Co-mutagenicity of coumarin (1,2-benzopyrone) with aflatoxin B1 and human liver S9 in mammalian cells.

Coumarin (1,2-benzopyrone), a natural dietary constituent and drug currently under evaluation for treatment of certain cancers and lymphedema, reduces polycyclic aromatic hydrocarbon-induced neoplasms in rodents. Because most rodents metabolize coumarin through 3,4-epoxidation, whereas 7-hydroxylation predominates in humans, their suitability as a model for coumarin effects in humans has been questioned. We examined coumarin chemoprotection against the promutagen and dietary contaminant aflatoxin B1 with human liver S9 bioactivation in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyltransferase mutation assay. Coumarin in the absence of aflatoxin B1 was not mutagenic or cytotoxic up to 500 microM. When included with either 1 or 10 microM aflatoxin B1, coumarin produced a dose-dependent increase in mutant frequency and cytotoxicity. At concentrations greater than 50 microM, coumarin stimulated human liver S9 bioactivation of aflatoxin B1 to the mutagenic 8,9-epoxide. This increase was 12- and fivefold at 500 microM coumarin with 1 and 10 microM aflatoxin B1, respectively, compared with incubations with aflatoxin B1 alone. These findings differ from previous results with liver S9 from other species, and indicate that coumarin co-mutagenicity with aflatoxin B1 and human liver S9 is through increased aflatoxin B1 bioactivation.     

Comparative effect of dietary butylated hydroxyanisole and {beta}-naphthoflavone on aflatoxin B1 metabolism, DNA adduct formation, and carcinogenesis in rainbow trout

Butylated hydroxyanisole (BHA) and ß-naphthoflavone(BNF), both chemicals with anti-carcinogeneic properties insome experimental animals, were compared for effects on afiatoxinB1 (AFB1) metabolism, hepatic DNA adduct formation and carcinogenesisin the rainbow trout. Dietary BHA had no effect on the hepatictumor incidence when fed at 0.03 or 0.3% 4 weeks prior to andduring a 4 week dietary exposure of 10 p.p.b. AFB1. BNF, whenfed at 0.005 or 0.05% under similar conditions, significantlyreduced tumor response, which confirms previous results in trout(Nixon et al.9 Carcinogenesis, 5, 615–619, 1984). BHAfed at either 0.03 or 0.3% for 8 weeks had no post-initiationeffect on the 52 week hepatic tumor incidence of trout exposedto a 0.5 p.p.m. AFB1 solution as embryos. A similar post-initiationexposure to 0.05% BNF significantly enhanced AFB1 tumor response.The influence of dietary BHA and BNF on AFB1 metabolism andDNA adduct formation and persistence in trout were examined.A 3 week pre-treatment with 0.3% dietary BHA had no effect onin vivo hepatic nuclear AFB1-DNA adduct formation at 0.5, 1,2 and 7 days after AFB1 i.p. injection. By contrast 0.05% dietaryBNF reduced hepatic AFB1-DNA adducts to 33–60% of controllevels at 0.5, 1, 2 and 4 days after AFB1 exposure. This wasaccompanied by significantly lower blood and liver levels ofAFB1 during the first 24 h after i.p. injection. Livers of BNFtrout also contained 4-fold more of the less carcinogenic metabolite,aflatoxin M1, and 50% less aflatoxicol (AFL), a metabolite withsimilar carcinogenicity as AFB1. Bile AFL-glucuronide levelswere significantly decreased in BNF-fed trout, but total bileglucuronides were significantly increased due to a 15-fold increasein aflatoxicol-M1 glucuronide. Freshly isolated hepatocytesfrom BHA-fed fish, when incubated with AFB1 for 1 h, showedno difference in levels of AFB1-DNA adducts or ratios of AFB1metabolites when compared to hepatocytes isolated from fishfed a control diet only. By contrast, dietary BNF has been previouslyshown to greatly enhance AFM1 production, reduce AFL production,and significantly reduce AFB1-DNA adduct formation in isolatedtrout hepatocytes (Bailey et al., Natl. Cancer Inst. Monograph,65, 379–385, 1984). These results indicate that dietaryBHA up to 0.3% does not alter AFB1 metabolism or DNA adductionin trout, nor does it inhibit or promote AFB1 hepatocarcinogenesisin this species. This is in contrast to anti-oxidant enhancementof AFB1-glutathione conjugation, reduction of AFB1-DNA binding,and consequent reduction of tumor response in rats. The nullresults in trout thus support enhanced glutathione conjugationas the major mechanism for BHA inhibition of AFB1 cardnogenesisin mammalian models. By contrast, BNF dietary pre-treatmentappears to inhibit AFB1 carcinogenicity in trout by enchancingglucuronide formation and elimination of the carcinogen, leadingto reduced DNA adduct formation in target tissue.     

Porphyria Cutanea Tarda: Multiplicity of Risk Factors Including HFE Mutations, Hepatitis C, and Inherited Uroporphyrinogen Decarboxylase Deficiency

The coexistence of factors considered to contribute to development of porphyria cutanea tarda was studied in 39 consecutive patients. Highly prevalent factors were alcohol intake in 79%, smoking in 86%, hepatitis C virus infection in 74%, estrogen use in 73% of 11 females, and at least one mutation in the HFE (hereditary hemochromatosis) gene in 65%. The C282Y mutation was found in 29%, H63D in 47%, and S65C in 0%. HFE genotypes included C282Y/C282Y in 9%, H63D/H63D in 9%, C282Y/H63D in 12%, C282Y/wild type in 9%, and H63D/wild type in 26%. Less prevalent were HIV infection in 15% (or 25% of those tested, N = 24) and erythrocyte uroporphyrinogen decarboxylase deficiency, which distinguishes familial (type 2) from sporadic (type 1) porphyria cutanea tarda, in 19%. Multiple contributing factors coexisted in both types 1 and 2, with 92% of all patients having three or more factors. These observations indicate that this porphyria is multifactorial in the individual patient, and therefore is seldom attributable to a single identifiable cause. Profiling for all potentially contributing factors is important for individualizing management.     

Comparison of the toxicities of Senecio jacobaea,Senecio vulgaris and Senecio glabellus in rats

The toxicity of Senecio jacobaea, S. vulgaris and S. glabellus to rats was assessed in a feeding trial. The plants were of similar toxicity, with a plant dry matter intake of about 20% of initial body weight being a lethal dose. Gas chromatography-mass spectrometry analysis demonstrated the presence of seneciphylline, jacobine, jacozine and jacoline in S. jacobaea, senecionine and seneciphylline in S. vulgaris, and senecionine in S. glabellus. An unidentified alkaloid was also found in all three plants.     

Effect of consumption of milk from goats fed Senecio jacobaea on hepatic drug metabolizing enzyme activities in rats

Milk from lactating goats fed tansy ragwort (Senecio jacobaea) was evaluated for its ability to influence hepatic drug metabolism. The milk after being freeze-dried was fed to male rats for 1 week ad lib. A significant reduction in the activities of hepatic aminopyrine A'-demethylase and aryl hydrocarbon hydroxylase was obtained. There was no significant change in the activities of microsomal epoxide hydrolase and cytosolic glutathione S-transferase. The data suggest that consumption of milk from goats fed Senecio jacobaea produces a selective alteration of the activities of hepatic drug-metabolizing enzymes.     

Lophocladines, bioactive alkaloids from the red alga Lophocladia sp

Lophocladines A (1) and B (2), two 2,7-naphthyridine alkaloids, were isolated from the marine red alga Lophocladiasp. collected in the Fijian Islands. Their structures were deduced on the basis of high-resolution mass spectra and one- and two-dimensional NMR spectroscopy. Lophocladine A (1) displayed affinity for NMDA receptors and was found to be a delta-opioid receptor antagonist, whereas lophocladine B (2) exhibited cytotoxicity to NCI-H460 human lung tumor and MDA-MB-435 breast cancer cell lines. Immunofluorescence studies indicated that the cytotoxicity of lophocladine B (2) was correlated with microtubule inhibition. This is the first reported occurrence of alkaloids based on a 2,7-naphthyridine skeleton from red algae.     

The neurotoxic lipopeptide kalkitoxin interacts with voltage-sensitive sodium channels in cerebellar granule neurons

The marine neurotoxin kalkitoxin, a thiazoline-containing lipid derived from the pantropical marine cyanobacterium Lyngbya majuscula, was assayed for interaction with the tetrodotoxin-sensitive, voltage-sensitive sodium channel (TTX-VSSC) in cerebellar granule neuron cultures (CGN). The naturally occurring isomer of kalkitoxin (KTx-7) blocked veratridine-induced (30 microM) neurotoxicity in a concentration-dependent manner (EC50 22.7 nM [9.5-53.9 nM, 95% confidence interval {CI}]) in CGN. Kalkitoxin was a potent inhibitor (EC50 26.1 nM [12.3-55.0 nM, 95% CI]) of the elevation of intracellular Ca2+ concentration [Ca2+](i) that accompanies exposure of CGN to veratridine. To further explore the potential interaction of KTx-7 with TTX-VSSC, we assessed the influence of KTX-7 on the binding of [3H]batrachotoxin ([3H]BTX) to neurotoxin site 2 on the TTX-VSSC. Although kalkitoxin was without effect on the basal binding of [3H]BTX to intact cerebellar granule neurons, in the presence of the positive allosteric modulator, deltamethrin, [3H]BTX binding was inhibited by KTx-7 in a concentration-dependent manner (11.9 nM [IC50=3.8-37.2 nM, 95% CI]). These results provide both direct and functional evidence for an interaction of kalkitoxin with the neuronal TTX-VSSC.     

Neurotoxic meroditerpenoids from the tropical marine brown alga Stypopodium flabelliforme

Brine shrimp toxicity and TLC analysis guided the isolation of five new and biologically active meroditerpenoids [2beta,3alpha-epitaondiol (1), flabellinol (2), flabellinone (3), stypotriolaldehyde (4), and stypohydroperoxide (5)] along with five known compounds from the marine brown alga Stypopodium flabelliforme collected in Papua New Guinea. The planar structures of compounds 1-5 were determined by extensive spectroscopic analysis (1D and 2D NMR, LRMS, HRMS, IR, and UV), while relative configuration was determined by 1D and 2D NOE experiments. X-ray crystallography confirmed the relative configuration of 2beta,3alpha-epitaondiol (1), and the modified Mosher's ester method was used to establish its absolute configuration. All of the new metabolites were moderately toxic to murine neuro-2a cells (LC50 2-25 microM), and three [2beta,3alpha-epitaondiol (1), flabellinol (2), and flabellinone (3)] possessed potent sodium channel blocking activity. Stypotriolaldehyde (4) had a biphasic effect on the concentration of intracellular Ca2+ in rat cerebellar granule neurons (CGN). The previously known compound, stypoldione (6), also modulated intracellular calcium concentration and was cytotoxic in CGN. Metabolites 2beta,3alpha-epitaondiol (1), flabellinol (2), and flabellinone (3) displayed moderate cytotoxicity to the NCI-H460 human lung cancer cell line.     

Mechanisms of anti-carcinogenesis: the distribution and metabolism of aflatoxin B1 in rainbow trout fed Aroclor 1254

Polychlorinated biphenyls (PCBs) have previously been shownto be inhibitors of carcinogenesis in trout. The mechanism ofthis inhibition was investigated by studying the effects ofPCBs on aflatoxin B₁ (AFB₁) distribution, metabolism and DNAadduct formation, both in vivo and in vitro. A 24 h distributionstudy of injected tritiated AFB₁ showed more radioactivity inblood, liver and bile in fish fed PCBs, but less in residualcarcass. The metabolites of AFB₁ found in vivo in blood plasmaand liver homogenates were shifted by PCB pre-treatment towardsgreater production of the polar metabolite aflatoxin M₁ (AFL-M₁)and glucuronide conjugates. The major metabolite in bile ofPCBs fish was the glucuronide of aflatoxi-col M₁ (AFL-M₁), whichwas enhanced 15-fold over controls. Levels of aflatoxicol (AFL)glucuronide, the major conjugate in controls, were unalteredby PCBs. The pattern of AFB1 metabolism in isolated bepatocytesfrom PCB-prefed fish was consistent with in vivo metabolism.AFB₁-DNA adduct formation in a 1 h assay was similar in hepatocytesfrom PCB-fed and control fish. However, the total rate of AFB₁metabolism was significantly elevated in hepatocytes from PCB-fedfish such that the degree of AFB₁-DNA adduct formed per unitAFB₁ metabolized was 42% lower than control. Similarly, adductformation in vivo during the first 24 h post-AFB₁ injectionin PCB fish was not significantly different from controls. However,over a longer 21 day period, adduct levels in PCB fish wereonly 48–69% of controls (P < 0.005, analysis of variance),once peak adduct formation was reached. Thus, initial ratesof adduct formation may be misleading in the absence of furtherinformation on rates of carcinogen metabolism in vitro and/orpharmacokinetics of peak adduct formation in vivo. These resultsindicate that PCB inhibition of AFB₁ carcinogenesis in troutinvolves dramatic initial changes in carcinogen distribution,metabolism and elimination which, over time, results in a netreduction of DNA adduct formation.