR mutans; V79, Chinese hamster lung fibroblast cells; WT, wild type; xrs5, DNA repair-deficient CHO cells.

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irradiation (9 Gy) by modulating the macrophage and bone marrow population and triggering colony-stimulating factor production [184]. Posttreatment with b-glucan from Reishi (Ganoderma lucidum) (500 mg kg
1 body weight, oral) was highly effective in reducing DNA damage and subsequent chromosomal aberrations induced by radiation (4 Gy) in mice in a manner similar to that provided by amifostine, the only clinically approved radioprotective compound. Radioprotection produced by the mushroom glucan has been attributed to its ability not only to stimulate DNA repair mechanisms but also cellular antioxidants, such as glutathione, which favor biochemical repair processes [169]. At a concentration of 1%, b-glucan from S. cerevisiae protects against AFB1-induced DNA damage in broiler chicken lymphocytes. The genoprotective effects may be a consequence of chemical interaction with AFB1 due to the adsorbent ability of b-glucan or interference with DNA repair pathways. The effects are dose dependent since b-glucan at 10% potentiated ROS formation in the presence of AFB1 and increased DNA damage. However, exposure to high concentrations of b-glucan alone (up to 10%) did not induce genotoxicity [173]. Moreover, in wild-type and repair-deficient CHO cells, b-glucan (5–40 mg mL
1) is devoid of genotoxic and mutagenic properties. The chemopreventive effects of b-glucan against the clastogenic activity of alkylating agents, such as MMS, have been observed in all protocols used (pretreatment, simple simultaneous, simultaneous with preincubation, posttreatment) in wild-type CHO cells, with the lowest effectiveness in posttreatment. In repair-deficient CHO cells, posttreatment with b-glucan was ineffective at preventing the damage induced by MMS, while the percent reduction of DNA damage in the simultaneous protocol was lower than in wild-type cells (39% vs ca. 60%). These findings suggest that yeast b-glucan can act by both desmutagenesis and bioantimutagenesis, although the latter mechanism has proven to be less important. Desmutagenic compounds are known to protect against genotoxicants by binding and decreasing their bioavailability. Bioantimutagenic agents act by modulating DNA replication and repair [158]. In mice cotreatment with b-glucan prevents clastogenic damage induced by acute exposure of pregnant and nonpregnant females to cyclophosphamide, although it does not antagonize the teratogenicity of immunosuppressant drugs. b-Glucan appears to interfere with the metabolism of cyclophosphamide by altering CYP activity or binding to cyclophosphamide or its metabolites in the maternal organism, but not in the embryo [172]. Some water-soluble derivatives of b-glucan isolated from S. cerevisiae, such as carboxymethyl glucan (CM-G), sulfoethyl glucan (SE-G), and carboxymethyl chitin-glucan (CM-Ch-G), reduce oxidative DNA damage induced by H2O2 and visible light-excited methylene blue in V79 hamster lung cells. Antigenotoxic efficiency increases in the order CM-G < SE-G < CM-CG, the first compound exhibiting protective effects only at the highest concentration tested (1500 mg mL
1). The main genoprotective mechanism involves scavenging hydroxyl radicals and singlet oxygen [162].

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A wide range of biomodulatory properties have also been demonstrated for SE-G derivative. Its effects are dependent on combined administration with other bioactive compounds. SE-G protects against MMS-induced genotoxicity in repair-deficient algal strain (Chlamydomonas reinhardtii uvs10), HTC, and wild-type CHO cells due to its free radical scavenging and desmutagenic effects. The antimutagenic properties of SE-G have been proven against a large variety of mutagens in different experimental assays, such as ofloxacin in the S. cerevisiae mutagenicity assay, ofloxacin and acridine orange in the flagellate Euglena gracilis, hydrogen peroxide and visible light-excited methylene blue in V79 cells, 2-aminofluorene in the Ames assay, or hexavalent chromium in mice. Se-G exerts significant anticlastogenic activity not only against the toxicity of maleic hydrazide in Vicia faba and Vicia sativa assays but also against that of CPA in the polychromatic erythrocytes of mouse bone marrow. In addition, SE-G increases the cytotoxic/cytostatic effects of the cytostatic drug vumon in mouse leukemia cells [4,157]. Antimutagenic effects have been described for water-soluble derivatives of the chitin–glucan complex (Ch-G), such as carboxymethyl chitin-glucan (CM-Ch-G) and sulfoethyl chitin-glucan (SE-Ch-G). Chitin (poly-beta-(1–4)-N-acetyl-D-glucosamine) is a natural major polysaccharide that plays a key role in different biological structures (exoskeleton of arthropods, fungal cell walls, cyst wall of protozoa, nematode eggshells) and has multiple biopharmaceutical and biotechnological applications [185]. CM-Ch-G is able to reduce DNA damage brought on by H2O2 in human HepG2 and HeLa cells, and N-nitrosomorpholine in HepG2 cells and V79 hamster lung cells. Moreover, it exerts antimutagenic activity against the genotoxicity of acridine orange and ofloxacin in E. gracilis. Moreover, in rats a diet enriched with CM-Ch-G increases the resistance of lymphocytes, alveolar macrophages, epithelial cells, and testicular cells to diverse genotoxic agents, such as H2O2, B[a]P, visible light-excited methylene blue, N-nitrosomorpholine, and dimethyldibenzocarbazole. However, the compound does not protect against alkylating DNA lesions induced by MMS and MNG, although preincubation of damaged HepG2 cells with CM-Ch-G supports their rapid recovery [157]. An interesting aspect is that in vitro CM-Ch-G (0.25, 0.5, and 1 mg mL
1) exerts damaging effects on plasmid DNA, while SE-Ch-G derivative exerts the opposite effects depending on the genotoxicant. It inhibits the DNA-damaging process initiated by Fe2, but increases DNA nicking induced by H2O2. The protective effects of SE-Ch-G in the first case can be explained by its ferrous ion-chelating properties (sulfoethyl substituent appears to be responsible for this behavior) [174]. Botryosphaeran (BOT) (Fig. 7.6) is a water-soluble (1 ! 3)(1 ! 6)-b-Dglucan with a triple-helix conformation produced by the ascomycete Botryosphaeria rhodina [161,166]. It exhibits hypoglycemic, cholesterol-lowering, antiproliferative, and free radical scavenging properties. The sulfonated derivative of BOT exerts anticoagulant and antithrombotic effects [161]. BOT protects normal and tumoral human lymphocytes against MMS-induced

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FIG. 7.6 Chemical structure of fungal b-D-glucans.

genotoxicity. The posttreatment protocol was more effective in normal lymphocytes, while in tumoral cells BOT was active under both the simultaneous and posttreatment protocols. Posttreatment effectiveness suggests the possibility that this glucan acts on DNA repair mechanisms. Moreover, treatment of normal lymphocytes with BOT alone or in combination with DXR represses CCR5 gene transcripts likely as a result of the binding activity of glucan for the CCR5 receptor. It is known that high CCR gene expression is associated with promalignancy and tumor development [161]. Although BOT presented free radical scavenging abilities in vitro, it was not able to scavenge the ROS and RNS generated by H2O2 exposure. However, BOT attenuates the clastogenic effects of DXR, H2O2, and B[a]P in V79 and HTC cells in a dose-dependent manner in both the simultaneous and posttreatment protocols [166]. Lasiodiplodan (Fig. 7.6) is a soluble unbranched (1–6)-b-D-glucan obtained from the fungus Lasiodiplodia theobromae [155]. It reduces the genotoxic effects of DXR in rats via upregulation of the TP53 gene, which plays a significant role in DNA stability and cancer prevention [166].

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b-Glucan isolated from the mushroom A. brasiliensis is devoid of genotoxicity and protects human peripheral blood lymphocytes against oxidative DNA damage induced by H2O2 and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), a heterocyclic aromatic amine known to be a mutagen of cooked food. Its protective effects against Trp-P-2 occur at concentrations greater than 40mg mL
1. The glucan had no effect on genomic damage caused by BPDE, the main metabolite of B[a]P, one of the compounds with pronounced genotoxic and carcinogenic properties [165]. However, the glucan protects HepG2 cells against the toxicity of diol epoxide, probably due to the fact that these cells have greater detoxification capacity than lymphocytes [186]. Moreover, b-glucan from A. brasiliensis decrease the deleterious effects of B[a]P in HepG2 cells in a dose-dependent manner in every protocol tested (pretreatment, simultaneous, and presimultaneous). Presimultaneous treatment was the most effective. This might be explained by the inhibitory effect of glucan on CYP450 enzymes, its free radical scavenging activity, or direct binding to B[a]P [159]. Nevertheless, the major consensus is that the chemopreventive activity of mushroom b-glucan against B[a]P and its metabolites and such aromatic amines as Trp-P-2 is more likely to occur via the modulation of gene expression and the activity of phase I enzymes (CYP450/CYP1A1, CYP1A2) involved in the metabolism of carcinogens than direct interaction with them [166,186]. Moreover, it is possible that glucans can modulate bioenergetic cellular metabolism [186]. Protective properties have also been reported for the b-glucan found in A. brasiliensis against damage caused by H2O2, BLM, and DXR in HepG2 cells. It has been observed to be more effective against H2O2 than BLM and DXR, which are equally potent genotoxicants. The genoprotective properties of total polysaccharides from A. brasiliensis have been shown to be ineffective against DXR, as compared with purified glucans. The greatest reduction in DNA damage was noted for total polysaccharides isolated from mature mushrooms and was probably due to the higher presence of b-type (1 ! 3) and a-type (1 ! 4) linkages at the ripe stage. Moreover, purified b-glucans extracted from mature mushrooms have greater protective properties than compounds obtained from mushrooms at other stages, probably due to the greater number of b-(1 ! 3) linkages in the first case. It appears that b-(1 ! 3) and a-(1 ! 4) linkages are involved in the antitumor effect of glucans [2]. b-Glucan from cereals, such as barley (H. vulgare), is devoid of mutagenic properties at low concentrations (1, 5, 25 mg mL
1) in HepG2 cells, but genotoxic and cytotoxic effects were observed at concentrations of 100 and 200 mg mL
1, respectively [164]. Moreover, Oliveira et al. [163] have shown that barley b-glucan at a concentration of 20 mg mL
1 produces a mild mutagenic effect in wild-type CHO and HTC cells. The different effects may be explained by features of the cell lines used. It seems that the significant presence of b-(1 ! 3)(1 ! 4) linkages in glucans confers higher toxicity than b-(1 ! 3)(1 ! 6) linkages. The combined administration of barley glucan and B[a]P in two protocols (the simple simultaneous and simultaneous with

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preincubation) produces better genoprotection in HepG2 cells [164]. In addition, barley glucan protects against mutagenicity induced by B[a]P, MMS, and 2-aminoanthracene in Cho-k1 and HTC rodent cell lines [163,165]. Oliveira et al. [163] reported that simultaneous with preincubation administration provides the most effective protection against MMS and 2-aminoanthracene suggesting that b-glucan can react with chemical mutagens, thus preventing their harmful interaction with DNA [163,164]. These findings suggest that barley glucan acts mainly by a desmutagenic mechanism that implies sequestration of mutagens due to its binding, modulation of cell metabolism, and free radical scavenging properties. Although the effects observed using pretreatment and posttreatment protocols with barley glucan were less protective, they also suggest the existence of a mild bioantimutagenic mechanism [163,164] that is not achieved when DNA polymerase is involved [165]. Mannans are among the most important components of the hemicellulose family. They are structural constituents of microbial (yeasts, fungi), algal, and plant cell walls. Plant mannans consist of repeating units of D-mannose joined by b-1,4-glycosidic linkages [187]. Yeast mannans present a branched structure with a main chain consisting of units of mannose a-(1 ! 6) linked together and side chains with a-(1 ! 2) and a-(1 ! 3) linkages. Different mannans may contain other sugar molecules, such as glucose, galactose, xylose, or acetyl groups that are important for biological activity [180]. Glucomannans are among the major mannans and are composed of a D-mannosyl backbone and units of D-glucose randomly linked [187]. Like b-glucans, mannans are important biological response modifiers and exert a wide array of bioactivities, such as antiviral, antioxidant, antitumor, hematopoetic, radioprotective, cholesterol lowering, and wound healing [180,188]. The most investigated compounds regarding genoprotective potential were yeast mannans, their conjugates with proteins, and yeast glucomannans (Table 7.5). The addition of mannan in the diet (250 or 500 mg kg
1, 4 weeks) protects against AFB1-induced genotoxicity in mice. The protective effects are dose dependent and are mediated by the adsorptive capacity of mannan. The compound has been shown to reduce the bioavailability of genotoxicant by its binding in the digestive tract of the mouse [178]. S. cerevisiae mannan (Fig. 7.7) and its conjugates with human serum albumin and penicillin G acylase proteins exhibits significant antimutagenic activity against ofloxacin and acridine orange (AO)-induced damage in the flagellate E. gracilis. Mannan–protein conjugates are more effective than nonconjugated mannan in terms of genoprotection and antioxidant activity. The genotoxic effects of ofloxacin may be prevented by antioxidant intervention of mannan derivatives since the antibiotic works by generating ROS and inhibiting DNA gyrase. Protection against AO-induced damage may occur via direct physical adsorption of mutagen onto mannan conjugates because the toxic properties of AO are mediated by its intercalative DNA-binding abilities [179].

284 Studies in Natural Products Chemistry

FIG. 7.7 Chemical structure of mannan from Saccharomyces cerevisiae [189].

Candida utilis glucomannan (Fig. 7.8) has been found to exhibit antimutagenic, anticlastogenic, and bioprotective effects in different experimental models and various genotoxic agents. Glucomannan protects against the mutagenic effects of ofloxacin and acridine orange in E. gracilis, cyclophosphamide in polychromatic erythrocytes of mouse bone marrow, 9-aminoacridine, and sodium azide in S. typhimurium assay. It prevents the toxicity of MMS in repair-deficient Chlamydomonas reinhardtii algal strains and clastogenic action of N-nitroso-N0 -methylurea treatment in V. sativa assay. The free radical scavenging properties and absorptive capacity of glucomannan are the main mechanisms involved in its genoprotective effects. Biological activity is positively influenced by terminal glucopyranosyl residues of glucomannan. Compared with mannans from S. cerevisiae and Candida albicans, glucomannan from C. utilis has higher antioxidant activity. Moreover, some of the physicochemical properties of glucomannans are better than those of b-glucans, such as good solubility in water and low molecular weight (30 kDa), thus adding support for their clinical application [175].

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FIG. 7.8 Chemical structure of glucomannan from Candida utilis [190].

Overall, the genoprotective effects of polysaccharides may be related to: l

l

l

l l

binding of genotoxicants in supramolecular complexes and decreasing their bioavailability; antioxidant activity (free radical scavenging properties, ferrous ionchelating abilities, stimulation of SOD activity); inhibition of phase I metabolic enzymes involved in the bioactivation of xenobiotics; stimulation of DNA repair processes; and positive modulation of bioenergetic cellular metabolism.

The huge structural diversity of polysaccharides brings challenges in assessing and establishing the structure-bioactivity relationship. The primary structure,

286 Studies in Natural Products Chemistry

solubility (water solubility), branching degree (DB), molecular weight (MW), charge, and structure of polysaccharides in aqueous solutions are the most important parameters influencing their biological effects. b-Glucans with a triple-helix structure, 100  MW  200 kDa and 0.2 DB  0.33, are generally considered more biologically active [191]. However, no generally available criteria have been postulated on the relationship between chemical structure and biological activity; the different structural features of glucans seem to be important for each type of bioactivity [184]. The immunopharmacological effects of glucans are thought to be related to their triple-helix conformation. Introducing chemical groups into glucan macromolecules, such as carboxymethyl or sulfonyl, increases the capacity of such macromolecules to bind to immune cell receptors and the potency of immunomodulatory effects. Moreover, the presence of additional glycosidic linkages in the main chain and a more branched structure have been shown to stimulate the immune system more effectively [156,192]. Carboxymethyl groups enhance the stiffness of the chains and sulfonyl groups increase backbone flexibility and improve the functional activity of glucans (mainly antitumor activity). Replacing b-[1 ! 3] linkages in the backbone of glucans with b-[1 ! 6] and b-[1 ! 4] bonds has been found to be disadvantageous for their antitumor activity [193]. Antioxidant activity is one of the most important general genoprotective mechanisms. The structural characteristics and physicochemical properties that determine the antioxidant activity of polysaccharides are water solubility, a MW higher than 90 ku, a backbone chain of b-(1–3)-D-glucose and b-(1–6) side chains, the degree of branching, functional groups (acetyl, formyl, polyhydroxyl, carboxymethyl), and the triple-helix structure [154]. Conjugating polysaccharides with proteins increases their antioxidant potency, heightening their free radical scavenging activity [179]. There is a need for more studies on the relationship between the chemical structure/conformation and genoprotective effects of glucans to identify the functionalized active structures [193]. Apart from lentinan, schizophyllan, and krestin (polysaccharides that are approved for clinical use as immunoadjuvants in cancer), only a few clinical trials with extracts rich in b-glucans from mushrooms or yeast have been carried out on patients with cancer [156]. b-Glucan extracts contain many different compounds that may interact together. It is important to keep in mind that this does not reflect the real clinical effectiveness of pure b-glucans. Future studies should thus focus on purified compounds. Clinical application of glucans as chemopreventive agents needs a better understanding of the interaction between a DNA-damaging agent and glucans, or between conventional or targeted therapies and glucans. There is also a need to elucidate the mechanisms of action (desmutagenic and/or bioantimutagenic), and for much more information regarding the safety and efficacy of pure compounds.

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CONCLUSION Some dietary phytochemicals, such as organosulfur compounds, terpenoids, and polysaccharides, have important genoprotective potential as revealed by many different bacteria, plant, animal, and cell-based assays. They may act as scaffolds on which to build novel molecules for therapeutics and offer tremendous scope for further research (organosulfur compounds in particular are highly bioactive). The phytochemicals mentioned in this chapter can prevent or attenuate DNA damage and usually do so in a dose- and xenobiotic-dependent manner. Their effects are mediated by many different mechanisms, even for the same structural category of phytochemicals, but these activities have still not been fully elucidated. The use of these compounds in chemotherapeutic clinical practice needs more investigation such that their genoprotective mechanisms, differentiation between desmutagens and bioantimutagens, dose–biological response relationship, biotransformation, influence on other xenobiotics, and safety and efficacy for long-term administration can be better understood.

ABBREVIATIONS 2-AF 2-AmA 4-NQO 8-OH-dG 9-AA AAs AB AFAR AFB1 AFBO AFM1 AFP1 AFQ1 AGE AITC AKR AM ANT AO APS ARE AZT

2-aminofluorene 2-aminoanthracene 4-nitroquinoline-1-oxide 8-hydroxydeoxyguanosine 9-aminoacridine aromatic amines Agaricus blazei (a mushroom) aflatoxin B1 aldehyde reductase aflatoxin B1 aflatoxin B1-8,9-epoxide aflatoxin M1 aflatoxin P1 aflatoxin Q1 agarose gel electrophoresis allyl isothiocyanate aldo-keto reductase allyl mercaptan anethole acridine orange Aloe polysaccharides antioxidant response element azidothymidine, zidovudine

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B[a]P BD BITC BLM BOT BPDE BRN CAs CBMN CCl4 Ch-G CHO-k1 CM-Ch-G CM-G CPA CR CTR CU CVC DADS DAFA DAS DATS DES DM DMBA DMDS DNP DPDS DPS DXR EG EH ELP EMS EP EUC EUG GCS GL GLSs GPx GR

benzo[a]pyrene branching degree benzyl isothiocyanate bleomycin botryosphaeran benzo[a]pyrene-7,8-diol-9,10-epoxide borneol chromosome aberrations cytokinesis-blocked micronucleus carbon tetrachloride chitin–glucan complex Chinese hamster ovarian cell line (wild type) carboxymethyl chitin-glucan carboxymethyl glucan cyclophosphamide Chlamydomonas reinhardtii citral Candida utilis carvacrol diallyl disulfide DNA adduct formation diallyl sulfide diallyl trisulfide diethylstilbestrol Drosophila melanogaster 7,12-dimethylbenz[a]anthracene dimethyl disulfide 1,6 dinitropyrene dipropyl disulfide dipropyl sulfide doxorubicin Euglena gracilis epoxide hydrolase Enteromorpha linza polysaccharide ethyl methanesulfonate exopolysaccharides eucalyptol eugenol gamma-glutamylcysteine synthetase Ganoderma lucidum glucosinolates glutathione peroxidase glutathione reductase

Antigenotoxic Potential of Phytochemicals Chapter

GRAS GSH GST H2O2 HAAs HANs HB HDAC HepG2 HgCl2 HMC HO1 HPBLC HTC HWBC IQ ITCs Jurkat Keap1 LMN LS-174 MAH MB MCF-10F MeIQx MH MMC MMS MN MNNG MTBITC MTPITC MTPnITC NaN3 NAT NC–NC NCTC NDBA NDMA nitro-PAHs NPhIP NPIP NPYR

generally recognized as safe glutathione glutathione transferase hydrogen peroxide heterocyclic aromatic amines heterocyclic aromatic nitroderivatives high bioactivation histone deacetylase human hepatocellular liver carcinoma cells mercuric chloride human mesothelial cells heme oxygenase 1 human peripheral blood lymphocyte cells rat hepatoma cells human whole-blood lymphocytes 2-amino-3-methylimidazo[4,5-f]quinolone isothiocyanates leukemic human lymphocytes Kelch-like ECH-associated protein 1 limonene human colon adenocarcinoma cells monocyclic aromatic hydrocarbon methylene blue human normal breast epithelial cells 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline maleic hydrazide mitomycin C methyl methanesulfonate micronuclei N-methyl-N0 -nitro-N-nitrosoguanidine 4-methylthio-3-butenyl-1-isothiocyanate 3-methylthiopropyl-1-isothiocyanate 5-methylthiopentyl-1-isothiocyanate sodium azide N-acetyltransferase human B lymphoblast cells mouse normal liver cells N-nitrosodibutylamine N-nitrosodimethylamine nitro-polycyclic aromatic hydrocarbons N-hydroxy PhIP N-nitrosopiperidine N-nitrosopyrrolidine

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NQO1 Nrf2 OSCs OTM PCB PEITC PhIP PM ROS SAC SC SCEs SCGE SD Se-Ch-G SE-G SFN SMART SO ST STD T24 T5-1A2/2E1 TAA t-BHP TI TL TM TP Trp-P-2 UGT URE UV uv10 uv12 uv14 V79 VCR Vero VH10 WT xrs5 ZEN

NAD(P)H quinone oxido-reductase nuclear factor (erythroid-derived 2)-related factor 2 organosulfur compounds olive tail moment procarbazine phenethyl isothiocyanate 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine propyl mercaptan reactive oxygen species S-allyl cysteine Saccharomyces cerevisiae sister chromatid exchanges single-cell gel electrophoresis Sprague–Dawley sulfoethyl chitin-glucan sulfoethyl glucan sulforaphane somatic mutation and recombination test styrene-7,8-oxide Salmonella typhimurium standard bioactivation human bladder carcinoma cells T-antigen immortalized human liver epithelial cells expressing human CYP1A2/2E1 thioacetamide tert-butylhydroperoxide tail intensity tail length tail moment total polysaccharides 3-amino-1-methyl-5H-pyrido[4,3-b]indole 1 UDP-glucuronyl transferase urethane ultraviolet recombination repair-deficient CR mutans excision repair-deficient CR mutans mismatch repair-deficient CR mutans Chinese hamster lung fibroblast cells vincristine African green monkey kidney cells human fibroblasts wild type DNA repair-deficient CHO cells zearalenone

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