Modification of in vivo heterocyclic amine genotoxicity by dietary flavonoids

Female BALB/c mice were fed diets containing equimolar amountsof quercetin or its glycoside, rutin, for 5 weeks. These micewere used either in host-mediated bacterial mutation assaysor as sources of hepatic microsomes. In host-mediated bacterialmutation assays using radiolabelled mutagens, the heterocyclicamines 2-amino-3,5-dimethyl [4,5-f]imidazoquin-oline (MeIQ)and 3-amino-1-methyl-5H-pyrido[4,3-b] indole (Trp-P-2) inducedgreater numbers of revertants in mice fed either of the flavonoiddiets compared with control. Experiments using hepatic microsomesrevealed that although feeding mice either flavonoid producedslight changes in some parameters of hepatic xenobiotic metabolism(mixed function oxidase and glutathione transferase activities),microsomes from quercetin-fed mice were more potent activatorsof both MeIQ and Trp-P-2 compared with microsomes from controlor rutin-fed mice. This difference in microsomal ability maybe due to the different biological availability of the two flavonoidswithin the gastrointestinal tract. ¹To whom correspondence to be addressed     

Effects of Maillard reaction products on mutagen formation in boiled pork juice

The three IQ (2-amino-3-methylimidazo[4,5-f]quinoline) compoundsIQ, MeIQx (2-amino-3, 4-dimethyl[4, 5-f]quinoxaline) and MeIQ(2-amino-3, 4-dimethylimidazo[4, 5-f]quinoline) have been foundin boiled pork juice. To determine which Maillard reaction productsare important in the formation of IQ-type mutagens in boiledpork juice, six Maillard reaction products were separately addedto the pork juice before reflux boiling and then the mutagenicityof each sample was examined with Salmonella typhe murium TA98in the presence of S9 mix. The addition of four Maillard reactionproducts enhanced the mutagenicity of pork juice 1.2–2.9-foldafter reflux boiling. The highest level of enhancement was observedwith tetrahydrothiophene, followed by 2, 3-dimethylpyrazine,3-methylpyridine and 2-methylpyridine. However, the additionof 2-acetylpyrrole and imidazole greatly inhibited the mutagenicityof pork juice. To confirm which IQ-type mutagenes were changed,four major mutagenic fractions were monitored after HPLC separationby their mutagenicity with Salmonella typhimurium TA98. By comparingthe retention time of authentic IQ compounds from boiled porkjuice with added tetrahydrothiophene or 2, 3-dimethylpyrazine,we show that four major HPLC fractions have significantly increasedmutagenicity compared with the same fractions in boiled porkjuice alone. In contrast, the mutagenicity of these fractionswas considerably reduced with the addition of imidazole or 2-acetylpyrroleto the pork juice before refluxing. The residual amounts oftetrahydrothiophene and 2, 3-dimethylpyrazine added to the boiledpork juice after heating were measured by gas chromatographyand found to be inversely correlated with the mutagenicity ofthe pork juice. These data suggest that these two Maillard reactionproducts are probably involved in the formation of IQ-type mutagensin boiling pork juice. ¹To whom correspondence should be addressed     

Characterization of the mutagen 2-amino-3-methylimidazo[4,5-f]quinoline prepared from a 2-methylpyridine/creatinine/acetylformaldehyde model system

A mixture of 2-methylpyridine, creatinine and aldehydes washeated in diethylene glycol containing 5% water for 1 h at 140°C.The mutagenic compounds were purified by XAD-2 column chromatography,acid/base partition, blue cotton treatment, thin layer chromatographyand HPLC. The active substances purified from each step weremonitored by their mutagenicity with Salmonella typhimuriumTA98 in the presence of S9 mix. Among the mutagens collected,2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was isolated fromHPLC, and was identified by its UV and mass spectrum using aphotodiode array detector and mass spectrometry. Our findingsappear to be the first experimental evidence to substantiatethe hypothetical pathway for the formation of IQ mutagens froma heated model system consisting of a pyridine or pyrazine derivative,an aldehyde and creatinine or pyrazine derviative, an aldehydeand creatinine or creatine. ²To whom correspondence should be addressed     

Contribution of theafulvins to the antimutagenicity of black tea: their mechanism of action

Theafulvins were isolated from black tea aqueous infusions andtheir antimutagenic activity was evaluated against a numberof food carcinogens. Theafulvins gave rise to a concentration-dependentinhibition of the mutagenicity of 2-amino-3-methylimidazo-[4,5-f]quinoline,2-amino-lmethyI-6-phenylimidazo[4,5-b]pyridine, benzo[a]pyrene,7,12-dimethylbenz[a]anthracene,nitrosopyrrolidine and nitrosopiperidine, but, in contrast,the mutagenicity of aflatoxin B₁ was enhanced. The mutagenicityexhibited by N'-methyl-N'-nitro-N-nitrosoguanidine and 9-aminoacridinewas not influenced and weakly inhibited by theafulvins, respectively.The p-hydroxylation of aniline and the O-dealkylations of methoxy-,ethoxy- and, to a lesser extent, pentoxyresorufin were inhibitedby theafulvins in a concentration- dependent manner. When microsomalmetabolism was terminated after metabolic activation of thepromutagens, incorporation of the theafulvins into the activationsystem did not modulate the mutagenic response. It is concludedthat theafulvins play an important role in the antimutagenicactivity of black tea by inhibiting cytochrome P450-dependentbioactivation of the carcinogens. ¹To whom correspondence should be addressed. Tel: +44 1483 259709; Fax: +44 1483 576978; Email: c.ioannides{at}surrey.ac.uk     

Naturally occurring diallyl disulfide inhibits the formation of carcinogenic heterocyclic aromatic amines in boiled pork juice

Three heterocyclic aromatic amines, 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), 2-amino-3, 4-dimethylimid-azo[4, 5-f]quinoxalineand 2-amino-3, 4-dimethylimidazo-[4, 5-f)quinoline, have beenfound in boiled pork juice. We have investigated the effectof naturally occurring organo-sulfur compounds, which are presentin garlic and onion, on mutagen formation in boiled pork juice.Six organosulfur compounds-diallyl disulfide (DAD), dipropyldisulfide (DPD), diallyl sulfide (DAS), allyl methyl sulfide(AMS), allyl mercaptan (AM) and cysteine-were added separatelyto the pork juice before reflux boiling and then the muta-genicityof each sample was examined with the Salmonella typhimuriumstrain TA98 in the presence of S9 mix. All six compounds werefound to inhibit the mutagenicity of boiled pork juice. Thegreatest inhibitory effect was observed with DAD and DPD, andthis was 111-fold higher than that of the lowest, cysteine.To elucidate the inhibitory effect of DAD on mutagen formationin boiled pork juice, the major mutagenic fractions were monitoredafter HPLC separation by their mutagenicity with S.typhimuriumTA98. By comparing the retention times of authentic IQ compoundsfrom boiled pork juice with those following the addition ofDAD, we showed that the mutagenicity of three major fractionswas significantly inhibited compared with those same fractionsin boiled pork juice alone. In addition, the Maillard reactionproducts (MRPs) in the boiled pork juice with and without theaddition of DAD were quantified and identified by capillarygas chromatography and gas chromatography-mass spectrometry.The results show that the reduction in the total amount of MRPs(pyridines, pyrazines, thiophenes and thiazoles) in boiled porkjuice after boiling for 12 h is correlated with their mutagenicity.Among the MRPs, tetrahydrothiophene-3-one exhibited the strongestcorrelation. These data suggest that the inhibition of IQ mutagenformation by DAD is mediated through the reduction of MRPs production. ³To whom correspondence should be addressed     

Relationship between antimutagenic activity and major components of various teas

The objectives of this study were to determine the major componentsin tea leaves and tea extracts and to study the relationshipbetween chemical content and antimutagenic activity of varioustea extracts. The amount of catechins in various tea extractswas in the order: green tea (26.7%) > oolong tea (23.2%)> pouchong tea (15.8%) > black tea (4.3%). The amountsof caffeine and phenolic compounds in oolong tea extracts were8.3 and 32.4%, respectively; these amounts were greater thanthose in the other three tea extracts. The ascorbic acid ingreen tea extracts was a little higher than in oolong and pouchongtea extracts. The amount of catechins in tea leaves also showedthe order: nonfermented (green tea) > semifermented (pouchongtea and oolong tea) < fermented tea (black tea). The amountsof caffeine and phenolic compounds in oolong tea leaves arealso higher than in other tea leaves. Besides water solublecomponents, tea leaves also contain several lipid soluble chemicalssuch as (ß-carotene and tocopherols. The tea extracts,especially oolong and pouchong teas, markedly inhibited themutagenicity of 2-amino-3-methylimidazo( 4,5-f)quinoline (IQ),3-amino-l,4-dimethyl-5H-pyrido- (4,3-b)indole (Trp-P-1), 2-amino-6-methyl-dipyrido(l,2-a:3',2'-d)imidazole(Glu-P-1), benzo[a]pyrene (B[a]P) and aflatoxin B₁ (AFB₁). Theinhibitory effect of tea extracts against the mutagenicity ofIQ and Glu-P-1 in Salmonella typhimurium TA100 showed a significant(P < 0.05) correlation to the contents of catechins and ascorbicacid. The antimutagenic activity of tea extracts to Trp-P-1in TA98 or TA100 was well correlated (P < 0.05) to the caffeinecontents. No significant (P > 0.05) correlation was foundbetween the antimutagenicity of tea extracts to B[a]P and AFB₁in TA100 and the content of major components in tea extracts. ¹To whom correspondence should be addressed     

Modulation of the potency of promutagens and direct acting mutagens in bacteria by inhibitors of the multidrug resistance mechanism

Multiple drug resistance (MDR) mechanisms are known to limitthe effectiveness of some cancer chemotherapies, probably throughenhancing P-glycoprotein-mediated drug efflux from mammaliancells. Similar mechanisms appear to act in other organisms,including bacteria, and may affect not only the toxicity butalso the mutagenicity of certain chemicals. At least in someexperimental situations, MDR can be overcome through concomitanttreatment of the cells with various types of inhibitors. TwoMDR inhibitors, verapamil, a calcium channel blocker, and trifluoperazine,a calmodulin inhibitor, were assayed for their ability to modulatethe potency of nine mutagens with varying mechanisms of actionin various Salmonella typhimurium his^– strains. Neitherverapamil nor trifluoperazine affected the direct mutagenicityof sodium dichromate and 2-methoxy-6-chloro-9[3-(2-chloroethyl)amino-propylamino]dihydrochloride (ICR 191) or the S9-mediated mutagenicity ofbenzol[a]pyrene and 2-amino-3, 4-dimethylamidazo[4,5-f]quinoline(MelQ). Both modulators enhanced the direct mutagenicity ofdoxorubicin. Moreover, trifluoperazine sharply increased theS9-mediated mutagenicity of cyclophosphamide and 2-aminofluorene,while it consistently decreased the mutagenicity of 3-amino-l-methyl-5H-pyrido[4,3-b]indole(Trp-P-2). The contrasting effect towards the aromatic amine2-aminofluorene and the heterocyclic amine Trp-P-2, representativeof important chemical families responsible for the bacterialmutagenicity of cigarette smoke, may explain the observed lackof influence of trifluoperazine on the mutagenicity of a cigarettesmoke condensate. These observations extend the known rangeof chemical types whose mutagenicity can be modulated by inhibitorsof MDR and suggest that there may be value in adding MDR inhibitors,especially trifluoperazine, to optimize the detection of mutagenicityby certain types of chemicals in the Salmonella/manunahan microsomemutagenicity test. ³To whom correspondence should be addressed. Tel: +39 10 353 8500; Fax: +39 10 353 8504; Email: sdf{at}unige.it     

Mutagenic nitrenes/nitrenium ions from azido-imidazoarenes and their structure--activity relationships

New heterocyclic arylazides (azido-imidazoarenes) structurallyrelated to the food mutagen/carcinogen 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) have been prepared. Their photolysis yieldsnitrenes and/or nitrenium ions which induce mutations in Salmonellatyphimurium. The relationships between the chemical structureand mutagenic activity of these species are the same as thosefound in our previous studies of the amino- and nitro-imidazoarenes.Therefore the efficiency of the reaction with DNA of the ultimatemetabolite, the nitrene/nitrenium ion, is the critical stepgoverning the mutagenic potency of the amino- and nitro-imidazoarenes.The efficiency of DNA-binding depends on the delocalizationof the positive charge of the nitrenium ion or of the electrondeficiency of the nitrene. It is typical of the N-1-substitutedand N-3-substituted arenimidazolyl-nitrenium ions that theycan form another nitrenium resonance structure very similarto the parent nitrenium ion structure. We suggest that thisproperty of the nitrene/nitrenium ion, in combination with itsaromatic structure facilitating carbonium ion resonance structures,is the basic reason for the extremely potent mutagenic activityof IQ and related food mutagens/carcinogens. ^*This publication is dedicated to Professor Dr D.Henschler onthe occasion of his 65th birthday in November. ¹To whom correspondence should be addressed      

Genotoxicity of compounds from cooked beef in repair-deficient CHO cells versus Salmonella mutagenicity

A series of compounds isolated on the basis of their muta-genicityin the Ames/Salmonella reversion assay were previously identifiedin fried beef and chemically synthesized for further evaluation.In this study three of these compounds were tested for genotoxiceffects in the UV5 line of Chinese hamster ovary (CHO) cells,which is deficient in nucleotide excision repair. Both 2-amino-3,4-dimethyl-imidazo]4, 5-f]quinoline (MeIQ) and 2-amino-3, 8-dimethyl-imidazo[4,5-f]quinoxaline (MelQx) gave very weak responses for cell killing,hprt mutation induction and sister chromatid exchange. Theseeffects occurred at doses in the range of 100–800 µg/ml( solubility limit), and dose-dependent increases were notobserved. Induction of chromosomal aberrations did not occurwith either compound. Nor did either of these compounds producedifferential cytotoxicity in normal CHO cells versus UV5 cells,indicating that potentially repairable DNA damage was not responsiblefor the observed cell killing. In contrast to these results,2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP), whichconstitutes > 90% of the mass of bacterial mutagens in beef,was strongly positive for all endpoints at doses in the range1–3 µg/ml. PhIP also gave marked differential cytotoxicity(ratio of 6) and cell survival curves that were strongly dependenton repair capacity. Because PhIP is 50- to 300-fold less mutagenicthan MelQ and MelQx in Salmonella TA1538, these results pointto major differences between the bacterial and mammalian assaysin terms of the relative potency of these food-related compounds.     

The structure--activity relationships of flavonoids as inhibitors of cytochrome P-450 enzymes in rat liver microsomes and the mutagenicity of 2-amino-3-methyl-imidazo[4,5-f]quinoline

The antimutagenicity of 19 naturally occurring flavonoids andtheri derivatives including flavones, flavonols, flavanoes,inoflavones and flavanols were determined using Salmonella typhimuriumTA98 against 2-amino-3-methylimidazo[4,5-f] quinoline (IQ) inthe presence of Aroclor 1254-induced rat hepatic S9. In general,a relationship between the chemical structure of flavonoidsand their antimutagenicity was found for compounds containingone or more of the following featutes: (i) C4 keto group, (ii)aglycone, (iii double bond at positions C2 and C3, (iv) phenylgroup at position C2, and (v) three hydroxyl substituents atpositons C4', C5 and C7. The inhibitory effects of flavonoidson activities of 7-ethoxycoumarin deethylase (ECD) and 7-ethoxyresorufindeethylase (ESD) of Aroclor 12540-induced hepatic microsomeswere also examined. In addition, we studied the effects of flavonoidson the metabolism of IQ by Aroclor 1254-induced microsomes usinghigh-performance liquid chromatography. The antimutagenicitycorrelated with the inhibition of cytochrome P-450IA1-linkedESD and P-450IA2-linked ECD activity in hepatic microsomes,and with an inhibition of N-hydroxy-IQ fromation from IQ metabolismby hepatic microsomes. These reslute indicated that flavonesor flavonols that contasin C5, C7 and C4' hydroxyl groups arepotent inhibitors of P-450 enzyme activities induced by Aroclor1254 (P-450IA1 and P-450IA2), and masy potentially be usefulas chemopreventive agents against heterocyclic amine-inducedmutagenesis or carcinogenesis. ¹To whom correspondence sould be addressed     

Genotoxicity of the cooked-food mutagens IQ and MeIQ in primary cultures of rat, hamster, and guinea pig hepatocytes

To investigate possible interspecies differences in the hepatocellular genotoxicity of the food-borne mutagens 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (MeIQ), primary cultures of rat, hamster, and guinea pig hepatocytes were established. The induction of DNA repair activity in cultures exposed to various concentrations of IQ and MeIQ was determined by liquid scintillation spectrometry. DNA repair responses to MeIQ were, in general, greater than those elicited by IQ. In all three preparations of rat and hamster hepatocytes and in two of three preparations of guinea pig cells, MeIQ produced statistically significant (p less than 0.05) repair responses. IQ stimulated significant levels of repair in all three rat hepatocyte preparations and in two of three hamster cell preparations. In guinea pig cells exposed to IQ, no significant repair activity was observed. These results indicate that the genotoxicity of IQ and MeIQ in hepatic cells in species-dependent.     

In vitro reaction of hydroxyamino derivatives of MelQx, Glu-P-1 and Trp-P-1 with DNA: 32P-postlabelling analysis of DNA adducts formed in vivo by the parent amines and in vitro by their hydroxyamino derivatives

The synthetic hydroxyamino derivatives of three mutagenic andcarcinogenic heterocyclic amines present in cooked foods andamino acid pyrolysates, 2-amino-3,8-dimethylimidazo-[4,5-f)quinoxaIine(MeIQx), 2-amino-6-methyldipyrido[l,2-a:3',2'-d]imidazole (Glu-P-1)and 3-amino-l,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), werereacted with DNA in vitro. Their reactivities were increasedby addition of 10-fold excess of acetic anhydride. ³²P-Postlabellinganalysis of the adducts formed in these in vitro reactions revealedthat almost all the adducts of the hydroxyamino derivativesof MeIQx and Glu-P-1 were the same as those formed in liverDNA of rats intragastrically treated with the parent amines.In contrast, analysis of Trp-P-1 -DNA adducts showed that theadducts formed in vitro were minor components of those formedin vivo; the two main adducts formed in vivo were not formedin vitro. Thus, MeIQx and Glu-P-1 may be metabolized in vivoto hydroxyamino derivatives and/or their esterified forms, suchas N-acetoxy derivatives that form DNA adducts. Formation ofadducts by Trp-P-1, however, may occur through more complicatedmetabolic pathways. Elucidation of the structures of DNA adductsin vivo is necessary to clarify this problem. ¹Present addresses: Department of Chemistry, Swedish Universityof Agricultural Sciences, PO Box 7015, S-750 07 Uppsala, Sweden      

Biotransformation of genotoxic agents in marine sponges. Mechanisms and modulation

Marine sponges do not appear to suffer from neoplastic diseases,in spite of possible high exposures resulting from their natureas sessile bottom filter feeders which pump large volumes ofsea water. The assessment of several parameters related to thebiotransformation of mutagens/carcinogens showed that the metabolicmachinery of sponge medulla cells is mainly oriented towardsdetoxification, with some differences depending on species (Geodiacydonium or Tethya aurantium). Glutathione (GSH) levels wereunexpectedly high in these cells, especially in Geodia, in whichthe concentration of this tripeptide was more than twice thatmeasured in liver preparations from untreated rats, at leastwhen related to the protein content. The oxidoreductive enzymeactivities involved in the glutathione cycle were balanced insuch a way as to favour a high GSH: oxidized glutathione (GSSG)ratio. GSH S-transferase activity was conversely rather low,compared to that of rat liver, and the dehydrogenases involvedin the hexose monophosphate shunt were high in Tethya but lowin Geodia. The metabolism of mutagens was investigated by usingthe Salmonella typhimurium his strains TA100, TA98 and YG1024.Sponge S12 fractions failed to activate aflatoxin B1, benzo[a]pyreneand the two heterocyclic amines 3-amino-1-methyl-5H-pyrido[4,3-b)indole and 2-amino-3, 4-dimethyl-imidazo[4, 5-flquinoline.Although far less efficiently than untreated rat liver S12 fractions,Geodia and especially Tethya preparations weakly activated thethree aromatic amines 2-acetylaminoflurence, 2-aminofluorenceand 2-aminoanthracene. On the other hand, sponge S12 fractionswere remarkably efficient in degreasing the mutagenic potencyof the direct acting mutagens 4-nitroquinoline 1-oxide and sodiumdichromate. The possibility of modulating biochemical parametersand metabolism of mutagens was explored by exposing sponge cubesto a variety of agents, either individually or in combinationor sequentially. The most significant effects were a depletionof GSH produced by treatment with the buthionine sulfoximineand, at least in part of the experiments, an increase of GSHstores produced by treatment with the thiol N-acetylcysteine.Irrespective of combination with other agents, exposure of spongesto 2-acetylaminofluorene resulted in an autoinduction of metabolicactivation of the same compound and of its related compound2-aminofluorene. Treatments with methimazole or verapamil hadminor effects on the investigated parameters. However, in separateexperiments verapamil, a calcium channel blocker inhibitingthe P-glycoprotein pump, was found to enhance the accumulationof radioactive vincristine in Geodia and even more in Tethya,thus confirming the occurrence of a multixenobiotic resistancemechanism in marine sponges.     

Intracellular calcium increase and acrosome reaction in response to progesterone in human spermatozoa are correlated with in-vitro fertilization

In this study we have investigated responsiveness to progesteronein spermatozoa from a group of unselected male partners of couplesundergoing in-vitro fertilization (IVF). We evaluated progesterone-stimulatedintracellular Ca²⁺ ([Ca²⁺]_i) and percentage increase in acrosomereaction in the same sperm sample used for oocyte inseminations.[Ca²⁺]_i was measured with a fluorimetric method, while the acrosomereaction was assessed using a fluorescent probe (fluoresceinisothiocyanate-labelled peanut lectin). The average percentage[Ca²⁺]_i as well as the rate of increase in the frequency ofacrosome reaction following progesterone challenge were significantlylower (P < 0.005) in the group of patients with a fertilizationrate <50%. In addition, significant correlations betweenthe fertilization rate and the progesterone-stimulated [Ca²⁺]_iand acrosome reaction increases (r = 0.78 and r = 0.79 respectively)were observed. Furthermore, in cases of fertilization failure,no increase of [Ca²⁺]_i or acrosome reaction was observed inresponse to progesterone with the exception of one case. Ourresults indicate that [Ca²⁺]_i and acrosome reaction increasesin response to progesterone can be of value in the predictionof sperm fertilizing ability. As the two parameters were significantlycorrelated to each other (r = 0.86), the two assays have similarIVF predictive value and might be used interchangeably as adiagnostic tool in the assignment of male patients to the differentkinds of assisted fertilization techniques.     

Effect of tryptamine on the mutagenic activity of 2-amino-3-methylimidazo(4,5-f) quinoline (IQ) and related azaarenes in the Ames test

The bioactivation of the azaarenes 2-amino-3-methylimid-azo(4,5-f) quinoline (IQ), 2-amino-3, 4-dimethylimidazo(4, 5-f) quinoline(MeIQ) and 2-amino-3, 8-dimethylimidazo(4, 5-f) quinoxaline(MeIQx) to mutagens by hepatic S9 preparations derived fromAroclor-pretreated Wistar rats was inhibited by tryptamine (2–50µM).However, with similar prepations derived from Sprague-Dawleyrats, bioactivation of IQ and MeIQx was less markedly inhibitedby tryptamine while metabolic activation of MeIQ was enhanced.In the absence of cytosol, activation of IQ by microsomal preparationsof both rat strains was inhibited by tryptamine. Cytosolic fractionsfrom both rat strains were incapable of activation of IQ perse but increased the mutagenicity of the microsomal metabolite(s).This potentiation of the mutagenic activity by cytosol derivedfrom Wistar rats was also inhibited by tryptamine whereas nosignificant inhibition was observed with cytosolic preparationsfrom Sprague-Dawley rats. There appear to be two alternativepathways of microsomal metabolism of IQ: a tryptamine-sensitivepathway, probably involving the formation of the N-hydroxymetabolite;and a tryptamine-insensitive pathway producing weakly mutagenicor non-mutagen metabolites which are activated to a potent mutagenby the cytosol. The tryptamine-insensitive pathway appears tobe the major route of activation of the azaarenes in microsomalpreparations from Sprague-Dawley rats and the principal activationroute for MeIQ in both rat strains.     

Endocrinology and paracrinology: Activation of elements of the phosphatidylinositol pathway in the primate corpus luteum by prostaglandin E2

The current study was designed to examine the effects of prostaglandin(PG) E² on progesterone production by primate luteal cells collectedduring the late luteal phase. PGE₂ inhibited basal and humanchorionic gonadotrophin (HCG)-stimulated progesterone production(P <0.01) in late luteal phase corpora lutea. The abilityof PGE² to activate a second messenger system (phosphatidylinositolpathway) in corpora lutea of rhesus monkeys was also assessed.PGE² significantly increased the accumulation of inositol phosphates(P <0.05). This stimulation was not apparent in the earlyluteal phase but was manifested in the mid-late luteal phase.PGE² also caused a rapid, yet transient, increase (P <0.01)in intracellular free calcium ion concentrations ([Ca²⁺]_i) ina large proportion of primate luteal cells. The proportion ofluteal cells that responded to PGE² with an increase in [Ca²⁺]_iwas smaller (P <0.05) in corpora lutea collected during theearly luteal phase (12%) in comparison with those collectedduring the latter half of the luteal phase (63–66%). Changesin [Ca²⁺]_i in response to PGE₂ were similar in small and largeluteal cells. This study demonstrates that PGE₂ activates elementsof the phosphatidylinositol pathway in primate corpora lutea.This activation is augmented as the luteal phase progresses.Thus, the inhibitory effects of PGE₂ on luteal progesteroneproduction observed in the late luteal phase are associatedwith activation of elements of the phosphatidylinositol pathway. corpus luteum/phosphatidylinositol/progesterone/prostaglandin/rhesus monkey     

Development of short-term mutagenicity test systems in vitro: metabolic activation of indirectly acting mutagens by three immortal rat hepatocyte lines

The metabolic capacity to activate the indirectly acting promutagensaflatoxin B₁, cyclophosphamide, benzo[a]pyrene, 7,12-dimethylbenz[a]anthraceneand dimethylnitrosamine into DNA-reactive metabolites was investigatedin three immortalized rat hepatocyte cell lines (NRL cl-B, NRLcl-C and ARL) by analysing chromosome aberrations and sisterchromatid exchange (SCE). In all three cell lines a significantclastogenic and SCE inducing response was observed after exposureto each test compound. Furthermore, activities of the two enzymesaryl hydrocarbon hydroxylase and aldrinepoxidase, which playmajor roles in the cytochrome P450-dependent metabolism, couldbe determined in all cell lines. In contrast to the hepatocytelines in V79 Chinese hamster cells which were used as a referencecell line without any cytochrome P450 metabolizing capacity,no arylhydrocarbon hydroxylase or aldrinepoxidase activitieswere detected. A cytogenetic response to the test compoundswas only observed in the presence of the exogenous activatingsystem S9 mix. Due to the wide efficient and stable spectrumof their metabolizing capacities the tested rat hepatocyte linesoffer promising perspectives as alternative assay systems forthe detection of indirectly acting mutagens. ²To whom correspondence should be addressed     

Human oocyte activation after intracytoplasmic sperm injection

The concentration of acetoxymethyl ester of fluo-3 given inthe subsection ‘Measurement of [Ca²⁺]_i’ of the ‘Materialsand methods’ on page 512 of the above paper was incorrect.The corresponding sentence should read as follows: Oocytes were loaded with the [Ca²⁺]_i indicator fluo-3 (Mintaet al., 1989) by incubation for 45 min at 37°C with 2 µMacetoxymethyl ester of fluo-3 (fluo-3/AM, Sigma) dissolved inB2 medium.     

Induction of SCE by indirect mutagens in cultured rat hepatoma cells and in Chinese hamster V79 cells co-cultivated with hepatocyte primary cultures

The continuous rat hepatoma cell line H4IIEC3/G^– and rathepatocyte primary cultures (hpc) were compared with regardto their capacity to metabolize structurally different promutagens.The sensitivities of both activation systems were evaluatedby comparing the induction of SCE in H4IIEC3/G^– cellsthemselves with that in V79 cells co-cultured with hpc. Of thesix chemicals tested, aflatoxui B₁ (AFB1), cyclo-phosphamide,dimethylnitrosamine and nitrosomorpholine (NM) were shown tobe inducers of SCE in H4HEC3/G^– cells as well as in V79cells with hepatocyte activation. 7,12-Dimethylbenzanthracenegave positive responses in hpc/V79 co-cultures but not in H4IIEC3/G^–cells whereas benzo[a]pyrene was negative in both systems. Theseresults suggest that H4IIEC3/G^– cells retain metabolicactivities to convert different indirect mutagens into theiractive forms and clearly indicate the presence of liver specificcytochrome P-450-dependent mono-oxygenases. However, freshlyisolated hepatocytes are more efficient in metabolizing thetest compounds. Although hpc provide only external activation,the V79 cells system appears to be more sensitive for the detectionof promutagens.     

Uptake and DNA photodamage induced in plant cells in vivo by two cationic porphyrins

The in vivo uptake of two cationic porphyrins: mesotetra (4-N-methylpyridyl)porphine (T₄MPyP) and its zinc complex (ZnT₄MPyP) was determinedin Allium cepa meristematic cells. Both photosensitizers (10^–7M for 4 h) penetrated into the nucleus producing a red fluorescenceof chromatin under blue-violet (436 nm) exciting light. Theability of T₄MPyP and ZnT₄MPyP to induce DNA photodamage wasmeasured by the sister chromatid exchange (SCE) test. 5-Bromo-2'-deoxyuridine-substitutedchromosomes treated with both the porphyrins (10^–8 M for4 h) showed increased frequencies of SCE when they were postirradiatedwith 436 nm light. A higher genotoxic effect was observed forZnT₄MPyP than the other compound. ¹To whom correspondence should be addressed