The toxic effects in rats of some synthanecine carbamate and phosphate esters analogous to hepatotoxic pyrrolizidine alkaloids

Five synthetic compounds analogous to pyrrolizidine alkaloids have been tested for toxicity in rats. These were the bis-N-ethylcarbamate esters of synthanecines A, B, C and D (Compounds I–IV) and the bis-diethylphosphate ester (V) of synthanecine A. The amino alcohol moiety in each of these had a single 5-membered heterocyclic ring in place of the pyrrolizidine amino alcohol (necine) moiety of natural pyrrolizidine alkaloids.The toxicity of these compounds differed considerably. The synthanecine A carbamate (I) was the most toxic, male and female rats being similarly susceptible. Like many hepatotoxic pyrrolizidine alkaloids, a single dose of compound I caused acute centrilobular necrosis of the liver, chronic hepatotoxicity involving the development of persistent giant hepatocytes, and chronic lung injury. Compound III had similar actions but was less toxic. The synthanecine D carbamate (IV) caused acute liver necrosis but no chronic hepatotoxicity, whereas the synthanecine A phosphate (V) had the opposite effect, with only chronic hepatotoxicity.The different toxic effects were related to the structure and metabolism of the compounds. Doses of compounds I, III and IV associated with a similar degree of acute hepatotoxicity led to similar levels of pyrrolic metabolites in the liver. Compound II, which was not hepatotoxic, gave very little liver pyrrole. The liver level of pyrrolic metabolite from the phosphate ester (V) decreased more rapidly than that from (I), and was not associated with acute toxicity.Antimitotic activity, indicated by the appearance of bizarre giant cells, was shown by compounds capable of forming pyrrolic metabolites which were bifunctional alkylating agents, but not by compound IV, which could only form a monofunctional alkylating agent. Pretreatment with phenobarbitone lowered the susceptibility of rats to compound I and greatly increased the liver level of pyrrolic metabolites associated with acute hepatotoxicity. Some rats given compounds I and III had kidney lesions primarily involving the glomerulus. The results confirm that toxic effects characteristic of many natural pyrrolizidine alkaloids can be reproduced using simplified synthetic analogues, and that such toxicity is associated with pyrrolic metabolites.     

Comparative HILIC/ESI-QTOF-MS and HPTLC studies of pyrrolizidine alkaloids in flowers of Tussilago farfara and roots of Arnebia euchroma

The utility of HPTLC and HILIC/ESI-QTOF-MS for the determination of pyrrolizidine alkaloids (PAs) and their N-oxides (PANOs) was compared in the selected plant species: Tussilago farfara L. (TF, flower) and Arnebia euchroma (Royle) I.M. Johnst. (AE, root). HPTLC confirmed the postulated presence of PAs (saturated and unsaturated) or PANOs in the tested extracts. In accordance with previous studies, HILIC/ESI-Q-TOF-MS confirmed the presence of the toxic PA senkirkine and the saturated otonecine-type PAs, tussilagine and isotussilagine in the TF extract and 7-angeloylretronecine and 9-angeloylretronecine in AE extract. Moreover, the following alkaloids were identified in AE root: intermedine, intermedine-N-oxide, leptanthine-N-oxide, echimidine-N-oxide (or their corresponding stereoisomers) and traces of 7-angeloylretronecine and 9-angeloylretronecine-N-oxide. The study demonstrates the HILIC/ESI-Q-TOF-MS method to be a very useful tool for monitoring PAs and PANOs in the test samples, even when not all of the necessary standards are available. Quantitative analysis of senkirkine in TF flower by HILIC/ESI-QTOF-MS featured high resolution, high precision, high mass accuracy, and very high sensitivity with limit-of-detection (LOD) of 27.50 fg/μL and limit-of-quantitation (LOQ) of 91.60 fg/μL. The results from both methods may be used for the development or rejection of European Pharmacopoeia (X) monographs of both investigated species.     

Major components in the hairpencil secretion of a butterfly, Euploea mulciber (Lepidoptera, Danaidae): their origins and male behavioral responses to pyrrolizidine alkaloids

Two compounds, 9,10-epoxytetrahydroedulan (ET) and viridiflorine beta-lactone (VL), were identified as major components from the hairpencils of field-caught males of a danaid butterfly, Euploea mulciber. By contrast, laboratory-reared males entirely lacked VL, but possessed a significant quantity of ET. Various feeding experiments with larvae and indoor adult males strongly suggested that ET is biosynthesized de novo only after eclosion from nutrients ingested during the larval development. Since VL was suspected to be derived from pyrrolizidine alkaloids (PAs) acquired as an adult, tests for feeding response to and oral administration of four PAs (a 4:1 mixture of intermedine/lycopsamine, heliotrine, monocrotaline, and retronecine) were conducted. When the tarsi or proboscis were stimulated with PA solutions, males showed positive feeding responses (proboscis extension and sucking movements) to intermedine/lycopsamine, heliotrine, and retronecine in decreasing order of responsiveness, thereby providing evidence that male adults are endowed with taste receptor(s) specific to PAs on the legs as well as on the proboscis. Differently from gustatory responsiveness, only males fed with intermedine/lycopsamine produced a significant quantity of VL (ca. 35 microg/male), whereas those that ingested heliotrine or monocrotaline hydrochloride produced traces of VL (<0.18 microg/male). Uptake of retronecine did not lead to VL formation at all. In behavioral bioassays to test the attractivity of PAs to males, all individuals tested were attracted exclusively to intermedine/lycopsamine. This shows that certain PA(s) per se serve as attractant(s) for males in locating PA sources, and further suggests that in the field, males will seek particular PA(s) that are indispensable as precursors for the efficient biosynthesis of VL.     

Relation of structural features to pyrrolic metabolites in livers of rats given pyrrolizidine alkaloids and derivatives

Levels of pyrrolic metabolites have been measured in the livers of rats given some pyrrolizidine alkaloids and semisynthetic derivatives. Structural and chemical features favouring the formation of such metabolites have been defined. The most important of these were: steric hindrance or chemical properties giving resistance to ester hydrolysis; lipophilic character, allowing access to hepatic microsomal enzymes; a conformation favoring microsomal oxidation of the pyrroline ring in preference to N-oxidation. In addition, the presence of ester groups gave the resulting pyrroles high chemical reactivity, leading to tissue binding.Amounts of pyrroles bound to liver were very low when animals were given either highly water soluble pyrrolizidine derivatives, including non-esterified bases or more-lipophilic esters if these were easily hydrolysed. Compounds prone to hydrolysis gave increased pyrrole levels in rats pretreated to deplete their esterase activity. Whereas heliotridine-based alkaloids usually give more pyrrole than similar retronecine esters, heliotridine ditiglate gave less pyrrole than retronecine ditiglate because the former was more open to hydrolytic attack.Among the carboxylic diesters, the cyclic retronecine diesters, in which the pyrrolizidine nucleus is more exposed to oxidative metabolism, gave the highest pyrrole levels in rats.Liver pyrrole measurements are useful for studying relationships between molecular structure, metabolism and toxicity of pyrrolizidine derivatives. They can be used for screening alkaloids for potential toxicity and for assessing dose levels suitable for toxicity tests when limited material is available.     

Evolution of pyrrolizidine alkaloids in Phalaenopsis orchids and other monocotyledons: Identification of deoxyhypusine synthase, homospermidine synthase and related pseudogenes

In order to study the evolution of pathways of plant secondary metabolism, we use the biosynthesis of pyrrolizidine alkaloids (PAs) as a model system. PAs are regarded as part of the plant’s constitutive defense against herbivores. Homospermidine synthase (HSS) is the first specific enzyme of PA biosynthesis. The gene encoding HSS has been recruited from the gene encoding deoxyhypusine synthase (DHS) from primary metabolism at least four times independently during angiosperm evolution. One of these recruitments occurred within the monocot lineage. We have used the PA-producing orchid Phalaenopsis to identify the cDNAs encoding HSS, DHS and the substrate protein for DHS, i.e., the precursor of the eukaryotic initiation factor 5A. A cDNA identified from maize was unequivocally characterized as DHS. From our study of Phalaenopsis, several pseudogenes emerged, of which one was shown to be a “processed pseudogene”, and others to be transcribed. Sequence comparison of the HSS- and DHS-encoding sequences from this investigation with those of monocot species taken from the databases suggest that HSS and probably the ability to produce PAs is an old feature within the monocot lineage. This result is discussed with respect to the recent discovery of structural related PAs within grasses.     

Characterization of tannic acid metabolites formed in vitro by rat liver microsomes and assay of their carcinogenicity by the microsomal degranulation technique

Tannic acid is converted to four metabolites on incubation with isolated rat liver microsomes. Bis-(3,4,5-trihydroxyphenyl)methanone (I); 3,4,6,7,9,10-hexahydroxy-2,11-epoxy-1,12-(epoxy-methano)-14H , 16H-benzo[b](1,4)benzodioxepino [3,2-g](1,4)benzodioxepin-14,16,17-trione (II) and 1,1,2-trimethyl-1-ethanyl-2-ylidene tris(3,4,5-trihydroxybenzoate) (III) were isolated from the post-microsomal supernatant of the incubate while 6,12,18,26,27,29,31,33-octahydroxy-22,22-dimethyl-2,8,14,20-tet raoxapentacyclo (22.2.2.2(4,7).2(10,13).2(16,19) tetratriaconta-4,6,10,12,16,18,24,26,27,29,31,33-dodecene-3, 9,15,21,23-pentone (IV) was found attached with the microsomal fraction. Metabolite (III) was found to be a potential carcinogen on the basis of microsomal degranulation technique.     

Use of repair-deficient strains of escherichia coli and liver microsomes to detect and characterise DNA damage caused by the pyrrolizidine alkaloids heliotrine and monocrotaline

E. coli WP2 and its repair-deficient derivatives were treated with the pyrrolizidine alkaloids, heliotrine and monocrotaline in the presence of a liver microsomal fraction. The doubly repair-deficient strains WP100 uvrA recA and CM611 uvrA exrA showed considerable killing. The singly repair-deficient strains WP2 uvrA, CM561 exrA and CM571 recA showed slight killing. In strains WP2 and WP2 uvrA induced reversion to Trp⁺ was not detected with either monocrotaline or mitomycin C. These results are entirely consistent with liver activation converting pyrrolizidine alkaloids into bifunctional alkylating agents.     

Uptake and metabolism of pyrrolizidine alkaloids in<Emphasis Type="Italic"> Longitarsus</Emphasis> flea beetles (Coleoptera: Chrysomelidae) adapted and non-adapted to alkaloid-containing host plants

Several Longitarsus flea beetle species sequester pyrrolizidine alkaloids acquired from their Asteraceae and Boraginaceae host plants. We carried out feeding and injection experiments using radioactively labeled pyrrolizidine alkaloids to investigate the physiological mechanisms of uptake, metabolism and storage of alkaloids in adult beetles. We examined six Longitarsus species belonging to different phylogenetic clades in a comparative approach. All species that accepted pyrrolizidine alkaloids in a preceding food choice study showed the ability both to store pyrrolizidine alkaloid N-oxides and to metabolize tertiary pyrrolizidine alkaloids into their N-oxides. Regardless of whether the beetles' natural host plants contain pyrrolizidine alkaloids or not, these species were found to possess an oxidizing enzyme. This oxygenase appears to be specific to pyrrolizidine alkaloids: [³H]Atropine and [¹⁴C]nicotine, two alkaloids not related to pyrrolizidine alkaloids, were neither stored nor N-oxidized by any of the tested species. One species, L. australis, that strictly avoids pyrrolizidine alkaloids behaviorally, exhibited a lack of adaptations to pyrrolizidine alkaloids on a physiological level as well. After injection of tertiary [¹⁴C]senecionine, beetles of this species neither N-oxidized nor stored the compounds, in contrast to L. jacobaeae, an adapted species that underwent the same treatment. L. jacobaeae demonstrated the same efficiency in N-oxidation and storage when fed or injected with tertiary [¹⁴C]senecionine.Communicated by G. Heldmaier     

Synthesis and/or accumulation of bioactive molecules in the in vivo and in vitro root

Abstract

Roots of many species are studied because of the presence of high-value bioactive molecules, yet few studies have attempted to determine the biosynthetic pathways of these compounds or the way in which synthesis is regulated. The presence of secondary metabolites in the root does not necessarily mean that this organ is also the site of synthesis. Thus the identification of organ-specific intermediate precursors and key enzymes is important for understanding the biosynthetic pathway and the regulation of bioactive molecules. This knowledge could allow researchers to predict the suitability of in vitro systems, such as regenerated roots and hairy roots, for producing the molecules of interest. In the present review, the production of bioactive molecules in in vivo roots is compared to that in in vitro untransformed and transformed roots, concentrating on recent developments in the study of the biosynthesis of the anti-cancer alkaloid camptothecin in Camptotheca acuminata Decne. The results of a recent study performed in our laboratory on the production of camptothecin and other secondary metabolites in in vivo and in vitro C. acuminata roots are also presented.     

Inhibition of carbon tetrachloride-induced lipid peroxidation by novel antioxidants in rat hepatic microsomes: dissociation from hepatoprotective effects in vivo

The ability of two novel antioxidants, U-74,006F and U-78,517G, as well as the known antioxidant N,N'-diphenyl-p-phenylenediamine to inhibit lipid peroxidation induced by carbon tetrachloride (CCl4) was investigated in Aroclor 1254-induced rat hepatic microsomes. All three compounds completely inhibited lipid peroxidation in microsomes as measured by the formation of thiobarbituric acid reactive substances (TBARS). Inhibition of lipid peroxidation was not a function of decreased bioactivation of CCl4, as the compounds did not substantially inhibit benzphetamine N-demethylase activity or covalent binding of [14-C]CCl4 to lipid or protein. Parallel studies examined the hepatoprotective effects of the compounds in vivo. Rats were pretreated with antioxidant or vehicle prior to administration of CCl4 (300 or 600 microL/kg i.p.). Sera were collected 24 h postadministration of CCl4 and analyzed for alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities and total bilirubin. Administration of CCl4 produced elevations in ALT, moderate changes in bilirubin, and no change in ALP activities. Histological examination of CCl4-treated livers revealed lipidosis and centrilobular necrosis. The antioxidants partially improved the clinical chemistry parameters, but had minimal effects on the histological lesion. In contrast to the complete inhibition of lipid peroxidation observed in the in vitro studies, none of the antioxidants markedly protected against CCl4-induced toxicity in vivo.     

Chemometric analysis applied to a chemophenetics approach in Simaroubaceae family: The relevance of alkaloids and quassinoids

Simaroubaceae family belongs to the order Sapindales, which in turn is part of Eurosides II (APG IV). It consists of 23 genera: Ailanthus; Amaroria; Brucea; Castela; Eurycoma; Gymnostemon; Hannoa; Holacantha; Homalolepsis; Iridosma; Laumoniera; Leitneria; Nothospondias; Odyendeya; Perriera; Picrasma; Picrolemma; Pierreodendron; Quassia; Samadera; Simaba; Simarouba; Soulamea. Over the years, the classification of Simaroubaceae has changed and there is still a lot to evolve in this direction since there are still not many studies on some of its genera. In this sense, the present work discusses the chemistry of Simaroubaceae through the micromolecular chemical data, evolutionary indexes and chemometric analysis in order to assess affinities between its genera. The chemistry of this family is diverse and includes quassinoids, alkaloids, triterpenes, flavonoids, simple phenolics, coumarins, lignans and quinones. In addition to presenting specificity, quassinoids and alkaloids are prevalent among the substances already identified in the family and can be considered the chemical markers of it. Quassionoids are degraded triterpenes and can be classified as c25, c22, c20, c 19 and c18; c20 being the most common type in the family. The alkaloids already isolated in Simaroubaceae are predominantly beta-carboline and canthinone, both originating from the amino acid tryptophan. The evolutionary indexes calculated for the chemical markers showed great variation between the genera of Simaroubaceae and showed no correlation among them. The chemometric analysis positioned Simaroubaceae close to Rutaceae within the order Sapindales and, among the genera, alkaloids and quassinoids were very important for their distribution in Simaroubaceae considering the evolutionary proximity, corroborating their selection as chemical markers.     

Protective effects of the apigenin-O/C-diglucoside saponarin from Gypsophila trichotoma on carbone tetrachloride-induced hepatotoxicity in vitro/in vivo in rats

This study investigated the hepatoprotective activity of saponarin, isolated from Gypsophila trichotoma Wend., using in vitro/in vivo hepatotoxicity model based on carbone tetrachloride (CCl₄)-induced liver damage in male Wistar rats. The effect of saponarin was compared with those of silymarin. In vitro experiments were carried out in primary isolated rat hepatocytes. Cell incubation with CCl₄ (86 μmol l⁻¹) led to a significant decrease in cell viability, increased LDH leakage, decreased levels of cellular GSH and elevation in MDA quantity. Cell pre-incubation with saponarin (60–0.006 μg/ml) significantly ameliorated CCl₄-induced hepatic damage in a concentration-dependent manner. These results were supported by the following in vivo study. Along with decreased MDA quantity and increased level of cell protector GSH, seven day pre-treatment of rats with saponarin (80 mg/kg bw; p.o.) also prevented CCl₄ (10%, p.o.)-caused oxidative damage by increasing antioxidant enzyme activities (CAT, SOD, GST, GPx, GR). Biotransformation phase I enzymes were also assessed. Administered alone, saponarin decreased EMND and AH activities but not at the same extent as CCl₄ did. However, pre-treatment with saponarin significantly increased enzyme activities in comparison to CCl₄ only group. The observed biochemical changes were consistent with histopathological observations where the hepatoprotective effect of saponarin was comparative to the effects of the known hepatoprotecor silymarin. Our results suggest that saponarin, isolated from Gypsophila trichotoma Wend., showed in vitro and in vivo hepatoprotective and antioxidant activity against CCl₄-induced liver damage.     

Activation of osteoclasts by interleukin-1: divergent responsiveness in osteoclasts formed in vivo and in vitro

Recently, it has been found that osteoclasts are induced and activated by osteoblastic cells through expression of receptor activator NF-kB ligand (RANKL), and that soluble recombinant RANKL, with M-CSF, can replace the need for osteoblastic cells in osteoclast formation. We exploited this opportunity to compare the responsiveness of osteoclast-like cells (OCL) formed in vitro in the absence of osteoblasts, with that of osteoclasts ex vivo. We found that while OCL responded to several hormones and cytokines like ex vivo osteoclasts, their responsiveness to interleukin-1 (IL-1) was fundamentally different: IL1 directly stimulated actin ring formation in OCL, but had no effect on actin rings or survival in osteoclasts ex vivo unless osteoblastic cells were present. This difference could not be attributed to the use of plastic culture substrates for OCL formation, nor to osteoblastic contamination, and did not seem to be mediated by the macrophages that form in OCL cultures. To understand the mechanisms by which IL-1 induces bone loss, it will need to be determined whether or not IL-1-responsive OCLs have a counterpart in vivo. Whichever is the case, our data suggest that the behavior of osteoclasts formed in culture will not always predict that of osteoclasts in vivo.     

New perspective on the metabolism of AD-1 in vivo: Characterization of a series of dammarane-type derivatives with novel metabolic sites and anticancer mechanisms of active oleanane-type metabolites

20(R)-25-methoxyl-dammarane-3β,12β,20-triol (AD-1, CN Patent: 201010107476.7) is a novel derivative of dammarane-type ginsenoside. AD-1 has been shown to inhibit cancer cell proliferation without significant host toxicity in vivo, and has excellent development potential as a new anti-cancer agent. This study was designed systematically to explore the metabolic pathway of ginseng sapogenins. The metabolism of drugs in the body is a complex biotransformation process where drugs are structurally modified to different molecules (metabolites) through various metabolizing enzymes. The compounds responsible for the effects of orally administered ginseng are believed to be metabolites produced in the gastrointestinal tract, so understanding the metabolism of the drug candidate can help to optimize its pharmacokinetics. In this study, faeces samples were collected and extracted after oral administration of AD-1. The 16 metabolites of AD-1 were isolated and identified for the first time with various chromatographic techniques, including semi-preparative high performance liquid chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry; of these 16 metabolites, 10 were novel compounds. We first discovered the biotransformation of dammarane-type sapogenins into oleanane-type sapogenins in rats and found a series of metabolites that changed, mainly at C-25 and C-29. This study provides new ideas for the metabolic pathway of ginseng sapogenins. The isolated compounds were screened for their effect on the viability and proliferation against cancer cell lines (Human A549, MCF-7, HELA, HO-8901 and U87). The discovery of novel active metabolites 3β,12β,21α,22β-Hydroxy-24-norolean-12-ene (M6) may lead to a new or improved drug candidate. For one, M6 could inhibit the growth of all the tested cancer cells. Among the tested cell lines, M6 exhibited the most remarkable inhibitory effect on ovarian cancer HO-8901 cells, with IC₅₀ value of 2.086 μM. On this basis, we studied the anticancer mechanisms of M6. The results indicated that the pro-apoptotic feature of M6 acts via a mitochondrial pathway. Our results indicated that M6 exhibited a higher inhibitory effect on cancer-cell proliferation than AD-1 by inducing cell apoptosis. Our work provides data for future investigations on the metabolic mechanism of AD-1 in vivo and the potential for future research on developing a new drug.     

Identification and comparative oridonin metabolism in different species liver microsomes by using UPLC-Triple-TOF-MS/MS and PCA

Oridonin (ORI) is an active natural ent-kaurene diterpenoid ingredient with notable anti-cancer and anti-inflammation activities. Currently, a strategy was developed to identify metabolites and to assess the metabolic profiles of ORI in vitro using ultra-high-performance liquid chromatography-Triple/time-of-flight mass spectrometry (UPLC-Triple-TOF-MS/MS). Meanwhile, the metabolism differences of ORI in the liver microsomes of four different species were investigated using a principal component analysis (PCA) based on the metabolite absolute peak area values as the variables. Based on the proposed methods, 27 metabolites were structurally characterized. The results indicate that ORI is universally metabolized in vitro, and the metabolic pathway mainly includes dehydration, hydroxylation, di-hydroxylation, hydrogenation, decarboxylation, and ketone formation. Overall, there are obvious inter-species differences in types and amounts of ORI metabolites in the four species. These results will provide basic data for future pharmacological and toxicological studies of ORI and for other ent-kauranes diterpenoids. Meanwhile, studying the ORI metabolic differences helps to select the proper animal model for further pharmacology and toxicological assessment.     

Improvement of hairy root cultures and plants by changing biosynthetic pathways leading to pharmaceutical metabolites: Strategies and applications

A plethora of bioactive plant metabolites has been explored for pharmaceutical, food chemistry and agricultural applications. The chemical synthesis of these structures is often difficult, so plants are favorably used as producers. While whole plants can serve as a source for secondary metabolites and can be also improved by metabolic engineering, more often cell or organ cultures of relevant plant species are of interest. It should be noted that only in few cases the production for commercial application in such cultures has been achieved. Their genetic manipulation is sometimes faster and the production of a specific metabolite is more reliable, because of less environmental influences. In addition, upscaling in bioreactors is nowadays possible for many of these cultures, so some are already used in industry. There are approaches to alter the profile of metabolites not only by using plant genes, but also by using bacterial genes encoding modifying enzymes. Also, strategies to cope with unwanted or even toxic compounds are available. The need for metabolic engineering of plant secondary metabolite pathways is increasing with the rising demand for (novel) compounds with new bioactive properties. Here, we give some examples of recent developments for the metabolic engineering of plants and organ cultures, which can be used in the production of metabolites with interesting properties.     

Aporphine alkaloids and lignans formed in response to injury of sapwood in Liriodendron tulipifera

Two new phenolic aporphine alkaloids, (+)-lirioferine and (+)liriotulipiferine, were isolated from discolored sapwood of L. tulipifera. Injury to the tree stem greatly stimulated biosynthesis of glaucine and phenolic alkaloids related to glaucine including thaliporphine, predicentrine, N-methylaurotetanine and corunine as well as the above two compounds. Injury also stimulated synthesis of oxoaporphine related and other polymeric pigments. Corunine was responsible for at least part of the color of discolored sapwood. None of the above compounds except glaucine was detected in normal sapwood or heartwood of L. tulipifera. Thus, formation of alkaloids and lignans in discolored sapwood differs both quantitatively and qualitatively from that observed during the normal transition of sapwood to heartwood in this tree. The compounds formed in response to injury differed substantially from one zone of injury to another within the same tree.     

DNA adduction by polychlorinated biphenyls: adducts derived from hepatic microsomal activation and from synthetic metabolites

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants and complete carcinogens in rodents. Metabolism of lower chlorinated congeners with rat liver microsomes was investigated in earlier studies and DNA adduction was also reported. The current study was designed to compare DNA adducts formed after bioactivation of PCBs with rat, mouse and human hepatic microsomes, and to investigate the role of quinoid PCB metabolites in DNA adduct formation. Eight congeners ranging from mono- to hexachlorinated biphenyls were tested. Metabolites obtained through microsomal bioactivation as well as synthetic quinoid metabolites of 4-monochlorobiphenyl (4-CB) were incubated with calf-thymus DNA (CT-DNA), and the resulting adducts were analyzed by the 32P-post-labelling method. DNA adducts were formed with mono- di- and tri-chlorinated congeners, but not with higher chlorinated congeners. Similar adduct patterns were observed for 2-monochlorobiphenyl (2-CB) activated with hepatic microsomes from rat, mouse and human, while 4-CB, 3,4-dichlorobiphenyl (3,4-CB) and 3,4,5-trichlorobiphenyl (3,4,5-CB) showed similar patterns for two out of the three microsomal systems tested. 4,4' -trichlorobiphenyl (4,4' -CB) showed different adduct patterns in all microsomal systems. Higher adduct levels were obtained with the rodent microsomes compared with human microsomes and were related to higher cytochrome P450 activity. When adducts derived from microsomal activation of 4-CB were compared by co-chromatography with those derived from the incubation of DNA with synthetic 2-(4' -chlorophenyl)-1,4-benzoquinone (4-BQ), one adduct co-migrated in three different chromatography systems. This study demonstrates that rodents as well as human hepatic enzymes metabolize lower chlorinated biphenyl congeners to reactive intermediates that form DNA adducts in vitro and shows that the para-quinone metabolites of PCBs are, in part, involved in direct DNA adduction.     

Monoterpenoid indole alkaloids biosynthesis and its regulation in Catharanthus roseus: a literature review from genes to metabolites

Phytochemistry Reviews - As the only source for the low-abundance antitumor agents vinblastine and vincristine, Catharanthus roseus is highly valued and has been studied extensively as a model for...