Formation of DHP-DNA Adducts from Rat Liver Microsomal Metabolism of 1,2-Unsaturated Pyrrolizidine Alkaloid-Containing Plant Extracts and Dietary Supplements.

1,2-Unsaturated pyrrolizidine alkaloids (PAs) are carcinogenic phytochemicals. We previously determined that carcinogenic PAs and PA N-oxides commonly form a set of four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA adducts, namely, DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4. This set of DHP-DNA adducts has been implicated as a potential biomarker of PA-induced liver tumor initiation from metabolism of individual carcinogenic PAs. To date, it is not known whether this generality occurs from metabolism of PA-containing plant extracts. In this study, we investigate the rat liver microsomal metabolism of nine PA-containing plant extracts and two PA-containing dietary supplements in the presence of calf thymus DNA. The presence of carcinogenic PAs and PA N-oxides in plant extracts was first confirmed by LC-MS/MS analysis with selected reaction monitoring mode. Upon rat liver microsomal metabolism of these PA-containing plant extracts and dietary supplements, the formation of this set of DHP-DNA adducts was confirmed. Thus, these results indicate that metabolism of PA-containing plant extracts and dietary supplements can generate DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts, thereby potentially initiating liver tumor formation.

Liquorice Extract and 18ß-Glycyrrhetinic Acid Protect Against Experimental Pyrrolizidine Alkaloid-Induced Hepatotoxicity in Rats Through Inhibiting Cytochrome P450-Mediated Metabolic Activation.

Misuse of pyrrolizidine alkaloid (PA)-containing plants or consumption of PA-contaminated foodstuffs causes numerous poisoning cases in humans yearly, while effective therapeutic strategies are still limited. PA-induced liver injury was initiated by cytochrome P450 (CYP)-mediated metabolic activation and subsequent formation of adducts with cellular proteins. Liquorice, a hepato-protective herbal medicine, is commonly used concurrently with PA-containing herbs in many compound traditional Chinese medicine formulas, and no PA-poisoning cases have been reported with this combination. The present study aimed to investigate hepato-protective effects of liquorice aqueous extract (EX) and 18ß-glycyrrhetinic acid (GA, the primary bioactive constituent of liquorice) against PA-induced hepatotoxicity and the underlying mechanism. Histopathological and biochemical analysis demonstrated that both single- and multiple-treatment of EX (500 mg/kg) or GA (50 mg/kg) significantly attenuated liver damage caused by retrorsine (RTS, a representative hepatotoxic PA). The formation of pyrrole-protein adducts was significantly reduced by single- (30.3% reduction in liver; 50.8% reduction in plasma) and multiple- (32.5% reduction in liver; 56.5% reduction in plasma) treatment of GA in rats. Single- and multiple-treatment of EX also decreased the formation of pyrrole-protein adducts, with 30.2 and 31.1% reduction in rat liver and 51.8 and 53.1% reduction in rat plasma, respectively. In addition, in vitro metabolism assay with rat liver microsomes demonstrated that GA reduced the formation of metabolic activation-derived pyrrole-glutathione conjugate in a dose-dependent manner with the estimated IC50 value of 5.07 µM. Further mechanism study showed that GA inhibited activities of CYPs, especially CYP3A1, the major CYP isoform responsible for the metabolic activation of RTS in rats. Enzymatic kinetic study revealed a competitive inhibition of rat CYP3A1 by GA. In conclusion, our findings demonstrated that both EX and GA exhibited significant hepato-protective effects against RTS-induced hepatotoxicity, mainly through the competitive inhibition of CYP-mediated metabolic activation of RTS.

Phototoxicity of herbal plants and herbal products.

Plants are used by humans in daily life in many different ways, including as food, herbal medicines, and cosmetics. Unfortunately, many natural plants and their chemical constituents are photocytotoxic and photogenotoxic, and these phototoxic phytochemicals are widely present in many different plant families. To date, information concerning the phototoxicity and photogenotoxicity of many plants and their chemical constituents is limited. In this review, we discuss phototoxic plants and their major phototoxic constituents; routes of human exposure; phototoxicity of these plants and their constituents; general mechanisms of phototoxicity of plants and phototoxic components; and several representative phototoxic plants and their photoactive chemical constituents.

Phototoxicity of kava - formation of reactive oxygen species leading to lipid peroxidation and DNA damage.

Kava is one of the most widely sold herbal dietary supplements in the United States. It has been reported that, besides exhibiting hepatotoxicity, kava also possesses photosensitivity and induces dermopathy in humans. In this study, we determined that UVA irradiation of kava in the presence of a lipid, methyl linoleate, generated lipid peroxidation which was mediated by singlet oxygen generated during photoirradiation. The six major kavalactones(yangonin, 7,8-dihydrokawa in, kawain, 7,8-dihydromethysticin, methysticin, and 5,6-dehydrokawain) were also studied in parallel; only 5,6-dehydrokawain and yangonin-induced a low level of lipid peroxidation. UVA irradiation of kava in human HaCaT skin keratinocytes induced cytotoxicity which was mediated by oxidative stress, led to DNA strand cleavage, and produced 8-hydroxy-2'-deoxyguanosine (8-OHdG) adduct. Study by the electron spin resonance (ESR) method revealed that UVA irradiation of kava produced singlet oxygen and carbon-centered radicals. The overall results suggest that kava is photocytotoxic and photogenotoxic, both mediated by free radicals generated during photoirradiation.

Nanoscale ZnO induces cytotoxicity and DNA damage in human cell lines and rat primary neuronal cells.

The toxic effects of ZnO nanoparticles (nano-ZnO) (1-100 microg/mL) suspended in DMEM were examined in human A549 cells, HepG2 cells, human skin fibroblast cells, human skin keratinocytes, and rat primary neuronal cells for 24 h. Nano-ZnO induced dose dependent cytotoxicity and damaged cell membranes. Cell death was not mediated by reactive oxygen species (ROS) or apoptosis. Nano-ZnO induced DNA damage in rat primary neuronal cells, human fibroblasts, and A549 cells. The cytotoxicity of nano-ZnO in DMEM supplemented with 10% FBS, instead of serum free DMEM, was also examined in the A549 cells, human skin fibroblast cells, and human skin keratinocytes. The levels of cytotoxicity induced were similar to those tested without FBS; in addition, ROS was observed. These results indicate that the cause of cytotoxicity is medium dependent and imply that cellular growth conditions may play a significant role in induction of cytotoxicity and DNA damage by nano-ZnO.

Characteristic ion clusters as determinants for the identification of pyrrolizidine alkaloid N-oxides in pyrrolizidine alkaloid-containing natural products using HPLC-MS analysis.

Pyrrolizidine alkaloid (PA)-containing plants are widely distributed in the world. PAs are hepatotoxic, affecting livestock and humans. PA N-oxides are often present together with PAs in plants and also exhibit hepatotoxicity but with less potency. HPLC-MS is generally used to analyze PA-containing herbs, although PA references are unavailable in most cases. However, to date, without reference standards, HPLC-MS methodology cannot distinguish PA N-oxides from PAs because they both produce the same characteristic ions in mass spectra. In the present study, the mass spectra of 10 PA N-oxides and the corresponding PAs were systemically investigated using HPLC-MS to define the characteristic mass fragment ions specific to PAs and PA N-oxides. Mass spectra of toxic retronecine-type PA N-oxides exhibited two characteristic ion clusters at m/z 118-120 and 136-138. These ion clusters were produced by three unique fragmentation pathways of PA N-oxides and were not found in their corresponding PAs. Similarly, the nontoxic platynecine-type PA N-oxides also fragmented via three similar pathways to form two characteristic ion clusters at m/z 120-122 and 138-140. Further application of using these characteristic ion clusters allowed successful and rapid identification of PAs and PA N-oxides in two PA-containing herbal plants. Our results demonstrated, for the first time, that these characteristic ion clusters are unique determinants to discriminate PA N-oxides from PAs even without the availability of reference samples. Our findings provide a novel and specific method to differentiate PA N-oxides from PAs in PA-containing natural products, which is crucial for the assessment of their intoxication.

Hepatotoxicity and tumorigenicity induced by metabolic activation of pyrrolizidine alkaloids in herbs.

In the recent decades, the use of herbal products has been rapidly growing in the Western countries. While their use in many cases causes adverse effects, to date, safety issues of herbal products have not been adequately addressed. It is rarely determined whether the non-purported bioactive constituents in the herbs and the metabolites of the bioactive components can lead to adverse effects. In this review, we discuss, using pyrrolizidine alkaloids (PAs) as an example, the hepatotoxicity and tumorigenicity induced by metabolic activation of herbal components and by herb-herb and herb-drug interactions with other herbal ingredients and synthetic drugs. PAs are constitutively produced by plants as the secondary metabolites. There are more than 600 PAs and PA N-oxides identified in over 6000 plants, and more than half of them exhibit hepatotoxicity. Toxic PA-containing plants grow in many geographical regions worldwide, rendering it highly possible that PA-containing plants are the most common poisonous plants affecting livestock and humans. PAs require metabolic activation mediated by cytochrome P450 enzymes to generate reactive pyrrolic metabolites that react with cellular proteins and DNA leading to hepatotoxicity and genotoxicity. PAs can also modulate both phase I and phase II metabolizing enzymes, which may alter the metabolic fate of endogenous and exogenous chemicals. Alteration and/or competition of the metabolizing enzymes by PAs upon the co-administered herbal medicines or drugs can potentially result in serious clinical and toxicological consequences through decreased pharmacological activities or increased toxic effects.

Gene expression profiling as an initial approach for mechanistic studies of toxicity and tumorigenicity of herbal plants and herbal dietary supplements.

Dietary supplements are consumed by more than 300 million people worldwide, and herbal dietary supplements represent the most rapidly growing portion of this industry. Even though adverse health effects of many herbal dietary supplements have been reported, safety assurances are not being addressed adequately. Toxicological data on the identification of genotoxic and tumorigenic ingredients in many raw herbs are also lacking. Currently, more than 30 herbal dietary supplements and active ingredients have been selected by the National Toxicology Program (NTP) for toxicity and tumorigenicity studies. Due to the complexity of the chemical components present in plant extracts, there are no established methodologies for determining the mechanisms of toxicity (particularly tumorigenicity) induced by herbs, such as Gingko biloba leaf extract (GBE) and other herbal plant extracts. Consequently, the understanding of toxicity of herbal dietary supplements remains limited. We have proposed that application of DNA microarrays could be a highly practical initial approach for revealing biological pathways and networks associated with toxicity induced by herbal dietary supplements and the generation of hypotheses to address likely mechanisms. The changes in expression of subsets of genes of interest, such as the modulation of drug metabolizing genes, can be analyzed after treatment with an herbal dietary supplement. Although levels of gene expression do not represent fully the levels of protein activities, we propose that subsequent biochemical and genomic experiments based on these initial observations will enable elucidation of the mechanisms leading to toxicity, including tumorigenicity. This review summarizes the current practices of microarray analysis of gene expressions in animals treated with herbal dietary supplements and discusses perspectives for the proposed strategy.

Gene expression profiling in male B6C3F1 mouse livers exposed to kava identifies--changes in drug metabolizing genes and potential mechanisms linked to kava toxicity.

The association of kava products with liver-related health risks has prompted regulatory action in many countries. We used a genome-wide gene expression approach to generate global gene expression profiles from the livers of male B6C3F1 mice administered kava extract by gavage for 14 weeks, and identified the differentially expressed drug metabolizing genes in response to kava treatments. Analyses of gene functions and pathways reveal that the levels of significant numbers of genes involving drug metabolism were changed and that the pathways involving xenobiotics metabolism, Nrf2-mediated oxidative stress response, mitochondrial functions and others, were altered. Our results indicate that kava extract can significantly modulate drug metabolizing enzymes, potentially leading to herb-drug interactions and hepatotoxicity.

Quality assurance and safety of herbal dietary supplements.

Since the U.S. Congress passed the Dietary Supplement Health and Education Act (DSHEA) in 1994, use of herbal products has been growing rapidly worldwide. To ensure consumer health protection, the quality and safety of herbal plants, particularly those used for dietary supplement preparations, must be determined. To date, toxicological data on the identification of genotoxic and tumorigenic ingredients in many raw herbs and their mechanisms of action are lacking. Thus, identification of carcinogenic components in herbal plants is timely and important. In this review, the issues of quality control and safety evaluation of raw herbs and herbal dietary supplements are discussed. Two examples of tumorigenicity and mechanism of tumor induction are discussed: aristolochic acid and riddelliine, both of which have been detected in Chinese herbal plants. It is proposed that an organized effort with international participation on cancer risk assessment should be actively pursued so that the safety of commercial herbal plants and herbal dietary supplements can be ensured.

Analysis of gene expression changes of drug metabolizing enzymes in the livers of F344 rats following oral treatment with kava extract.

The association of kava product use with liver-related risks has prompted regulatory action in many countries. We studied the changes in gene expression of drug metabolizing enzymes in the livers of Fischer 344 male rats administered kava extract by gavage for 14 weeks. Analysis of 22,226 genes revealed that there were 14, 41, 110, 386, and 916 genes significantly changed in the 0.125, 0.25, 0.5, 1.0, and 2.0 g/kg treatment groups, respectively. There were 16 drug metabolizing genes altered in all three high-dose treatment groups, among which seven genes belong to cytochrome P450 isozymes. While gene expression of Cyp1a1, 1a2, 2c6, 3a1, and 3a3 increased; Cyp 2c23 and 2c40 decreased, all in a dose-dependent manner. Real-time PCR analyses of several genes verified these results. Our results indicate that kava extract can significantly modulate drug metabolizing enzymes, particularly the CYP isozymes, which could cause herb-drug interactions and may potentially lead to hepatotoxicity.

Toxicity of kava kava.

Kava is a traditional beverage of various Pacific Basin countries. Kava has been introduced into the mainstream U.S. market principally as an anti-anxiety preparation. The effects of the long-term consumption of kava have not been documented adequately. Preliminary studies suggest possible serious organ system effects. The potential carcinogenicity of kava and its principal constituents are unknown. As such, kava extract was nominated for the chronic tumorigenicity bioassay conducted by the National Toxicology Program (NTP). At present toxicological evaluation of kava extract is being conducted by the NTP. The present review focuses on the recent findings on kava toxicity and the mechanisms by which kava induces hepatotoxicity.

Ginkgo biloba leave extract: biological, medicinal, and toxicological effects.

Ginkgo biloba leave extract is among the most widely sold herbal dietary supplements in the United States. Its purported biological effects include: scavenging free radical; lowering oxidative stress; reducing neural damages, reducing platelets aggregation; anti-inflammation; anti-tumor activities; and anti-aging. Clinically, it has been prescribed to treat CNS disorders such as Alzheimer's disease and cognitive deficits. It exerts allergy and changes in bleeding time. While its mutagenicity or carcinogenic activity has not been reported, its components, quercetin, kaempferol and rutin have been shown to be genotoxic. There are no standards or guidelines regulating the constituent components of Ginkgo biloba leave extract nor are exposure limits imposed. Safety evaluation of Ginkgo biloba leave extract is being conducted by the U.S. National Toxicology Program.

Synthesis and photoirradiation of isomeric ethylchrysenes by UVA light leading to lipid peroxidation.

Polycyclic aromatic hydrocarbons (PAHs) are widespread genotoxic environmental pollutants. We have recently demonstrated that photoirradiation of PAHs leads to cytotoxicity, DNA damage, and induction of lipid peroxidation. In this paper we report the synthesis of all the six isomeric ethylchrysenes and the study of light-induced lipid peroxidation by these ethylchrysenes. 5-Ethylchrysene was synthesized by reaction of 5-keto-5,6,6a,7,8,9,10,10a-octahydrochrysene with CH3CH2MgBr followed by dehydration catalyzed by p-toluenesulfonic acid and dehydrogenation with DDQ in benzene. 1- and 4-Ethylchrysenes were similarly prepared by reaction of 1-keto-1,2,3,4,5,6-hexahydrochrysene and 4-keto-1,2,3,4-tetrahydrochrysenes, respectively with CH3CH2MgBr followed by dehydration and dehydrogenation. Direct acetylation of chrysene followed by Wolff-Kishner or Clemmensen reduction resulted in the formation of 2-, 3-, and 6-ethylchrysenes in 4%, 16%, and 43% yields, respectively. Photoirradiation of these compounds with 7 and 21 J/cm2 UVA light in the presence of methyl linoleate all resulted in lipid peroxidation. For comparison, photoirradiation of 4-methylchrysene and 5-methylchrysene was similarly conducted. For irradiation at a UVA light dose of 21 J/cm2, the level of induced lipid peroxidation is in the order 4-methylchrysene = 5-methylchrysene = 5-ethylchrysene = 4-ethylchrysene = chrysene > 1-ethylchrysene = 2-ethylchrysene > 3-ethylchrysene > 6-ethylchrysene. Compared with chrysene, these results indicate that the ethyl group at C4 or C5 position either slightly enhances or has no effect on the light-induced lipid peroxidation, while at C1-, C2-, C3-, or C6 position reduces light-induced lipid peroxidation.

UVA photoirradiation of methylated benzo[a]pyrene and benzo[e]pyrene leading to induction of lipid peroxidation.

Polycyclic aromatic hydrocarbons (PAHs) are widespread genotoxic environmental pollutants and potentially pose a health risk to humans. Although the biological and toxicological activities, including metabolism, mutagenicity and carcinogenicity of PAHs have been thoroughly studied, their phototoxicity and photo-induced biological activities have not been well examined. In this research, we studied the photoirradiation of isomeric methylbenzo[a]pyrene (MBaP) and methylbenzo[e]pyrene (MBeP) by UVA light in the presence of a lipid, methyl linoleate, and evaluated the potential of these compounds to induce lipid peroxidation. The compounds chosen for study included BaP, 3-MBaP, 4-MBaP, 6-MBaP, 7-MBaP, 10-MBaP, BeP, 4-MBeP, and 9-MBeP. The results indicate that upon photoirradiation by UVA at 7 and 21 J/cm2, these compounds induced lipid peroxidation. The levels of the induced lipid peroxidation were similar among BaP and the isomeric MBaPs, and among the BeP and MBePs, with the BaP group higher than the BeP group. There was also a co-relation between the UV A light dose and the level of lipid peroxidation induced. Lipid peroxide formation was inhibited by NaN3 (singlet oxygen and free radical scavenger) and was enhanced by the presence of deuterium oxide (D2O) (extends singlet oxygen lifetime). These results suggest that photoirradiation of MBaPs and MBePs by UVA light generates reactive oxygen species (ROS), which induce lipid peroxidation.

Photo-irradiation of Aloe vera by UVA--formation of free radicals, singlet oxygen, superoxide, and induction of lipid peroxidation.

Aloe vera whole leaf extracts are incorporated into a wide variety of topically applied commercial products. Aloe vera whole leaf extracts may contain anthraquinones, which have been shown to generate reactive oxygen species in the presence of ultraviolet A (UVA) light. Exposure to UVA light alone can also generate reactive oxygen species and is associated with photo-damaged and photo-aged skin in humans. This paper examines the photochemical properties of two Aloe vera whole leaf extracts that differed in their anthraquinone content. In the presence of methyl linoleate, the UVA irradiation of Aloe vera leaf extracts induced lipid peroxidation. The amounts of lipid peroxides formed were higher in the Aloe vera leaf extract that contained lower amounts of anthraquinones. Superoxide dismutase and sodium azide inhibited and deuterium oxide enhanced the formation of lipid peroxides, suggesting that singlet oxygen and superoxide were involved in the mechanism. Spin trapping electron spin resonance (ESR) spectroscopy was used to investigate the generation of free radicals by the UVA photo-irradiated Aloe vera plant extracts. ESR measurements indicated that the UVA photo-irradiation of Aloe vera plant extracts produced carbon-centered free radicals. These results suggest that humans exposed to products that contain Aloe vera whole leaf extracts may have enhanced sensitivity to ultraviolet light.

Metabolic formation of DHP-derived DNA adducts from a representative otonecine type pyrrolizidine alkaloid clivorine and the extract of Ligularia hodgsonnii hook.

Plants that contain pyrrolizidine alkaloids (PAs) are widely distributed, and PAs have been shown to be genotoxic and tumorigenic in experimental animals. Our recent mechanistic studies indicated that riddelliine, a tumorigenic retronecine type PA, induced tumors via a genotoxic mechanism mediated by the formation of a set of eight 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts. However, it is not known if this mechanism is general to PAs of other types. In this study, we report that the metabolism of clivorine, a tumorigenic otonecine type PA, by F344 rat liver microsomes results in DHP formation. When incubations were conducted with clivorine in the presence of calf thymus DNA, eight DHP-derived DNA adducts were formed. The Ligularia hodgsonnii Hook plant, an antitussive traditional Chinese medicine, was found to contain otonecine type PAs with clivorine being predominant. DHP and DHP-derived DNA adducts were also obtained when microsomal incubations were conducted with extracts of L. hodgsonnii Hook. This is the first report that DHP-derived DNA adducts are formed from the metabolic activation of otonecine type PA and that these DHP-derived DNA adducts are potential biomarkers of PA exposure and PA-induced tumorigenicity. These results also provide evidence that the principal metabolic activation pathway of clivorine leading to liver genotoxicity and tumorigenicity is (i) formation of the corresponding dehydropyrrolizidine (pyrrolic) derivative through oxidative N-demethylation of the necine base followed by ring closure and dehydration and (ii) binding of the pyrrolic metabolite to DNA leading to the DNA adduct formation and tumor initiation.