Annonaceae acetogenins: A potential treatment for gynecological and breast cancer.

Breast and gynecological cancers are major health concerns due to their increasing incidence rates, and in some cases, their low survival probability. In recent years, multiple compounds of natural origin have been analyzed as alternative treatments for this disease. For instance, Acetogenins are plant secondary metabolites from the Annonaceae family, and its potential anticancer activity has been reported against a wide range of cancer cells both in vitro and in vivo. Several studies have demonstrated promising results of Acetogenins' antitumor capacity, given their selective activity of cellular inhibition at low concentrations. This review outlines the origin, structure, and antineoplastic activities in vitro and in vivo of Acetogenins from Annonaceae against breast cancer and gynecological cancers reported to date. Here, we also provide a systematic summary of the activity and possible mechanisms of action of Acetogenins against these types of cancer and provide references for developing future therapies based on Acetogenins and nanotechnologies.

Nanoarchitectonics of an acetogenin-enriched nanosystem mediated by an aqueous extract ofAnnona cherimolaMill with anti-inflammatory and proapoptotic activity against HepG2 cell line.

For the first time, this study shows the nanoarchitectonic process to obtain an acetogenin-enriched nanosystem (AuNPs-Ac) using an aqueous extract fromAnnona cherimolaMill (ACM) composed of gold nanoparticles embedded in an organic matrix that acts as stabilizing agent and presents anti-inflammatory activity and cytotoxical effect against HepG2 cell line, promoting apoptosis. The synthesis of AuNPs-Ac was confirmed by x-ray diffraction analysis, showing metallic gold as the only phase, and the scanning transmission microscope showed an organic cap covering the AuNPs-Ac. Fourier-transformed infrared suggests that the organic cap comprises a combination of different annonaceous acetogenins, alkaloids, and phenols by the presence of bands corresponding to aromatic rings and hydroxyl groups. High-Performance Liquid Chromatography has demonstrated the presence of annonacin, a potent acetogenin, in the extract of ACM. Anin vitroanti-inflammatory activity of the extract of ACM and the AuNPs-Ac was performed using the albumin denaturation method, showing a nonlinear response, which is better than sodium diclofenac salt in a wide range of concentrations that goes from 200 to 400µg ml-1with both samples. The viability assay was studied using trypan blue, treating IMR90 and HepG2 at different concentrations of AuNPs-Ac. The results defined a median lethal dose of 800µg ml-1against HepG2 through apoptosis according to the ratio of caspase-cleaved 9/alpha-tubulin evaluated. It was also demonstrated that the nanosystem presents a higher cytotoxic effect on the HepG2 cell line than in IMR90, suggesting a targeted mechanism. In addition, the nanosystem performs better than using only the extract of ACM in the anti-inflammatory or antiproliferative test, attributed to their higher surface area.

In Vitro and In Silico Evaluation of Red Algae Laurencia obtusa Anticancer Activity.

Studies estimate that nearly 2 million new cases of gastric cancer will occur worldwide during the next two decades, which will increase mortality associated with cancer and the demand for new treatments. Marine algae of the Laurencia genus have secondary metabolites known for their cytotoxic action, such as terpenes and acetogenins. The species Laurencia obtusa has demonstrated cytotoxicity against many types of tumors in previous analyses. In this study, we determined the structure of terpenes, acetogenins, and one fatty acid of Laurencia using mass spectrometry (ESI-FT-ICR/MS). In vitro cytotoxicity assays were performed with adenocarcinoma gastric cells (AGS) to select the most cytotoxic fraction of the crude extract of L. obtusa. The Hex:AcOEt fraction was the most cytotoxic, with IC50 9.23 µg/mL. The selectivity index of 15.56 shows that the Hex:AcOEt fraction is selective to cancer cells. Compounds obtained from L. obtusa were tested by the analysis of crystallographic complexes. Molecular docking calculations on the active site of the HIF-2α protein showed the highest affinity for sesquiterpene chermesiterpenoid B, identified from HEX:AcOEt fraction, reaching a score of 65.9. The results indicate that L. obtusa presents potential compounds to be used in the treatment of neoplasms, such as gastric adenocarcinoma.

Antileishmanial Effects of Acetylene Acetogenins from Seeds of Porcelia macrocarpa (Warm.) R.E. Fries (Annonaceae) and Semisynthetic Derivatives.

As part of our continuous studies involving the prospection of natural products from Brazilian flora aiming at the discovery of prototypes for the development of new antiparasitic drugs, the present study describes the isolation of two natural acetylene acetogenins, (2S,3R,4R)-3-hydroxy-4-methyl-2-(n-eicos-11'-yn-19'-enyl)butanolide (1) and (2S,3R,4R)-3-hydroxy-4-methyl-2-(n-eicos-11'-ynyl)butanolide (2), from the seeds of Porcelia macrocarpa (Warm.) R.E. Fries (Annonaceae). Using an ex-vivo assay, compound 1 showed an IC50 value of 29.9 µM against the intracellular amastigote forms of Leishmania (L.) infantum, whereas compound 2 was inactive. These results suggested that the terminal double bond plays an important role in the activity. This effect was also observed for the semisynthetic acetylated (1a and 2a) and eliminated (1b and 2b) derivatives, since only compounds containing a double bond at C-19 displayed activity, resulting in IC50 values of 43.3 µM (1a) and 23.1 µM (1b). In order to evaluate the effect of the triple bond in the antileishmanial potential, the mixture of compounds 1 + 2 was subjected to catalytic hydrogenation to afford a compound 3 containing a saturated side chain. The antiparasitic assays performed with compound 3, acetylated (3a), and eliminated (3b) derivatives confirmed the lack of activity. Furthermore, an in-silico study using the SwissADME online platform was performed to bioactive compounds 1, 1a, and 1b in order to investigate their physicochemical parameters, pharmacokinetics, and drug-likeness. Despite the reduced effect against amastigote forms of the parasite to the purified compounds, different mixtures of compounds 1 + 2, 1a + 2a, and 1b + 2b were prepared and exhibited IC50 values ranging from 7.9 to 38.4 µM, with no toxicity for NCTC mammalian cells (CC50 > 200 µM). Selectivity indexes to these mixtures ranged from >5.2 to >25.3. The obtained results indicate that seeds of Porcelia macrocarpa are a promising source of interesting prototypes for further modifications aiming at the discovery of new antileishmanial drugs.

Evaluation of Genotoxicity and Toxicity of Annona muricata L. Seeds and In Silico Studies.

Cancer is a multifactorial organic dysfunction for which great efforts are being devoted in searching for new treatments and therapeutic adjuvants. Annona muricata is a fruit that has promising activity against several types of cancer, as it contains acetogenins, the metabolite group associated with this action. Thus, the objective of this study was to evaluate, in experimental models, the toxic behavior of an extract and fraction rich in acetogenins from A. muricata seeds and study the acetogenin, Annonacin, in silico. Phytochemical characterization was made by thin layer chromatography, spectroscopy in the infrared region and nuclear magnetic resonance. Toxicity was evaluated by tests of Allium cepa and Artemia salina, and in silico studies using the SwissDock servers DockThor, PharmMapper, ADMETLab, PreADME, Osiris and ProTox. The extract and fraction showed genotoxic activity against meristematic cells of A. cepa, reducing the mitotic index; however, the extract produced great deleterious effects on the system, even causing cell necrosis. In A. Saline, the extract was more toxic than the fraction, but both samples were considered toxic. Annonacin was effectively linked to complex I, and presented different activities regarding toxicity. Thus, the results of this study are promising, highlighting the anticancer potential of acetogenins.

Effects of Acetogenins from Annona coriacea on the in Vitro Reactions of Photosynthesis.

Our search for candidates for photosynthesis inhibitors is allowing us to report the effect of two acetogenins identified in Annona coriacea Mart. leaves, ACG-A and ACG-B, a non-adjacent bis-THF and a mono-THF types, respectively. This is an important class of natural products which presents biological properties such as anticancer, neurotoxic, larvicidal and insecticidal. However, this is only the second report associated to its herbicidal activity. Their mechanisms of action on the light reactions of the photosynthesis were elucidated by polarographic techniques. Compounds inhibited the noncyclic electron transport on basal, phosphorylating, and uncoupled conditions from H2 O to methyl viologen (MV); therefore, they act as Hill reaction inhibitors. Studies on fluorescence of chlorophyll a (ChL a) indicated that they inhibited the acceptor side of PSII between P680 and PQ-pool, exactly as the commercial herbicide DCMU does.

Acetogenin-Based Formulated Bioinsecticides on Anastrepha fraterculus: Toxicity and Potential Use in Insecticidal Toxic Baits.

The present study evaluated the lethal toxicity and oviposition deterrence of ethanolic extracts of Annona mucosa Jacq., Annona muricata L., and Annona sylvatica A. St.-Hil on Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) compared with those of a limonoid-based bioinsecticide (Azamax™ 1.2 EC-azadiractin +3-tigloyl-azadiractol) and a synthetic spinosyn-based insecticide (Delegate™ 250 WG-spinetoram). In addition, the efficacy of the selected toxic bait formulations was evaluated by mixing them with food attractants (Anamed™, 3% Biofruit and 7% sugarcane molasses). In the topical application and ingestion bioassays (2000 mg L-1), the aqueous emulsion of the A. mucosa extract caused greater than 80% mortality of A. fraterculus adults in a similar manner to the spinosyn-based synthetic insecticide. Concentration-response curves were performed for the most promising treatments and showed an activity level dependent on the mode of contamination, exposure time, and applied concentration. In bioassays with and without choice, the A. mucosa (77%), A. muricata (51%), A. sylvatica (60%), Azamax™ (74%), and Delegate™ 250 WG (100%) significantly reduced the number of punctures and galleries in grape berries. In combination with the food attractants Anamed™, 3% Biofruit, and 7% sugarcane molasses, the emulsion of the A. mucosa extract had a residual effect similar to that of the spinetoram insecticide, with a mortality rate of over 80% of A. fraterculus adults up to 14 days after application (DAA) in the absence of rain. Thus, acetogenin-rich formulations, especially from A. mucosa seeds, are useful alternatives for the integrated management of A. fraterculus in agricultural orchards.

Chemical Profile and Safety Assessment of a Food-Grade Acetogenin-Enriched Antimicrobial Extract from Avocado Seed.

Acetogenins are bioactive fatty acid derivatives found in avocado tissues. Their efficacy as antimicrobials has been documented and initiated interest to use them as replacements of synthetic food additives. The present work focused on evaluation of multiple analytical methodologies for detection and quantification of organic solids present in a food-grade acetogenin-enriched extract (Avosafe®), and on its safety evaluations using bacterial reverse mutation (AMES) tests and acute oral toxicity to rat assays. Results confirmed chemical structures of two acetogenins as present in Avosafe® (AcO-avocadyne-(0) and AcO-avocadiene B-(3)), and together with seven other previously known compounds, quantified 94.74 ± 5.77% w/w of its solids as acetogenins. Safety evaluations indicated that Avosafe® was non-mutagenic and had an acute median lethal oral dose (LD50) to rats higher than the maximum concentration tested (>2000 mg·kg-1), with no signs of macroscopic abnormalities in organs. Mean body weight and hematological and biochemical parameters were normal after 14 days of a single oral dose of 2000 mg·kg-1. The results advance scientific information on the safety of avocado seed acetogenins and also generate new knowledge on profiles and concentrations of individual acetogenins found in avocado tissues (seed, pulp, and leaves) and in Avosafe®.

Antitrypanosomal Activity of Acetogenins Isolated from the Seeds of Porcelia macrocarpa Is Associated with Alterations in Both Plasma Membrane Electric Potential and Mitochondrial Membrane Potential.

As part of a drug discovery program aimed at the identification of anti- Trypanosoma cruzi metabolites from Brazilian flora, four acetogenins (1-4) were isolated from the seeds of Porcelia macrocarpa and were identified by NMR spectroscopy and HRESIMS. The new compounds 1 and 2 displayed activity against the trypomastigote (IC50 = 0.4 and 3.6 µM) and amastigote (IC50 = 23.0 and 27.7 µM) forms. The structurally related known compound 3 showed less potency to the amastigotes, with an IC50 value of 58 µM, while the known compound 4 was inactive. To evaluate the potential mechanisms for parasite death, parameters were evaluated by fluorometric assays: (i) plasma membrane permeability, (ii) plasma membrane electric potential (ΔΨp), (iii) reactive oxygen species production, and (iv) mitochondrial membrane potential (ΔΨm). The results obtained indicated that compounds 1 and 2 depolarize plasma membranes, affecting ΔΨp and ΔΨm and contributing to the observed cellular damage and disturbing the bioenergetic system. In silico studies of pharmacokinetics and toxicity (ADMET) properties predicted that all compounds were nonmutagenic, noncarcinogenic, nongenotoxic, and weak hERG blockers. Additionally, none of the isolated acetogenins 1-4 were predicted as pan-assay interference compounds.

Cytotoxic Acetogenins from the Roots of Annona purpurea.

Annona purpurea, known in Mexico as "cabeza de negro" or "ilama", belongs to the Annonaceae family. Its roots are employed in folk medicine in several regions of Mexico. Taking that information into account, a chemical and biological analysis of the components present in the roots of this species was proposed. Our results demonstrated that the dichloromethane (DCM) extract was exclusively constituted by a mixture of five new acetogenins named annopurpuricins A-E (1-5). These compounds have an aliphatic chain of 37 carbons with a terminal α,ß unsaturated γ-lactone. Compounds 1 and 2 belong to the adjacent bis-THF (tetrahydrofuran) α-monohydroxylated type, while compounds 3 and 4 belong to the adjacent bis-THF α,α'-dihydroxylated type; only compound 5 possesses a bis-epoxide system. Complete structure analysis was carried out by spectroscopy and chemical methods. All compounds were evaluated for their antiproliferative activity on three human tumor cell lines (MSTO-211H, HeLa and HepG2). Compounds 1-4 inhibited significantly the growth of HeLa and HepG2 cells, showing GI50 values in the low/subnanomolar range, while 5 was completely ineffective under the tested conditions. The investigation of the mechanism of action responsible for cytotoxicity revealed for the most interesting compound 1 the ability to block the complex I activity on isolated rat liver mitochondria (RLM).

A set of biogenetically interesting polyhalogenated acetogenins from Ptilonia magellanica.

Ptilonines A-F, pyranosylmagellanicus D-E and magellenediol are previously undescribed acetogenins isolated from the red alga Ptilonia magellanica. Their structures were determined from spectroscopic evidence. The absolute configuration of the known pyranosylmagellanicus A, was established by derivatization with (R)- and (S)-α-methoxy -α-phenylacetic acids (MPA). Ptilonines exhibit an unusual halogenation pattern, that may confer evolutionary advantages to Ptilonia magellanica, for which a biogenetic origin is proposed. The antimicrobial effect of some of these compounds was evaluated.

Isolation and chemical identification of lipid derivatives from avocado (Persea americana) pulp with antiplatelet and antithrombotic activities.

Platelets play a pivotal role in physiological hemostasis. However, in coronary arteries damaged by atherosclerosis, enhanced platelet aggregation, with subsequent thrombus formation, is a precipitating factor in acute ischemic events. Avocado pulp (Persea americana) is a good source of bioactive compounds, and its inclusion in the diet as a source of fatty acid has been related to reduced platelet aggregability. Nevertheless, constituents of avocado pulp with antiplatelet activity remain unknown. The present study aims to characterize the chemical nature of avocado constituents with inhibitory effects on platelet aggregation. Centrifugal partition chromatography (CPC) was used as a fractionation and purification tool, guided by an in vitro adenosine diphosphate (ADP), arachidonic acid or collagen-platelet aggregation assay. Antiplatelet activity was initially linked to seven acetogenins that were further purified, and their dose-dependent effects in the presence of various agonists were contrasted. This process led to the identification of Persenone-C (3) as the most potent antiplatelet acetogenin (IC50=3.4 mM) among the evaluated compounds. In vivo evaluations with Persenone A (4) demonstrated potential protective effects against arterial thrombosis (25 mg kg⁻¹ of body weight), as coagulation times increased (2-fold with respect to the vehicle) and thrombus formation was attenuated (71% versus vehicle). From these results, avocado may be referred to as a functional food containing acetogenin compounds that inhibit platelet aggregation with a potential preventive effect on thrombus formation, such as those that occur in ischaemic diseases.

In vivo antimalarial efficacy of acetogenins, alkaloids and flavonoids enriched fractions from Annona crassiflora Mart.

Annona crassiflora and Annonaceae plants are known to be used to treat malaria by traditional healers. In this work, the antimalarial efficacy of different fractions of A. crassiflora, particularly acetogenin, alkaloids and flavonoid-rich fractions, was determined in vivo using Plasmodium berghei-infected mice model and toxicity was accessed by brine shrimp assay. The A. crassiflora fractions were administered at doses of 12.5 mg/kg/day in a 4-day test protocol. The results showed that some fractions from woods were rich in acetogenins, alkaloids and terpenes, and other fractions from leaves were rich in alkaloids and flavonoids. The parasitaemia was significantly (p < 0.05, p < 0.001) reduced (57-75%) with flavonoid and alkaloid-rich leaf fractions, which also increased mean survival time of mice after treatment. Our results confirm the usage of this plant in folk medicine as an antimalarial remedy.

Activity-guided identification of acetogenins as novel lipophilic antioxidants present in avocado pulp (Persea americana).

Avocado fruit is a rich source of health-related lipophilic phytochemicals such as monounsaturated fatty acids, tocopherols, carotenes, acetogenins and sterols. However, limited information is available on the contribution of specific phytochemicals to the overall antioxidant capacity (AOC) of the fruit. Centrifugal partition chromatography was used as fractionation tool, guided by an in vitro chemical assay of oxygen radical absorbance capacity (ORAC). Subsequent experiments focused on isolation and characterization of the chemical nature of the main contributors to lipophilic AOC of avocado pulp. ORAC values obtained for acetogenins were contrasted with results from an isolated kidney mitochondria membrane lipid peroxidation bioassay. The present study established that lipophilic AOC of the pulp was significantly higher than its hydrophilic AOC. Our results confirmed the presence of acetogenins in the fractions with highest lipophilic AOC, and for the first time linked them as contributors to lipophilic-ORAC values. Further HPLC-PDA/MS-TOF analysis led to structural elucidation of two novel acetogenins, not previously reported as present in avocado pulp, along with five already known related-compounds. Antioxidant properties observed for avocado pulp acetogenins by the ORAC assay suggested that, in the presence of an emulsifying agent, acetogenins could serve as novel lipophilic antioxidants in a food matrix. Results from isolated mitochondria lipid peroxidation bioassay, indicated that L-ORAC values which may have relevance for food matrix applications, should not be interpreted to have a direct relevance in health-related claims, compounds need to be evaluated considering the complexity of biological systems.

Chemoenzymatic synthesis of trans-tetrahydrofuran cores of annonaceous acetogenins from bromobenzene.

Two types of trans-THF cores, present in acetogenins, have been synthesized by an intramolecular iodoetherification reaction. The starting alkenol was obtained in a few steps from a chiral cis-diol resulting from microbial oxidation of bromobenzene. The cyclization gave complete stereoselectivity for trans-THF cores with either (S,S) or (R,R) configurations at the THF chiral carbons.

Dynamic function of the alkyl spacer of acetogenins as potent inhibitors of mitochondrial complex I. A molecular dynamics simulation approach.

Acetogenins are among the most potent of the known inhibitors of complex I (NADH-ubiquinone oxidoreductase) in mitochondrial electron transfer system. Elucidation of the dynamic function of the alkyl spacer linking the two toxophores (i.e., the hydroxylated tetrahydrofuran and the γ-lactone rings) is critical for fully understanding their inhibition mechanism. To this end, using molecular dynamics simulations a structure-activity relationship study of a series of acetogenins was performed for the first time using this approach. Our results clearly indicated that both, the length and the molecular flexibility of the spacer, were crucial for taking an active conformation. A partially folded conformation with an optimal length (bis-tetrahydrofuran rings and 13 carbon atoms) of about 16 Šwith a high molecular flexibility might depict an active form of the spacer. In addition, we demonstrated that the bis-tetrahydrofuran derivatives are able to overcome the shortage of the length of the spacer more efficiently than the mono-tetrahydrofuran derivatives with the help of the additional tetrahydrofuran, which acts as a pseudospacer. Our results obtained from molecular dynamics calculations supported the use of a combined decane/water system as a good solvent model to simulate the biological environment of acetogenins acting as inhibitor of complex I.

Insecticidal properties of annonaceous acetogenins and their analogues. Interaction with lipid membranes.

The interactions were studied by FTIR and DSC of the terminal lactone of annonaceous acetogenins (ACGs) and synthetic analogues, such as THF, with POPC bilayers, as well as the toxic effect produced by these compounds on Spodoptera frugiperda larvae. The aim of this work was to find a relationship between ACG insecticidal properties and the specific sites of interaction with lipid membranes. ACGs interact to different extents with the phosphate of lipid membranes and differences in the antisymmetric stretching of the phosphate groups were found in the presence of water that indicate water loss and further hydrogen bonding.The ACG tested produced more than 70% larval mortality. Rolliniastatin-1 (3) proved to have the most toxic effects (100%) on early larval instars when incorporated in the larval diet at a dose of 100 microg per g of diet. Additionally, it produced a significant decrease in growth rate (GR) and consumption index (CI), and reduced the efficiency with which larvae converted ingested food into biomass (ECI). The destabilization that occurs in the membrane due to dehydration around the phosphate groups caused by interaction with ACGs and their synthetic analogues would account for ACGs' insecticidal action.

Attenuation of experimental TPA-induced dermatitis by acetylenic acetogenins is associated with inhibition of PLA2 activity.

Topical application of acetylenic acetogenins (AAG) from avocado (0.01-1.0mg/ear), was effective in inhibiting both 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema, and in decreasing tissue myeloperoxidase activity (indicative of polymorphonuclear leukocyte influx). Maximum edema inhibition of 72% was achieved by AAG at lower concentration (0.6 mg/ear) than that of the anti-inflammatory drug indomethacin (2mg/ear). The maximum myeloperoxidase inhibition of 60% was obtained at AAG concentration 0.1mg/ear. Chemical reduction of unsaturated bonds in aliphatic chain of AAG molecules almost abrogated inhibition effect of AAG at high concentration. In vitro AAG administration reduced secretion of PGE(2) in TPA-induced keratinocytes, and inhibited total PLA(2) and sPLA(2) activities in HaCaT cells. The results indicate a topical anti-inflammatory effect of acetylenic acetogenins which is associated with inhibition of PLA(2) activity in skin.

A quarter century of pharmacognostic research on Panamanian flora: a review.

Panama is a unique terrestrial bridge of extreme biological importance. It is one of the "hot spots" and occupies the fourth place among the 25 most plant-rich countries in the world, with 13.4 % endemic species. Panamanian plants have been screened for a wide range of biological activities: as cytotoxic, brine shrimp-toxic, antiplasmodial, antimicrobial, antiviral, antioxidant, immunosuppressive, and antihypertensive agents. This review concentrates on ethnopharmacological uses of medicinal plants employed by three Amerindian groups of Panama and on selected plants with novel structures and/or interesting bioactive compounds. During the last quarter century, a total of approximately 390 compounds from 86 plants have been isolated, of which 160 are new to the literature. Most of the work reported here has been the result of many international collaborative efforts with scientists worldwide. From the results presented, it is immediately obvious that the Panamanian flora is still an untapped source of new bioactive compounds.

Insecticidal, mutagenic and genotoxic evaluation of annonaceous acetogenins.

Annonaceous acetogenins represent a class of bioactive compounds whose primary mode of action is the inhibition of NADH-ubiquinone oxidoreductase (Mitochondrial Complex I). Given the potential pesticidal use of these compounds, we evaluated the effects of seven acetogenins: squamocin (1), molvizarin (2), itrabin (3), almuñequin (4), cherimolin-1 (5), cherimolin-2 (6), and tucumanin (7) isolated from Annona cherimolia Mill. against Ceratitis capitata Wiedemann (Tephritidae). These acetogenins did not display insecticidal action at 250 microg of treatment per g of adult diet. However, the oviposition capacity of C. capitata females was significantly altered by some of the acetogenins at this concentration. The most potent compounds were itrabin, molvizarin and squamocin. Moreover, significant differences were detected in the preference of oviposition sites when itrabin and squamocin were spread on the surface of artificial fruits at doses of 30 microg/cm2. Additionally, we investigated the mutagenic effects displayed by itrabin, as well as the phytotoxic and genotoxic action of squamocin and itrabin. Both compounds displayed slight phytotoxic and genotoxic effects on roots of Allium cepa at 2.5 microg/mL though no mutagenic effects were detected at 0.25, 0.5 and 2.5 microg/mL on Salmonella typhimurium strains TA98 and TA100.