Metabolic Activation Pathways Leading to Liver Tumor Initiation

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Pyrrolizidine Alkaloids: Metabolic Activation Pathways Leading to Liver Tumor Initiation Peter P. Fu Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00297 • Publication Date (Web): 07 Nov 2016 Downloaded from http://pubs.acs.org on November 14, 2016

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Chemical Research in Toxicology

Pyrrolizidine Alkaloids: Metabolic Activation Pathways Leading to Liver Tumor Initiation Peter P. Fu National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079 USA

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Table of Contents Graphic:


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ABSTRACT: Pyrrolizidine alkaloids (PAs) and PA N-oxides are a class of phytochemical carcinogens contained in over 6000 plant species widespread in the world. It has been estimated that approximately half of the 660 PAs and PA-N-oxides that have been characterized are cytotoxic, genotoxic, and tumorigenic. It was recently determined that a genotoxic mechanism of liver tumor initiation mediated by PA-derived DNA adducts is a common metabolic activation pathway of a number of PAs. We proposed this set of PA-derived DNA adducts could be a common biological biomarker of PA exposure and a potential biomarker of PA-induced liver tumor formation. We have also found that several reactive secondary pyrrolic metabolites can dissociate and inter-convert to other secondary pyrrolic metabolites, resulting in the formation of the same exogenous DNA adducts. This present perspective reports the current progress on these new findings and proposes the future research needed for obtaining a greater understanding on the role of this activation pathway, and validating the use of this set of PA-derived DNA adducts as a biological biomarker of PA-induced liver tumor initiation.



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■ CONTENTS 1. Introduction 1.1. Livestock and Human Poisoning 1.2. Mechanism of Toxicity 1.3. Carcinogenicity 2. Current Understanding on Metabolic Activation 2.1. Mechanism of Liver Tumor Initiation 2.1.1. Formation of DNA Cross-linking and DNA–Protein Cross-linking 2.1.2. Exogenous DNA Adduct Formation 2.2. DNA Adducts as Biomarkers 2.3. Quantitation of Protein Adducts 3. Perspectives and Future Research 3.1. DHP-DNA Adducts – Liver Tumor Initiation 3.1.1. More PAs for metabolic activation studies 3.1.2. Other potential carcinogenic metabolites 3.1.3. Multiple activation pathways 3.1.4. Inter-convertibility and reversibility between pyrrolic metabolites 3.1.5. DHP-DNA adducts – A surrogate of PA carcinogenicity determination 3.1.6. Site and gene sequence-specificity of DNA adducts formation 3.2. DHP-Protein Adducts for Clinical Assay 3.3. Regulation of PA-Containing Products 3.4. DNA-DNA Cross Links and DNA Protein-Cross Links 3.5. Other Type of Reactive Pyrrolic Metabolite(s) 4. Conclusion Author Information Corresponding Author Funding Notes Biograph Acknowledgements Abbreviations References


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1. INTRODUCTION Pyrrolizidine alkaloids (PAs), more formally by another nomenclature system called dehydropyrrolizidine alkaloids or 1,2-dehydro-PAs, and PA N-oxides are a class of rodent liver carcinogens.1-3 Recent findings have revealed that a number of PAs and PA N-oxides exert liver tumorigenicity mediated by a set of exogenous DNA adducts. In this present review, we address this mechanism, the discovery of the new reactive pyrrolic metabolites, their dissociation and interconversion leading to multiple metabolic activation pathways of PAs, and proposed future research directions. Part of the experimental results discussed in this perspective was generated from our laboratory.

1.1. Livestock and Human Poisoning PAs and PA N-oxides are common plant secondary metabolites.2, 4 There are over 660 PAs and PA N-oxides present in many plants around the world.5, 6 It has been estimated that approximately half of the identified PAs and PA N-oxides are hepatotoxic, mutagenic, carcinogenic, teratogenic, and occasionally pneumotoxic.2, 6-9 The acute hepatotoxicity caused by PAs results in haemorrhagic necrosis, hepatomegaly, ascites, necrosis, dysfunction, and death.2, 4, 6-8, 10, 11 The PA-induced liver damage results in the veno-occlusion disease (VOD).2, 7, 8, 11

Both PAs and PA-containing plant extracts exhibit a variety of genotoxicities in different

systems in vivo.2, 3, 7, 8, 12-24 There are two major routes of human exposure to PAs; (i) the consumption of food staples which contain the PAs constituents; including herbal medicines,18, 25-28 herbal dietary supplements,10, 11, 18, 29 teas,25, 30 herbal teas,18, 27, 31, 32 honey,7, 25, 33-37 and milk;38, 39 and (ii) the intake of food contaminated with toxic PAs7, 31, 40 Exposure of humans and livestock to toxic PAs through the food contamination pathway has resulted in numerous severe poisoning epidemics, with high mortality and morbidity rates.1, 2, 6, 7, 9, 18, 31, 38, 40-44 The first report on livestock poisoning by PAs was in 1787.31 Since then, many PAcontaining plant species have been found to cause numerous poisoning events in humans and livestock, including in cattle, sheep, chickens, ducks, and pigs.2, 9, 31, 43-45 The first large scale food poisoning in humans was recognized in 1920 in South Africa.46 The use of herbal products has been progressively increasing. Human poisonings caused by intake of PA-containing medicinal plants and herbal dietary supplements have been reported.28, 32 In 1989 the 5 ACS Paragon Plus Environment


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International Program on Chemical Safety (IPCS) concluded that ‘‘consumption of contaminated grain or the use of PA-containing plants as herbal medicine, beverages, or food by man, or grazing on contaminated pastures by animals, may cause acute or chronic disease’’.40

1.2. Mechanism of Toxicity PAs consist of a necic acid and a necine base.2, 4, 47 There are four common necine bases of PAs, the retronecine, heliotridine, otonecine, and platynecine types, among which the first three types of necine bases possess a double bond at the C1 and C2 positions and exhibit hepatotoxicity, genotoxic, and potentially carcinogenicity (Figure 1).2, 4, 6-8, 48 Among the PAs, macrocyclic diester PAs exhibit the highest hepatotoxicity, genotoxicity, and tumorigenicity, followed by monoester PAs.2, 4, 22 Retronecine-type PAs possess a 7R stereochemistry, while the heliotridine-type PAs have a 7S absolute configuration. Retronecinetype PAs are the most abundant PAs and therefore are most widely studied.2, 4, 40 Platynecinetype PAs which possess a saturated necine base are in general not toxic.2, 22, 49


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Figure 1. The common necine bases of PAs; structures of DHR, DHH, and DHP; and the necic acid and necine base of riddelliine. DHP is a molecule that contains DHR and DHH in an equal ratio.

PAs are biologically inactive and require metabolic activation to exert toxicity, including carcinogenicity.2, 4, 7, 8 The retronecine-type and heliotridine-type PAs have three major metabolic pathways: (i) hydrolysis of the ester groups to form the corresponding necines and necic acids; (ii) oxidation of the nitrogen atom of the necine bases to produce PA N-oxides; and (iii) enzymatic hydroxylation of the necine base to produce the 3- or 8-hydroxy-PAs which are rapidly dehydrated to the reactive (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine esters (DHP esters; or dehydro-PAs) (Figure 2). The otonecine-type PAs have only two major metabolic pathways: (i) hydrolysis of the ester groups to form the corresponding necine bases 7 ACS Paragon Plus Environment


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and necic acids; and (ii) enzymatic oxidative N-demethylation of the nitrogen atom of the necine base, resulting in the ring closure, and dehydration to form DHP esters (dehydro-PAs).2, 4

Figure 2. Major metabolic pathways of retronecine-type and heliotridine-type PAs.

Cytochrome P-450 (CYP) 2B and CYP3A are the principal metabolizing enzymes responsible for the metabolism of PAs.4, 7, 50, 51 The enzymes that catalyze the metabolism of PAs to PA N-oxides are both cytochrome P-450 and flavin-containing monooxygenases.4, 52 Hydrolysis of the ester groups of PAs to form necine base and necic acids is catalyzed by the hepatic microsomal carboxylesterases.4, 52 Because DHP esters (dehydro-PA) metabolites are biologically active and can lead to liver tumor initiation, the hepatic metabolism of PAs to DHP esters (dehydro-PAs) is considered the principal metabolic activation pathway (Figure 2).2 It has been found that the PA N-oxide 8 ACS Paragon Plus Environment

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metabolites, formed from metabolism of the parent PAs in vivo, can be enzymatically reduced to the parent PAs in the gut and liver (Figure 2).2, 4, 7, 8, 36, 53-55 Consequently, the metabolism of PAs to PA N-oxides should be considered a potential, but minor, activation pathway.2, 22, 53

1.3. Carcinogenicity In 1954, Schoental et al.56 reported that retrorsine and the PA-containing plant S. jacobaea lin induced liver tumors in rats. In the early 1970, a series of PAs were found to induce tumors, mainly the liver tumors, in rats and other experimental rodents. To date, more than 20 purified plant PAs, a PA N-oxide, dehydro-PAs, and plant extracts have been demonstrated to induce tumors in rodents. The PAs and retrorsine N-oxide that are known to induce liver tumors in rats are listed in Table 1, and their structures are shown in Figure 3. Since PAs are widespread in the world, human exposure to genotoxic and carcinogenic PAs is a concern. Among the carcinogenic PAs, lasiocarpine and riddelliine have been tested by the U.S. National Toxicology Program (NTP) in chronic tumorigenicity bioassays and shown to be rodent carcinogens.77, 78 Riddelliine is listed as a "reasonably anticipated to be human carcinogen” in the NTP 13rd Report on Carcinogens.79 The IARC listed monocrotaline, riddelliine, and lasiocarpine as Group 2B, possible human carcinogens.79-81



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Table 1. Carcinogenic Pyrrolizidine Alkaloids (1,2-Dehydro-PAs) and a Pyrrolizidine Alkaloid N-Oxide that Induce Liver Tumors in Rats. Pyrrolizidine Alkaloids Plant species (Family) References Retronecine - Type Pyrrolizidine Alkaloids 56-59 Retrorsine 1 Senecio (Compositae) 57, 58, 60 Riddelliine 2 Senecio (Compositae), Crotalaria (Leguminosae) 61, 62 Monocrotaline 3 Crotalaria (Leguminosae) 56, 58, 63 Senecionine 4 Senecio (Compositae) 57, 63 Seneciphylline 5 Senecio (Compositae) 56, 64 Jacobine 6 Senecio L. (Compositae) Symphytine 7 Symphytum officinale L (Boraginaceae) 65, 66 Retronecine – Type Pyrrolizidine Alkaloid N-Oxide 56, 58, 59 Retrorsine N-oxide Senecio (Compositae), (Isatidine) 8 Crotalaria (Leguminosae) Heliotridine - Type Pyrrolizidine Alkaloids 67-70 Lasiocarpine 9 Heliotropium (Boraginaceae) 71 Heliotrine 10 Heliotropium (Boraginaceae) Otonecine - Type Pyrrolizidine Alkaloids 72, 73 Ligularia dentata Hara (Compositae) Clivorine 11 63, 65, 74 Senecio (Compositae) Senkirkine 12 Petasites (Compositae) 74-76 Patasitenine 13 Senecio (Compositae)


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HO

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Figure 3. Structures and names of the liver carcinogens of PAs and retrorsine N-oxide.



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2. CURRENT UNDERSTANDING ON METABOLIC ACTIVATION 2.1. Mechanism of Liver Tumor Initiation Segall and co-workers determined that trans-4-hydroxy-2-hexenal was formed from the liver microsomal metabolism of senecionine.82-85 This metabolite was cytotoxic in primary cultures of rat hepatocytes83 and could bind with deoxyguanosine to form adducts.85 However, the mechanism of its formation was still uncertain. Further studies are required to find out whether or not the formation of this metabolite is involved in the senecionine-induced liver tumor initiation.

2.1.1. Formation of DNA Cross-linking and DNA–Protein Cross-linking Dehydro-PAs and DHP possess two electrophilic sites at the C7 and C9 positions of the necine base, both of which can bind to cellular DNA and proteins to form DNA-DNA cross-links and DNA-protein cross-links.13-15, 86-92 Coulombe and coworkers studied the formation of DNA-DNA and DNA-protein links using eight representative PAs in cultured bovine kidney epithelial cells in the presence of an external metabolizing system and determined the structure–activity relationships.14, 93 The eight PAs included five macrocyclic diesters, two open chain diesters, and one necine base (retronecine). The level of the DNA-protein cross-links was determined by Western immunoblotting, and the results indicated a positive structure-activity relationship, with the macrocyclic diester PAs producing higher quantities of DNA-protein cross linking than the other types of PAs. Dehydrosenecionine, dehydroseneciphylline, dehydroriddelliine, and dehydromonocrotaline are reactive primary DHP esters metabolites of the parent PAs senecionine, seneciphylline, riddelliine, and monocrotaline, all of which belong to macrocyclic diesters PAs. Kim et al.15 determined that, compared to DHR, these four DHP esters (dehydro-PAs) induced more proteinDNA cross-links. These results are in general consistent with mutagenicity and hepatocarcinogenicity of the parent PAs.15 Because the tested PAs are carcinogens, it is highly possible that DNA-DNA and DNA-protein cross-links can cause tumor initiation.

2.1.2. Exogenous DNA Adduct Formation It has long been determined that dehydromonocrotaline, dehydroretrorsine, DHR, and dehydroheliotridine (DHH) can bind to DNA, nucleosides, and nucleotides.2, 94-97 These results 12 ACS Paragon Plus Environment

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indicate that DHP esters metabolites could bind with cellular DNA in vivo, resulting in liver tumor initiation mediated by exogenous DNA adducts formation. The 2001 and the more recent research findings provided the first direct correlation demonstrating that PA induced liver tumors in experimental rodents are mediated by DNA-DHP adduct formation.23 Yang et al. determined that riddelliine induced liver tumors in rats and mice mediated by DHP-derived DNA adducts that were produced in the livers in vivo in a doseresponse manner.23, 98, 99 Described below is the mechanism-based evidence about this genotoxic mechanism, which should potentially be applicable to livestock and humans: 1. There is a good correlation between the levels of the DHP-DNA adducts and the tumorigenic potencies in rats fed different doses of riddelliine.23, 98, 99 2. The DHP-DNA adducts formation in vivo is cell type specific, generating more DHPDNA adducts in the hepatic endothelial cells than in the parenchymal cells.98 3. Using transgenic Big Blue rats for study, Mei et al.100 determined that riddelliine induced mutations at guanine bases in the liver; and mutations occurred mainly in the endothelial cells. 4. Hong et al.101 reported that B6C3F1 mice treated with riddelliine resulted in G to T mutations at codon 12 of the K-ras oncogene. 5. Using the cII gene mutation assay in transgenic Big Blue rats, Mei et al.100, 102 determined that riddelliine induced mutants, predominantly the G:C to T:A transversions. These mutation patterns suggested that metabolism of riddelliine generated DHP-dG adducts in the endothelial cells, which induce mutations leading to liver tumor initiation.100, 103 6. The metabolic pattern and DNA adduct profile produced from the human and rat liver microsomal metabolism of riddelliine were highly similar, which indicated that the studies using experimental rodents are highly relevant to humans and that riddelliine can be a human carcinogen.104 Subsequently, the U.S. NTP classified riddelliine as "reasonably anticipated to be a human carcinogen”.79 The success of the mechanistic studies on riddelliine relied on the development of a 32Ppostlabeling/HPLC method that could accurately quantify the exogenous DNA adducts formed in the liver of riddelliine-treated rats and mice.105 By using this analytical method, PA-DNA adducts were detected and quantified in vivo and in vitro from the metabolism of a series of



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PAs,23, 98, 99, 104, 106-112 PA N-oxides,53, 55, 112 PA-containing herbal plant extracts,29, 109 and dietary supplements extracts.29 Since the 32P-postlabeling/HPLC method could not provide information about the molecular structures of the PA-derived DNA adducts, an HPLC-ES-MS/MS method was then developed for the further studies.16, 19-22, 24 By this method, a set of four DHP-DNA adducts, designated as DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4, was detected and quantified in the livers of rats treated with riddelliine and monocrotaline (Figure 4).16, 22, 24

O

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Figure 4. Structures of DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts.

2.2. DNA Adducts as Biomarkers Xia et al.22 studied DHP-DNA adduct formation in the livers of rats treated with a series of PAs in vivo. The results indicated that all the seven hepatocarcinogenic PAs and riddelline Noxide produced the same set of DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts in the livers of treated rats in a dose-response manner. The levels of DHP-dG adducts were higher 14 ACS Paragon Plus Environment

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than those of DHP-dA adducts. The levels of DHP-DNA adducts formed in the liver of PAtreated rats were in the order: retrorsine > lasiocarpine > riddelliine ~ monocrotaline > riddelliine N-oxide > senkirkine > heliotrine ≥ clivorine >>> lycopsamine ~ retronecine ~ platyphylliine ~ control. Lycopsamine and retronecine, which are not liver tumorigens, formed the lowest levels of DHP-DNA adducts. These results indicate that the level of DHP-DNA adduct formation is in the order: di-ester PAs > mono-ester PAs ~ di-ester PA N-oxide >>> retronecine (which does not have an ester linkage).22 Based on these and our previously published results,16, 24, 104, 109, 110 a general metabolic activation pathway is proposed (Figure 5). This represents the ﬁrst finding that an identical set of exogenous (not endogenous) DNA adducts is generated from individual members of a large class of chemical carcinogens, and these DNA adducts can be potential biomarkers of PA exposure and liver tumor initiation.



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Figure 5. Proposed general mechanism of DHP-DNA adducts formation.

2.3. Quantitation of Protein Adducts Pyrrole-protein (DHP-protein) adducts have been proposed as a biological biomarker of exposure to toxic PAs.2, 7,113 A qualitative analytical method was developed by Mattocks and coworkers in the early 1990’s to detect these adducts in experimental animals and livestock.114-117 This method used silver nitrate in ethanol to cleave the thiol linkage of the adducts, replacing the protein moiety with ethoxyl groups. To enhance the detection sensibility, the 7,9-di-C2H5ODHP formed was reacted with the Ehrlich reagent to form the product that was measured colorimetrically.118 In 2011, Lin and co-workers modified this method, using the developed LC/MS/MS method to detect DHP-protein adducts in the blood of patients with liver disease.11 This method was further modified to allow quantification of DHP-protein adducts formed in vivo, including human blood samples.51


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Xia et al.119 recently developed an alternate LC/MS/MS method which was used to accurately quantify the DHP-protein adducts formed in the blood of rats treated with a series of PAs.22

3. PERSPECTIVES AND FUTURE RESEARCH 3.1. DHP-DNA Adducts - Liver Tumor Initiation Nearly half of the 660 PAs and PA N-oxides currently identified contain a unsaturated necine base and are cytotoxic, genototoxic, and tumorigenic.6, 8 As such, determination of the mechanism leading to liver tumor initiation for so many PAs is challenging. Recent findings have shown that the metabolic activation of a number of hepatocarcinogenic PAs is by the same mechanism mediated by DHP-DNA adducts formation leading to the liver tumor initiation. Consequently, much of the future research should focus on this mechanism. 3.1.1. More PAs for metabolic activation studies At the present, only seven hepatocarcinogenic PAs and retrorsine N-oxide have been shown to initiate liver tumors through this general metabolic activation pathway. In order to establish the generality of this genotoxic mechanism, more representative PAs and PA N-oxides will need to be studied.

3.1.2. Other potential carcinogenic metabolites To date, DHP esters (dehydro-PAs) and DHP are the only two reactive metabolites that have been shown to be able to cause liver tumor initiation.2, 7, 23 DHP esters are extremely unstable,120 and therefore, may not be the principal metabolites to initiate PA-induced carcinogenicity. On the other hand, although DHP is more stable, it is much less reactive and thus may not be the principal metabolite for liver tumor initiation.7, 23 Thus, other metabolites may be involved in generating DNA adducts in vivo and causing liver tumor initiation.2 7-Glutathione-DHP (7-GS-DHP) (Figure 6) is a metabolite formed in vivo and in vitro.21, 121125

Estep et al. reported that 7-N-acetylcysteine-DHP (Figure 6) was present in the urine of male

Sprague-Dawley rats treated with 14C-monocrotaline or 14C-senecionine.126 7-Cysteine-DHP (Figure 6) was formed from the rat liver microsomal metabolism of 7-GS-DHP.20 We have determined that these three metabolites (7-GS-DHP, 7-cysteine-DHP, and 7-N-acetylcysteine-



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DHP) can react with calf thymus DNA to form DNA adducts.19-21 Therefore, these three metabolites are potential carcinogenic metabolites, capable of initiating liver tumors.19-21 Segall et al.127 reported that 7-methoxy-DHP (7-CH3O-DHP) (Figure 6) was a metabolite formed from the mouse liver microsomal metabolism of senecionine.127 7-Lysine-DHP was recently found to be a metabolite formed in mice administered with monocrotaline.128 Chen et al. reported that 9-GS-DHP is a metabolite in the male Sprague-Dawley rats administered with isoline, retrorsine, or monocrotaline.129 7-CH3O-DHP, 7-lysine-DHP, and 9-GS-DHP all possess the same type of DHP moiety as that of the metabolites described above. It is important to determine whether or not they are hepatocarcinogens, and are also mediated by this activation pathway.

Figure 6. Structures of 7-GS-DHP, 9-GS-DHP, 7-cysteine-DHP, 7-N-acetylcysteine-DHP, 7CH3O-DHP, 9-CHO-DHP, and (3H-pyrrolizin-7-yl)methanol.

3.1.3. Multiple activation pathways 18 ACS Paragon Plus Environment

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At present, dehydro-PAs and DHP are the two types of metabolites known to bind to DNA in cells to form DHP-DNA adducts. As described previously, 7-GS-DHP, 7-cysteine-DHP, and 7N-acetylcysteine-DHP are able to bind to DNA to form DHP-DNA adducts, these secondary metabolites should be able to bind to cellular DNA in vivo, leading to liver tumor initiation. Similarly, both 7-CH3O-DHP and 7-lysine-DHP may also transfer the DHP moiety to cellular DNA. Consequently, it is highly possible that there exists multiple metabolic activation pathways resulting in the DHP-DNA adducts formation and PA-induced liver tumors.

3.1.4. Inter-conversion and reversibility between pyrrolic metabolites The primary DHP esters (dehydro-PAs) can react with water and cellular constituents to produce secondary pyrrolic metabolites. The reversibility of the secondary metabolites was first observed by Curtain and Edgar in 1976, who discovered that the binding of dehydroheliotridine (DHH) with DNA was reversible.97 In 1997, Sun et al. determined that the binding of DHR with albumin was reversible,130 and in 1983, Mattocks and Bird determined that the reaction product of DHR and nicotinamide was also reversible131 The inter-conversion and the reversible reactions proceed through an SN1 mechanism, and the resulting carbonium ion can be stabilized by resonance.2 Partly based on these observations, Edgar et al. proposed that in vivo, DHP, DHP-DNA adducts, and DHP-protein adducts can be dissociated in the liver, resulting in a pooled secondary metabolites and forming multiple activation pathways leading to the PA-induced liver tumor initiation.7 While this proposed mechanism is promising, the structures of the above-described secondary metabolites have not been fully characterized.7, 97, 130, 131 It is also not known whether or not these reaction products are indeed metabolites formed in vivo and are involved in the liver tumor initiation. As such, for the support of this proposed mechanism, it is timely and important to determine that some identified secondary metabolites can indeed undergo dissociation, interconversion, and reversible reactions. The following recently published experimental results support this proposed mechanism. 7-GS-DHP, 7-cysteine-DHP, and 7-N-acetylcysteine-DHP are secondary pyrrolic metabolites produced from the reactions of DHP esters (dehydro-PAs) with glutathione, cysteine, and Nacetylcysteine, respectively, in vivo and/or in vitro.19-21, 121-126, 132 We have demonstrated that upon incubation with human and rat liver microsomes, 7-GS-DHP, 7-cysteine-DHP, and 7-N19 ACS Paragon Plus Environment


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acetylcysteine-DHP dissociated to DHP.19-21 We have also demonstrated that reaction of 7-GSDHP, 7-cysteine-DHP, or 7-N-acetylcysteine-DHP with dG produced DHP-dG-3 and DHP-dG4; and reaction with dA generated DHP-dA-3 and DHP-dA-4. Upon incubation with calf thymus DNA, these three secondary metabolites were inter-converted into DHP-dG and DHP-dA adducts19-21 In all these reactions, the glutathionyl, cysteinyl, and N-acetylcysteinyl functional groups were disassociated (removed).19-21 We have determined that DHP can also react with glutathione, cysteine, and N-acetylcysteine to form the corresponding 7-GS-DHP, 7-cysteineDHP, and 7-N-acetylcysteine-DHP, but as expected, the reactions are much slower than the dissociation of 7-GS-DHP, 7-cysteine-DHP, and 7-N-acetylcysteine-DHP into DHP.19-21 Also, 7-GS-DHP was metabolically dissociated into 7-cysteine-DHP.20 In addition, we have found that DHP-dA-3 and DHP-dA-4, as well as DHP-dG-3 and DHP-dG-4, are inter-convertible; and these inter-conversion reactions were through an SN1 mechanism.24 Recently, Chen et al. found that 9-GS-DHP, a predominant glutathione-DHP metabolite in the bile of rats treated with PAs, was inter-converted to 7-GS-DHP.129 Thus, the inter-conversion and dissociation reactions of the secondary metabolites strongly support the mechanism proposed by Edgar et al.7 Similar to the mechanism proposed by Edgar at al., we herewith propose a mechanism using the identified secondary metabolites 7-GS-DHP, 9-GS-DHP, 7-cysteine-DHP, and 7-N-acetylcysteine-DHP as examples (Figure 7).


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R

R O

O

O

O

9

O 7

O

P450

CH2

CH2

1

8

6

2

N 4

5

Dehydro-PA GSH NA C

CH2OH

SG

HO

N

3

PA

NAC

O O

HO

H2O CH2OH

N

N

9-GS-DHP

GS H, CY S

DNA

SG

N

7-NAC-DHP

DHP DN A

DNA

+

N

A DN

CYS

CH2OH

7-GS-DHP

CH2OH

N

7-CYS-DHP

DHP-dG-3 DHP-dG-4 DHP-dA-3 DNP-dA-4

DNA

HO

CH2OH

N

DHP

Liver Tumors

Figure 7. Proposed multiple metabolic activation pathways of PA-induced tumor formation, involving the inter-conversion of secondary metabolites. Based on this proposed mechanism, secondary metabolites should play a critical role on the PA-induced liver tumor initiation. The significant new findings can provide inside of the mechanistic activation. The proposed new research directions are as follows: (i)

Besides DHP esters (dehydro-PAs) and DHP, other reactive carcinogenic metabolites are likely to exist.

(ii)

There are multiple activation pathways, with each pathway generating the same set of DHP-DNA adducts in vivo, leading to liver tumor initiation.

(iii) It is important to determine the principal carcinogenic metabolite(s) of PAs. (iv) The formation of 7-GS-DHP has long been considered as a detoxification pathway.121125, 133

Nevertheless, our findings suggest that metabolic pathways leading to the

formation of 7-GS-DHP, 9-GS-DHP, 7-cysteine-DHP, and 7-N-acetylcysteine-DHP are potential activation pathways leading to liver tumor initiation.



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(v)

As earlier described, DHP esters (dehydro-PAs) can bind to cellular DNA and proteins to produce DNA adducts,16, 19-24, 29, 98, 99, 104, 106-113 DHP-protein adducts,7, 11, 51, 114-119, 134 DNA-DNA cross links, and DNA-protein cross links in vivo and/or in vitro.13-15, 86-88, 90, 91, 119

It warrants to investigate whether or not DNA adducts, DHP-protein adducts,

DNA-DNA cross links, and DNA-protein cross links can undergo inter-conversion and reversible reactions.

3.1.5. DHP-DNA adducts – A surrogate of PA carcinogenicity determination It may not be possible to assess the carcinogenic potency for many PAs and PA N-oxides by animal tumorigenicity bioassays. As such, it is desirable to develop an alternative mechanismbased, convenient, and reliable bioassay for risk assessment. Since it has been shown that a series of carcinogenic PAs and a PA N-oxide (retrorsine N-oxide) induced liver tumors mediated by the same set of DHP-DNA adducts, we propose that this metabolic activation pathway can potentially serve as a mechanism-based biological assay for assessing the PAs-induced and PA N-oxides-induced carcinogenicity. For the method validation, it is necessary to use a variety of different types of carcinogenic PAs as substrates and many different biological systems for method development, so that a reliable and convenient in vitro system, such as liver microsomal metabolism or in the cultured cells, can be developed for use.

3.1.6. Site and gene sequence-specificity of DNA adducts formation It has been previously described that riddelliine induced hemangiosarcomas in B6C3F1 mice resulting in G to T mutations at codon 12 of the K-ras oncogene,101 and that G:C to T:A transversion was the major type of mutation in rats treated with riddelliine.103 These mutation patterns support that DHP-dG adducts can lead to mutations and progression of cells from normality to neoplasia. However, there are apparent knowledge gap concerning DNA adduct formation, its relationship with the specific gene mutations, and ultimately with the tumor initiation induced by PAs. These warrant further investigation.

3.2. DHP-Protein Adducts for Clinical Assay The analytical method developed by Lin et al. has been successfully used to quantify DHPprotein adducts present in the blood of HSOS patients.11, 51 Our developed LC/MS/MS analytical 22 ACS Paragon Plus Environment

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method can accurately quantify DHP-protein adducts formed in rodent blood samples.119 It is our belief that, with further study, this newly developed LC/MS/MS analytical method can be used to serve as a convenient clinical bioassay. Furthermore, since the level of DHP-protein adducts well correlated with that of DHP-DNA adducts,119 with the further study for method development, we propose that DHP-protein adducts may potentially be used to serve as an invasive biomarker of PA carcinogenesis.

3.3. Regulation of PA-containing Products Many PAs and PA N-oxides present in herbal plants and herbal products are chemical carcinogens. Several PAs have been listed as probable human carcinogens by IARC and the NTP. Regulatory agencies around the world have issued bans and alerts on products containing PAs.7, 28, 135 In 1992, the Federal Health Department of Germany restricted that the herbal plants “may be sold and used only if daily external exposure to no more than 100 µg PAs and internal exposure to no more than 1 µg per day for no more than six weeks a year”.28 However, hundreds of toxic PAs and PA N-oxides are present in thousands of plants worldwide and their carcinogenic potency is drastically different. At the present, no practical analytical methods are available for quantifying the total quantity of toxic PAs. Furthermore, the regulation to limit the intake of the carcinogenic PAs by weight is not scientifically justified. Therefore, practical and reliable mechanism-based analytical methods must be developed to assess the risk posed by PAs. We propose that, upon metabolism of the herbal products in vivo, in vitro, or in the cultured cells, quantitation of the DHP-DNA adducts should be an assessable, reliable, and mechanism-based bioassay. For regulatory purpose, we believe this is a highly significant and worthy goal to pursue.

3.4. DNA-DNA Cross Links and DNA-Protein Cross Links As previously addressed, PAs can bind to DNA and proteins to form DNA-DNA links and DNA-protein crosslinks in cultured cells.13-15, 87, 93, 136 The experimental results obtained by Coulombe and co-workers14, 93 support the concept that the DNA-DNA and DNA-protein links are potential biomarkers of PA-induced tumorigenicity. Nevertheless, the structures of the DNA crosslink adducts have not been fully elucidated. The correlation between the levels of these cross links formed in vivo with the tumor potency of 23 ACS Paragon Plus Environment


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treated animals has yet to be examined. It is important to validate the metabolic formation of DNA-DNA and DNA-protein crosslinks in vivo as another important metabolic activation pathway leading to the PA-induced liver tumor initiation. Future studies should include: (i) the structural characterization of these cross links formed in vivo; (ii) identification and quantitation of their formation in vivo; and (iii) study of the relationship between their formation and the liver tumor potency of PAs and PA N-oxides on a structure-activity basis.

3.5. Other Type of Reactive Pyrrolic Metabolite(s) Segall et al.127 found that 1-formyl-7-hydroxy-6,7-dihvdro-5H-pyrrolizine (9-CHO-DHP) was a metabolite formed from the mouse liver microsomal metabolism of senecionine (Figure 6). Fashe et al.137 reported that human liver microsomal metabolism of retrorsine generated (3Hpyrrolizin-7-yl)methanol which can form glutathione conjugates. These two metabolites contain a DHP moiety and may be able to transfer DHP to DNA to initiate liver tumor formation. In addition, there may be other active metabolites that can contribute to liver tumor initiation.

4. CONCLUSIONS The study of chemical carcinogens initially requires determination of the metabolic activation pathway leading to carcinogenesis, which involves the identification of the ultimate carcinogenic metabolite. In general, the most important and convincing pathway of cancer induction is the mechanism involving the exogenous DNA adduct formation. The detection and quantitation of the exogenous DNA adducts formed in vivo requires the development of analytical methods for detection and quantitation. With the analytical method developed available, the ultimate carcinogenic metabolite(s) can be determined. The determination of such an activation pathway that is responsible for tumor initiation is always challenging. Many carcinogenic PAs and PA N-oxides are carcinogenic, and PA-containing plants are the most common poisonous and possibly carcinogenic plants affecting livestock, wildlife, and humans. We recently determined that there is a common genotoxic mechanism mediated by DHP-DNA adducts. The levels of the DHP-DNA adducts correlated with the liver tumor potency of a series of PAs with different structural features. This finding is particularly important because this allows studying the mechanism for a whole family of PAs carcinogens, not a single carcinogenic PA. 24 ACS Paragon Plus Environment

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The new findings that several secondary metabolites can be dissociated and inter-convertible in vitro strongly support the mechanism proposed by Edgar et al.7 Because of the all abovedescribed significant new findings, we therefore consider that part of the future research will be focused on these research areas. At the same time, we also believe that the activation pathway mediated by DNA-DNA and DNA-protein cross links is equally important, and should be the focus of further investigations as well.

AUTHOR INFORMATION Corresponding Author *(P.P.F.) E-mail: [email protected].

Notes The author declares no competing ﬁnancial interest.

ACKNOWLEDGMENTS The author thanks Dr. Frederick A. Beland for critical review and Dr. Qingsu Xia and Ashley Groves for the preparation of this manuscript. The views presented in this paper do not necessarily represent those of the U.S. Food and Drug Administration (FDA). No official support or endorsement by the U.S. FDA is intended or should be inferred.

FUNDING SOURCES Funding was provided by an Internal FDA Fund.

ABBREVIATIONS PA, pyrrolizidine alkaloid; DHR, dehydroretronecine or (-)-R-6,7-dihydro-7-hydroxy-1hydroxymethyl-5H- pyrrolizine); DHP, (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5Hpyrrolizine; dG, 2’-deoxyguanosine; dA, 2’-deoxyadenosine; DHP-dG-3 and DHP-dG-4, a pair of epimers of 7-hydroxy-9-(deoxyguanosin-N2-yl)dehydrosupinidine adducts; DHP-dA-3 and DHP-dA-4, a pair of epimers of 7-hydroxy-9-(deoxyadenosin-N6-yl)dehydrosupinidine adducts; 25 ACS Paragon Plus Environment


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HPLC-ES-MS/MS, high-performance liquid chromatography electrospray ionization tandem mass spectrometry; NCTR, National Center for Toxicological Research; NTP, National Toxicology Program; MRM, multiple reaction monitoring.


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Biography Peter P. Fu received his Ph.D. degree in Chemistry at the University of Illinois at Chicago in 1973, he worked at the Ben May Institute for Cancer Research, University of Chicago on the mechanism by which polycyclic aromatic hydrocarbons induce cancer. Since working at the Biochemical Toxicology Division, National Center for Toxicological Research (NCTR) in 1979, his mechanistic studies on tumor induction provided mechanism-based information highly useful for subsequent carcinogenic risk assessments and regulatory decisions by FDA. His on-going research focuses on mechanistic studies of the genotoxicity and tumorigenicity of pyrrolizidine alkaloids, nanomaterials, and herbal dietary supplements of FDA interest.


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